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Antioxidants, Volume 13, Issue 8 (August 2024) – 143 articles

Cover Story (view full-size image): The linkage between antioxidants and calcification prevention. This study reveals how Nrf2, a key regulator of cellular antioxidant responses, fights ectopic calcification by upregulating ENPP1, an enzyme crucial for producing pyrophosphate, a calcification inhibitor. Through in vitro and in vivo experiments, including a mouse model of spinal ligament ossification, researchers show how Nrf2 activation effectively suppresses abnormal calcification. This work illuminates a novel connection between antioxidant systems and calcification, proposing innovative strategies for treating related diseases. By bridging oxidative stress research and calcification disorders, it opens new avenues for antioxidant studies, appealing to scientists and clinicians exploring ways to prevent harmful calcification. View this paper
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16 pages, 3469 KiB  
Article
Localization and Aggregation of Honokiol in the Lipid Membrane
by José Villalaín
Antioxidants 2024, 13(8), 1025; https://doi.org/10.3390/antiox13081025 - 22 Aug 2024
Viewed by 1124
Abstract
Honokiol, a biphenyl lignan extracted from bark extracts belonging to Magnolia plant species, is a pleiotropic compound which exhibits a widespread range of antioxidant, antibacterial, antidiabetic, anti-inflammatory, antiaggregant, analgesic, antitumor, antiviral and neuroprotective activities. Honokiol, being highly hydrophobic, is soluble in common organic [...] Read more.
Honokiol, a biphenyl lignan extracted from bark extracts belonging to Magnolia plant species, is a pleiotropic compound which exhibits a widespread range of antioxidant, antibacterial, antidiabetic, anti-inflammatory, antiaggregant, analgesic, antitumor, antiviral and neuroprotective activities. Honokiol, being highly hydrophobic, is soluble in common organic solvents but insoluble in water. Therefore, its biological effects could depend on its bioactive mechanism. Although honokiol has many impressive bioactive properties, its effects are unknown at the level of the biological membrane. Understanding honokiol’s bioactive mechanism could unlock innovative perspectives for its therapeutic development or for therapeutic development of molecules similar to it. I have studied the behaviour of the honokiol molecule in the presence of a plasma-like membrane and established the detailed relation of honokiol with membrane components using all-atom molecular dynamics. The results obtained in this work sustain that honokiol has a tendency to insert inside the membrane; locates near and below the cholesterol oxygen atom, amid the hydrocarbon membrane palisade; increases slightly hydrocarbon fluidity; does not interact specifically with any membrane lipid; and, significantly, forms aggregates. Significantly, aggregation does not impede honokiol from going inside the membrane. Some of the biological characteristics of honokiol could be accredited to its aptitude to alter membrane biophysical properties, but the establishment of aggregate forms in solution might hamper its clinical use. Full article
(This article belongs to the Special Issue Polyphenol-Lipid Interactions in Nutrition and Health)
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Figure 1

Figure 1
<p>(<b>A</b>) Molecular and chemical structures of honokiol and (<b>B</b>) molecular structures of the lipid molecules used in this study: POPC, POPE, POPS, PI-3P, PSM, and CHOL. The molecular structure of honokiol is shown in (<b>B</b>) in order to compare its size with the membrane lipids.</p>
Full article ">Figure 2
<p>Lateral and apical views of the initial, t = 0 ns, and lateral view of the final, t = 1 µs, dispositions of (<b>A</b>) system 1, (<b>B</b>) system 2, (<b>C</b>) system 3, and (<b>D</b>) system 4. The location of the HNK molecules in each one of the systems is also displayed. The HNK molecules and the phosphorous atoms of the phospholipids, which define the upper and lower boundaries of the membrane, are drawn in VDW representation. Lipids at t = 1 µs are drawn in transparent licorice representation. The lipid and water molecules and the chloride and sodium ions have been removed for clarity. The formation of a big oligomer can be clearly observed in system 4 (<b>D</b>).</p>
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<p>Time variation of the z-axis COM distance (middle of the membrane as a reference) for (<b>A</b>) system 1, (<b>B</b>) system 2, (<b>C</b>) system 3 and (<b>D</b>) system 4. Left panels represent the z-axis COM distance for the HNK molecules for the entire MD simulation, whereas the right panels represent the histograms corresponding to the z-axis COM distance for the last 30 ns of MD simulation time. The membrane upper and lower boundaries (z-axis distance of the phosphorous atoms of the phospholipids) is depicted in black, whereas the oxygen atoms of CHOL are depicted in orange. Red arrows in (<b>C</b>,<b>D</b>) mark the crossing of HNK molecules from the water layer into the membrane, green arrow in (<b>D</b>) marks the only HNK molecule which remained in the water solvent, whereas the red box in (<b>D</b>) marks the crossing of the HNK oligomer into the membrane.</p>
Full article ">Figure 4
<p>(<b>A</b>) Average z-COM for the oxygen (<span style="color:#2C05BB">■□</span>) and allyl carbon (<span style="color:#FF0000">■□</span>) atoms of the HNK molecules of systems 1, 2 and 3, and (<b>B</b>) average angle between the two phenyl groups of the HNK molecules with respect to the membrane surface for systems 1, 2 and 3 (■), as indicated. System 1 comprised 4 molecules, system 2 had 9 molecules, and system 3 had 8 molecules, as indicated. The average data has been obtained for each one of the molecules and for the last 30 ns of MD simulation. (<b>C</b>) Representation of the global mean position of HNK in the membrane (grey and orange boxes delimitate the phospholipid phosphorous and oxygen atoms of CHOL, respectively). See text for details.</p>
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<p>Average deuterium order parameter <span class="html-italic">S<sub>CD</sub></span> calculated for the hydrocarbon chains of the phospholipids in systems 1, 2 and 3. The (<b>A</b>,<b>C</b>,<b>E</b>,<b>G</b>) oleoyl and (<b>B</b>,<b>D</b>,<b>F</b>,<b>H</b>) palmitoyl acyl chains of (<b>A</b>,<b>B</b>) POPC, (<b>C</b>,<b>D</b>) POPE, (<b>E</b>,<b>F</b>) POPS and (<b>G</b>,<b>H</b>) PI-3P, as well as the sphingosyl (<b>I</b>) and palmitoyl (<b>J</b>) acyl chains of PSM. The data correspond to the bulk phospholipid acyl chains (-■-) and the phospholipid acyl chains within 5 Å of HNK molecules (-<span style="color:#FF0000">●</span>-). The analyses were carried out for the last 30 ns of simulation.</p>
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12 pages, 3814 KiB  
Article
Avicularin Attenuated Lead-Induced Ferroptosis, Neuroinflammation, and Memory Impairment in Mice
by Jun-Tao Guo, Chao Cheng, Jia-Xue Shi, Wen-Ting Zhang, Han Sun and Chan-Min Liu
Antioxidants 2024, 13(8), 1024; https://doi.org/10.3390/antiox13081024 - 22 Aug 2024
Viewed by 980
Abstract
Lead (Pb) is a common environmental neurotoxicant that results in abnormal neurobehavior and impaired memory. Avicularin (AVL), the main dietary flavonoid found in several plants and fruits, exhibits neuroprotective and hepatoprotective properties. In the present study, the effects of AVL on Pb-induced neurotoxicity [...] Read more.
Lead (Pb) is a common environmental neurotoxicant that results in abnormal neurobehavior and impaired memory. Avicularin (AVL), the main dietary flavonoid found in several plants and fruits, exhibits neuroprotective and hepatoprotective properties. In the present study, the effects of AVL on Pb-induced neurotoxicity were evaluated using ICR mice to investigate the molecular mechanisms behind its protective effects. Our study has demonstrated that AVL treatment significantly ameliorated memory impairment induced by lead (Pb). Furthermore, AVL mitigated Pb-triggered neuroinflammation, ferroptosis, and oxidative stress. The inhibition of Pb-induced oxidative stress in the brain by AVL was evidenced by the reduction in malondialdehyde (MDA) levels and the enhancement of glutathione (GSH) and glutathione peroxidase (GPx) activities. Additionally, in the context of lead-induced neurotoxicity, AVL mitigated ferroptosis by increasing the expression of GPX4 and reducing ferrous iron levels (Fe2+). AVL increased the activities of glycogenolysis rate-limiting enzymes HK, PK, and PYG. Additionally, AVL downregulated TNF-α and IL-1β expression while concurrently enhancing the activations of AMPK, Nrf2, HO-1, NQO1, PSD-95, SNAP-25, CaMKII, and CREB in the brains of mice. The findings from this study suggest that AVL mitigates the memory impairment induced by Pb, which is associated with the AMPK/Nrf2 pathway and ferroptosis. Full article
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Figure 1

Figure 1
<p>The schematic representation of timeline for the experiments.</p>
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<p>Avicularin (AVL) inhibited Pb-induced ferroptosis in the brains of mice. (<b>A</b>) Immunofluorescence analysis of ferroptosis in the cerebral cortex (200×) (GPX4, green; NeuN, red; Merged, yellow); (<b>B</b>) Western blot analysis of the GPX4 proteins in the brains. (<b>C</b>) Fe<sup>2+</sup> levels in the brains. Data are expressed as mean ± SEM (<span class="html-italic">n</span> = 3). Values not sharing a common superscript letter (a–d) differ significantly at <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>Effect of avicularin on Pb-induced oxidative stress in brains of mice. (<b>A</b>) MDA content; (<b>B</b>) GPx activity; (<b>C</b>) content. Data are expressed as mean ± S.E.M (<span class="html-italic">n</span> = 7). Values not sharing a common superscript letter (a–d) differ significantly at <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>The protein expression of the glucose metabolism in the brains of mice. (<b>A</b>) Western blotting was used to assess the expression levels of glucose metabolism-related proteins; (<b>B</b>–<b>D</b>) HK2, PKM2 and PYG protein quantification. Data are expressed as mean ± SEM (<span class="html-italic">n</span> = 3). Values not sharing a common superscript letter (a–d) differ significantly at <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>Avicularin (AVL) inhibited Pb-induced inflammation in the brains of mice. (<b>A</b>) Western blotting was used to assess the expression levels of inflammatory cytokines; (<b>B</b>,<b>C</b>) TNF-α and IL-1β protein quantification. Data are expressed as mean ± SEM (<span class="html-italic">n</span> = 3). Values not sharing a common superscript letter (a–d) differ significantly at <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>Avicularin (AVL) activated the AMPK/Nrf2 pathway in the brains of mice. (<b>A</b>–<b>D</b>) p-AMPK, nucleus Nrf2, HO-1, and NQO1 protein quantification. Data are expressed as mean ± SEM (<span class="html-italic">n</span> = 3). Values not sharing a common superscript letter (a–d) differ significantly at <span class="html-italic">p</span> &lt; 0.05.</p>
Full article ">Figure 7
<p>Avicularin (AVL) activated synaptic regulation pathway in the brains of mice. (<b>A</b>–<b>D</b>) PSD-95, SNAP-25, p-CREB, and CaMKII protein quantification. Data are expressed as mean ± SEM (<span class="html-italic">n</span> = 3). Values not sharing a common superscript letter (a–d) differ significantly at <span class="html-italic">p</span> &lt; 0.05.</p>
Full article ">Figure 8
<p>Schematic diagram showing the possible protective effects of avicularin (AVL) in Pb-induced brain injury. The → indicates activation or induction, and ┤ indicates inhibition or blockade.</p>
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38 pages, 9055 KiB  
Article
A Multi-Target Pharmacological Correction of a Lipoyltransferase LIPT1 Gene Mutation in Patient-Derived Cellular Models
by David Gómez-Fernández, Ana Romero-González, Juan M. Suárez-Rivero, Paula Cilleros-Holgado, Mónica Álvarez-Córdoba, Rocío Piñero-Pérez, José Manuel Romero-Domínguez, Diana Reche-López, Alejandra López-Cabrera, Salvador Ibáñez-Mico, Marta Castro de Oliveira, Andrés Rodríguez-Sacristán, Susana González-Granero, José Manuel García-Verdugo and José A. Sánchez-Alcázar
Antioxidants 2024, 13(8), 1023; https://doi.org/10.3390/antiox13081023 - 22 Aug 2024
Viewed by 1528
Abstract
Mutations in the lipoyltransferase 1 (LIPT1) gene are rare inborn errors of metabolism leading to a fatal condition characterized by lipoylation defects of the 2-ketoacid dehydrogenase complexes causing early-onset seizures, psychomotor retardation, abnormal muscle tone, severe lactic acidosis, and increased urine [...] Read more.
Mutations in the lipoyltransferase 1 (LIPT1) gene are rare inborn errors of metabolism leading to a fatal condition characterized by lipoylation defects of the 2-ketoacid dehydrogenase complexes causing early-onset seizures, psychomotor retardation, abnormal muscle tone, severe lactic acidosis, and increased urine lactate, ketoglutarate, and 2-oxoacid levels. In this article, we characterized the disease pathophysiology using fibroblasts and induced neurons derived from a patient bearing a compound heterozygous mutation in LIPT1. A Western blot analysis revealed a reduced expression of LIPT1 and absent expression of lipoylated pyruvate dehydrogenase E2 (PDH E2) and alpha-ketoglutarate dehydrogenase E2 (α-KGDH E2) subunits. Accordingly, activities of PDH and α-KGDH were markedly reduced, associated with cell bioenergetics failure, iron accumulation, and lipid peroxidation. In addition, using a pharmacological screening, we identified a cocktail of antioxidants and mitochondrial boosting agents consisting of pantothenate, nicotinamide, vitamin E, thiamine, biotin, and α-lipoic acid, which is capable of rescuing LIPT1 pathophysiology, increasing the LIPT1 expression and lipoylation of mitochondrial proteins, improving cell bioenergetics, and eliminating iron overload and lipid peroxidation. Furthermore, our data suggest that the beneficial effect of the treatment is mainly mediated by SIRT3 activation. In conclusion, we have identified a promising therapeutic approach for correcting LIPT1 mutations. Full article
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>The characterization of the physiopathology of mutant <span class="html-italic">LIPT1</span> fibroblasts. (<b>A</b>). C1 and C2: control cells. The Western blot analysis of the mutated protein LIPT1, E2 subunits of multienzyme complexes PDH and KGDH, and their lipoylated form. Actin expression and Ponceau S staining were used to demonstrate equal protein loading. (<b>B</b>). Band densitometry of Western blot data referred to actin and normalized to the mean of controls. (<b>C</b>). PDH complex activity was measured by PDH Enzyme Activity Dipstick Assay Kit. (<b>D</b>). Band intensity of PDH complex activity was obtained by ImageLab software. (<b>E</b>). KGDH activity was measured by α-Ketoglutarate Dehydrogenase Activity Assay Kit. Data represent the mean ± SD of 3 independent experiments. *** <span class="html-italic">p</span> &lt; 0.001 and **** <span class="html-italic">p</span> &lt; 0.0001 between the control and patient’s fibroblasts. a.u.: arbitrary units.</p>
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<p>The analysis of iron accumulation in mutant <span class="html-italic">LIPT1</span> fibroblasts. (<b>A</b>). The control and patient’s fibroblasts (LIPT1) were stained with Prussian Blue staining. Mutant cells were treated with 100 µM Deferiprone (DEF). Images were acquired by a Zeiss Axio Vert A1 microscope. Scale bar: 20 µm. (<b>B</b>). The quantification of Prussian Blue staining-integrated density. Images were analyzed by the ImageJ software (at least 30 images were analyzed per each condition and experiment). (<b>C</b>). The quantification of iron content by ICP-MS. (<b>D</b>). Lipofuscin accumulation was assessed by Sudan Black staining. Mutant cells were treated with 100 µM DEF. Images were acquired by a Zeiss Axio Vert A1 microscope. A PKAN (pantothenate kinase-associated neurodegeneration) cell line was used as a positive control of lipofuscin accumulation. Scale bar: 20 µm. (<b>E</b>). The quantification of Sudan Black staining-integrated density (at least 30 images were analyzed per each condition and experiment). Data represent the mean ± SD of 3 independent experiments. **** <span class="html-italic">p</span> &lt; 0.0001 between the control and patient’s fibroblasts. #### <span class="html-italic">p</span> &lt; 0.0001 between mutant fibroblasts untreated and treated with Deferiprone. a.u.: arbitrary units.</p>
Full article ">Figure 3
<p>The quantification of the proliferation ratio in the galactose medium. Control and mutant cells (LIPT1) were seeded in the glucose medium and treated with the active compounds individually (<b>A</b>) and in a combination cocktail (<b>B</b>) for seven days. Then, the glucose medium was changed to the galactose medium, the treatment was renewed, and images were taken in that moment (T0) and 72 h later (T72) by BioTek Cytation 1 Cell Imaging Multi-Mode Reader. The proliferation ratio was calculated as the number of cells in T72 divided by the number of cells in T0, in both control and mutant cells (values &gt; 1: cell proliferation; values = 1: number of cells unchanged; values &lt; 1: cell death). Representative images are included in <a href="#app1-antioxidants-13-01023" class="html-app">Supplementary Materials (Supplementary Figures S1 and S2)</a>. Data represent the mean ± SD of 3 independent experiments. **** <span class="html-italic">p</span> &lt; 0.0001 between control and mutant fibroblasts in the galactose medium (<b>A</b>). **** <span class="html-italic">p</span> &lt; 0.0001 between mutant fibroblasts in the glucose and in the galactose medium (<b>B</b>). #### <span class="html-italic">p</span> &lt; 0.0001 between untreated and CocT-treated mutant <span class="html-italic">LIPT1</span> cells in the galactose medium. a.u.: arbitrary units. ns: not significant.</p>
Full article ">Figure 4
<p><span class="html-italic">LIPT1</span> transcript levels in mutant (LIPT1) and control fibroblasts with and without treatment. Cells were treated with CocT for seven days. Data represent the mean ± SD of 3 independent experiments. **** <span class="html-italic">p</span> &lt; 0.0001 between control and mutant <span class="html-italic">LIPT1</span> fibroblasts. #### <span class="html-italic">p</span> &lt; 0.0001 between untreated and treated mutant fibroblasts. a.u.: arbitrary units.</p>
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<p>Expression levels of LIPT1, PDH E2, KGDH E2, and their lipoylated forms in control (C1, C2, and C) and mutant (LIPT1) fibroblasts before and after the supplementation with CocT. Cells were treated with CocT for seven days. (<b>A</b>). The Western blot analysis of the mutated protein LIPT1, E2 subunits of complexes PDH and KGDH, and their lipoylated forms. Actin expression and Ponceau S staining were used to demonstrate equal protein loading. (<b>B</b>). Band densitometry of Western blot data referred to actin and was normalized to the mean of controls. (<b>C</b>). PDH complex activity was measured by PDH Enzyme Activity Dipstick Assay Kit. (<b>D</b>). Band intensity of PDH complex activity was obtained by ImageLab software. (<b>E</b>). KGDH activity was measured by α-Ketoglutarate Dehydrogenase Activity Assay Kit. Data represent the mean ± SD of 3 independent experiments. *** <span class="html-italic">p</span> &lt; 0.001 and **** <span class="html-italic">p</span> &lt; 0.0001 between control and mutant LIPT1 fibroblasts. ### <span class="html-italic">p</span> &lt; 0.001 and #### <span class="html-italic">p</span> &lt; 0.0001 between untreated and treated mutant LIPT1 fibroblasts. a.u.: arbitrary units.</p>
Full article ">Figure 6
<p>The effect of CocT on iron accumulation in mutant <span class="html-italic">LIPT1</span> fibroblasts. (<b>A</b>). Untreated and treated (for seven days) control and patient (LIPT1) fibroblasts were stained with Prussian Blue staining. (<b>B</b>). Lipofuscin accumulation was assessed by Sudan Black staining. A PKAN cell line was used as a positive control of lipofuscin accumulation [<a href="#B39-antioxidants-13-01023" class="html-bibr">39</a>]. Mutant cells were treated with 100 µM DEF. Representative images were acquired by a Zeiss Axio Vert A1 microscope (at least 30 images were analyzed per each condition and experiment). Scale bar: 20 µm. (<b>C</b>). The quantification of Prussian Blue staining-integrated density. Images were analyzed by ImageJ software (at least 30 images were analyzed per each condition and experiment). (<b>D</b>). The quantification of iron content by ICP-MS. (<b>E</b>). The quantification of Sudan Black staining-integrated density. Data represent the mean ± SD of 3 independent experiments. **** <span class="html-italic">p</span> &lt; 0.0001 between control and mutant <span class="html-italic">LIPT1</span> fibroblasts. #### <span class="html-italic">p</span> &lt; 0.0001 between untreated and treated mutant <span class="html-italic">LIPT1</span> fibroblasts. aaaa <span class="html-italic">p</span> &lt; 0.0001 between untreated and treated mutant <span class="html-italic">LIPT1</span> fibroblasts with Deferiprone. a.u.: arbitrary units.</p>
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<p>Electron microscopy images of the control and patient’s (LIPT1) fibroblasts, both untreated and treated with CocT. Cells were treated with CocT for seven days. (<b>A</b>). Representative electron microscopy images. Scale bar: 2 µm. Red arrows: lipofuscin-like granules. (<b>B</b>). The quantification of lipofuscin-like aggregates per cell (at least 30 images were analyzed per each condition and experiment). Data represent the mean ± SD of 3 independent experiments. **** <span class="html-italic">p</span> &lt; 0.0001 between control and mutant <span class="html-italic">LIPT1</span> fibroblasts. #### <span class="html-italic">p</span> &lt; 0.0001 between untreated and treated mutant <span class="html-italic">LIPT1</span> fibroblasts. Magnified images are shown in <a href="#app1-antioxidants-13-01023" class="html-app">Supplementary Figure S5</a>.</p>
Full article ">Figure 8
<p>The effect of CocT on protein lipoylation. The immunofluorescence assay was performed in both untreated and treated control and mutant (LIPT1) fibroblasts. Cells were treated with CocT for seven days. (<b>A</b>). Cells were fixed and immunostained with the anti-LA antibody. TOMM20 was used as a mitochondrial marker and nuclei were visualized with DAPI staining. Scale bar: 15 µm. (<b>B</b>). The quantification of fluorescence intensity of the lipoic acid antibody. Images were analyzed by ImageJ software (at least 30 images were taken and analyzed from each condition and experiment). (<b>C</b>). The colocalization between lipoic acid and TOMM20 signals was analyzed by the Pearson correlation coefficient. The Pearson correlation coefficient was calculated by the DeltaVision system. Data represent the mean ± SD of 3 independent experiments. **** <span class="html-italic">p</span> &lt; 0.0001 between control and mutant <span class="html-italic">LIPT1</span> fibroblasts. #### <span class="html-italic">p</span> &lt; 0.0001 between untreated and treated mutant <span class="html-italic">LIPT1</span> fibroblasts. a.u.: arbitrary units.</p>
Full article ">Figure 9
<p>The effect of CocT on the expression levels of subunits of the mitochondrial electron transport chain (mtETC) complexes in control (C1 and C2) and mutant (LIPT1) cells. Cells were treated with CocT for seven days. (<b>A</b>). The Western blot analysis of proteins of complex I (NDUFA9 and mt-ND6), complex III (UQCRC1), complex IV (mt-CO2 and COX IV), and complex V (ATP5F1A). Actin expression and Ponceau S staining were used to demonstrate equal protein loading. VDAC1 was used as a mitochondrial mass marker. (<b>B</b>). Band densitometry of Western blot data referred to actin and was normalized to the mean of controls. Data represent the mean ± SD of 3 independent experiments. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and **** <span class="html-italic">p</span> &lt; 0.0001 between control and mutant <span class="html-italic">LIPT1</span> fibroblasts. # <span class="html-italic">p</span> &lt; 0.05, ### <span class="html-italic">p</span> &lt; 0.001, and #### <span class="html-italic">p</span> &lt; 0.0001 between untreated and treated mutant <span class="html-italic">LIPT1</span> fibroblasts. a.u.: arbitrary units. ns: not significant.</p>
Full article ">Figure 10
<p>The effect of CocT on mitostress bioenergetic assay control and mutant (LIPT1) fibroblasts. Cells were treated with CocT for seven days. The mitochondrial respiration profile was measured using a Seahorse XFe24 analyzer. Data represent the mean ± SD of 3 independent experiments. *** <span class="html-italic">p</span> &lt; 0.001, and **** <span class="html-italic">p</span> &lt; 0.0001 between control and mutant <span class="html-italic">LIPT1</span> fibroblasts. ## <span class="html-italic">p</span> &lt; 0.01, ### <span class="html-italic">p</span> &lt; 0.001 and #### <span class="html-italic">p</span> &lt; 0.0001 between untreated and treated mutant <span class="html-italic">LIPT1</span> fibroblasts. OCR: Oxygen Consumption Rate.</p>
Full article ">Figure 11
<p>The effect of CocT on complex I and complex IV activities in control and mutant (LIPT1) fibroblasts. Cells were treated with CocT for seven days. (<b>A</b>). Complex I activity was measured using Complex I Enzyme Activity Dipstick Assay Kit. Complex IV activity was measured using Complex IV Enzyme Activity Dipstick Assay Kit. (<b>B</b>). Band intensity was obtained using ImageLab software. Data represent the mean ± SD of 3 independent experiments. **** <span class="html-italic">p</span> &lt; 0.0001 between control and mutant <span class="html-italic">LIPT1</span> fibroblasts. ### <span class="html-italic">p</span> &lt; 0.001 and #### <span class="html-italic">p</span> &lt; 0.0001 between untreated and treated mutant <span class="html-italic">LIPT1</span> fibroblasts. a.u.: arbitrary units.</p>
Full article ">Figure 12
<p>The effect of CocT on the expression levels of proteins of the transcriptional canonical mtUPR axis in control (C1 and C2) and mutant (LIPT1) cells. Cells were treated with CocT for seven days. (<b>A</b>). The Western blot analysis of transcriptional canonical mtUPR proteins. Actin expression and Ponceau S staining were used to demonstrate equal protein loading. (<b>B</b>). Band densitometry of Western blot data referred to actin and was normalized to the mean of controls. Data represent the mean ± SD of 3 independent experiments. ** <span class="html-italic">p</span> &lt; 0.01 and **** <span class="html-italic">p</span> &lt; 0.0001 between control and mutant <span class="html-italic">LIPT1</span> fibroblasts. ## <span class="html-italic">p</span> &lt; 0.01, ### <span class="html-italic">p</span> &lt; 0.001, and #### <span class="html-italic">p</span> &lt; 0.0001 between untreated and treated mutant <span class="html-italic">LIPT1</span> fibroblasts. a.u.: arbitrary units.</p>
Full article ">Figure 13
<p>The effect of CocT on the expression levels of proteins of the SIRT3 mtUPR axis in control (C1 and C2) and mutant (LIPT1) cells. Cells were treated with CocT for seven days. (<b>A</b>). The Western blot analysis of SIRT3 mtUPR proteins. Actin expression and Ponceau staining were used to demonstrate equal protein loading. (<b>B</b>). Band densitometry of Western blot data referred to actin and was normalized to the mean of controls. Data represent the mean ± SD of 3 independent experiments. **** <span class="html-italic">p</span> &lt; 0.0001 between control and mutant <span class="html-italic">LIPT1</span> fibroblasts. #### <span class="html-italic">p</span> &lt; 0.0001 between untreated and treated mutant <span class="html-italic">LIPT1</span> fibroblasts. a.u.: arbitrary units.</p>
Full article ">Figure 14
<p>The effect of CocT on the expression levels of mitochondrial biogenesis proteins in control (C1 and C2) and mutant (LIPT1) cells. Cells were treated with CocT for seven days. (<b>A</b>). The Western blot analysis of mitochondrial biogenesis proteins. Actin expression and Ponceau S staining were used to demonstrate equal protein loading. (<b>B</b>). Band densitometry of Western blot data referred to actin and was normalized to the mean of controls. Data represent the mean ± SD of 3 independent experiments. **** <span class="html-italic">p</span> &lt; 0.0001 between control and mutant <span class="html-italic">LIPT1</span> fibroblasts. # <span class="html-italic">p</span> &lt; 0.05, ### <span class="html-italic">p</span> &lt; 0.001, and #### <span class="html-italic">p</span> &lt; 0.0001 between untreated and treated mutant <span class="html-italic">LIPT1</span> fibroblasts. a.u.: arbitrary units.</p>
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<p>The effect of CocT on SIRT3 activity and NAD<sup>+</sup>, NADH, NADt, and NAD<sup>+</sup>/NADH ratio levels in control and mutant (LIPT1) fibroblasts. Cells were treated with CocT for seven days. (<b>A</b>). Mitochondrial SIRT3 activity was determined by SIRT3 Activity Assay Kit (Fluorometric) in mitochondrial fractions. (<b>B</b>). The effect of CocT on cellular NAD<sup>+</sup> levels. (<b>C</b>). The effect of CocT on cellular NADH levels. (<b>D</b>). The effect of CocT on cellular NADt levels. (<b>E</b>). The effect of CocT on cellular NAD<sup>+</sup>/NADH ratio levels. (<b>F</b>). Consequences of SIRT3 inhibition in CocT positive effects. The quantification of the proliferation ratio of pharmacological screening in the galactose medium with 3-TYP, a SIRT3 inhibitor. Control and mutant (LIPT1) cells were seeded in the glucose medium and treated with CocT for seven days along with 50 nM 3-TYP (added for 72 h).Then, the glucose medium was changed to the galactose medium, treatment was refreshed, and photos were taken at that moment (T0) and 72 h later (T72) by BioTek Cytation 1 Cell Imaging Multi-Mode Reader. The proliferation ratio was calculated as the number of cells in T72 divided by the number of cells in T0, in both control and mutant cells (values &gt; 1: cell proliferation; values = 1: number of cells unchanged; values &lt; 1: cell death). Data represent the mean ± SD of 3 independent experiments. * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001, and **** <span class="html-italic">p</span> &lt; 0.0001 between control and mutant <span class="html-italic">LIPT1</span> fibroblasts. ## <span class="html-italic">p</span> &lt; 0.01 and #### <span class="html-italic">p</span> &lt; 0.0001 between untreated and treated mutant <span class="html-italic">LIPT1</span> fibroblasts. a.u.: arbitrary units.</p>
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<p>The effect of CocT supplementation on protein lipoylation in iNs. Control and mutant (LIPT1) cells, reprogrammed from fibroblasts to iNs, were treated with CocT for seven days. (<b>A</b>). iNs were fixed and immunostained with the anti-LA antibody. The mitochondrial network was assessed by MitoTracker<sup>TM</sup> Red CMXRos staining. Tau was used as a neuronal marker. Hoescht was used to stain nuclei. Scale bar: 15 μm. (<b>B</b>). The quantification of fluorescence intensity of the lipoic acid antibody. (<b>C</b>). The quantification of fluorescence intensity of Mitotracker<sup>TM</sup> Red CMXRos. Images were analyzed by Image J software (at least 30 images were analyzed per each condition and experiment). **** <span class="html-italic">p</span> &lt; 0.0001 between control and mutant <span class="html-italic">LIPT1</span> iNs. ### <span class="html-italic">p</span> &lt; 0.001 and #### <span class="html-italic">p</span> &lt; 0.0001 between untreated and treated mutant <span class="html-italic">LIPT1</span> iNs. a.u.: arbitrary units.</p>
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<p>The effect of CocT on iron accumulation in iNs generated from control and patient-derived fibroblasts by direct reprogramming. Control and mutant (LIPT1) iNs were treated with CocT for seven days. (<b>A</b>). Representative images were acquired by a Zeiss Axio Vert A1 microscope. Tau was used as a neuronal marker. Scale bar: 15 μm. (<b>B</b>). Quantification of Prussian Blue staining images were obtained by Image J software (at least 30 images were analyzed per each experimental condition). **** <span class="html-italic">p</span> &lt; 0.0001 between control and mutant <span class="html-italic">LIPT1</span> iNs. #### <span class="html-italic">p</span> &lt; 0.0001 between untreated and treated mutant <span class="html-italic">LIPT1</span> iNs. a.u.: arbitrary units.</p>
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20 pages, 3524 KiB  
Article
Can Rice Growth Substrate Substitute Rapeseed Growth Substrate in Rapeseed Blanket Seedling Technology? Lesson from Reactive Oxygen Species Production and Scavenging Analysis
by Kaige Yi, Yun Ren, Hui Zhang, Baogang Lin, Pengfei Hao and Shuijin Hua
Antioxidants 2024, 13(8), 1022; https://doi.org/10.3390/antiox13081022 - 22 Aug 2024
Viewed by 892
Abstract
Rapeseed (Brassica napus L.) seedlings suffering from inappropriate growth substrate stress will present poor seedling quality. However, the regulatory mechanism for the production and scavenging of reactive oxygen species (ROS) caused by this type of stress remains unclear. In the current study, [...] Read more.
Rapeseed (Brassica napus L.) seedlings suffering from inappropriate growth substrate stress will present poor seedling quality. However, the regulatory mechanism for the production and scavenging of reactive oxygen species (ROS) caused by this type of stress remains unclear. In the current study, a split plot experiment design was implemented with two crop growth substrates—a rice growth substrate (RIS) and rapeseed growth substrate (RAS)—as the main plot and two genotypes—a hybrid and an open-pollinated variety (Zheyouza 1510 and Zheyou 51, respectively)—as the sub-plot. The seedling quality was assessed, and the ROS production/scavenging capacity was evaluated. Enzymatic and non-enzymatic systems, including ascorbic acid and glutathione metabolism, and RNA-seq data were analyzed under the two growth substrate treatments. The results revealed that rapeseed seedling quality decreased under RIS, with the plant height, maximum leaf length and width, and aboveground dry matter being reduced by 187.7%, 64.6%, 73.2%, and 63.8% on average, respectively, as compared to RAS. The main type of ROS accumulated in rapeseed plants was hydrogen peroxide, which was 47.8% and 14.1% higher under RIS than under RAS in the two genotypes, respectively. The scavenging of hydrogen peroxide in Zheyouza 1510 was the result of a combination of enzymatic systems, with significantly higher peroxidase (POD) and catalase (CAT) activity as well as glutathione metabolism, with significantly higher reduced glutathione (GSH) content, under RAS, while higher oxidized glutathione (GSSH) was observed under RIS. However, the scavenging of hydrogen peroxide in Zheyou 51 was the result of a combination of elevated oxidized ascorbic acid (DHA) under RIS and higher GSH content under RAS. The identified gene expression levels were in accordance with the observed enzyme expression levels. The results suggest that the cost of substituting RAS with RIS is a reduction in rapeseed seedling quality contributing to excessive ROS production and a reduction in ROS scavenging capacity. Full article
(This article belongs to the Special Issue Oxidative Stress and Antioxidant Defense in Plants)
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<p>Phenotype of blanket rapeseed seedlings at 30 d old: Zheyouza 1510 and Zheyou 51 varieties under rice growth substrate (RIS) and rapeseed growth substrate (RAS) in the red box.</p>
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<p>ROS production under rice and rapeseed growth substrates in Zheyouza 1510 and Zheyou 51 and the correlations between ROS contents and seedling quality: (<b>A</b>) hydrogen peroxide; (<b>B</b>) superoxide anions; and (<b>C</b>) Pearson’s correlation coefficients between ROS and seedling quality. Different lowercase letters indicate a significant difference among treatments using Duncan’s method (<span class="html-italic">p</span> &lt; 0.05). “*” indicates a significant difference at <span class="html-italic">p</span> &lt; 0.05. Error bars indicate the standard deviation (SD) values.</p>
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<p>ROS scavenging capacity under rice growth substrate and rapeseed growth substrate in Zheyouza 1510 and Zheyou 51: (<b>A</b>) hydroxyl free radical scavenging capacity (HFRS); (<b>B</b>) superoxide peroxide anion scavenging capacity (SOAS); and (<b>C</b>) total antioxidant capacity (T-AOC). Different lowercase letters indicate significant difference among treatments using Duncan’s method (<span class="html-italic">p</span> &lt; 0.05). Error bars indicate standard deviation (SD) values.</p>
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<p>Alteration of enzyme activities in rapeseed leaves under rice growth substrate and rapeseed growth substrate in Zheyouza 1510 and Zheyou 51: (<b>A</b>) superoxide dismutase (SOD); (<b>B</b>) peroxidase (POD); and (<b>C</b>) catalase (CAT). Different lowercase letters indicate significant difference among treatments using Duncan’s method (<span class="html-italic">p</span> &lt; 0.05). Error bars indicate standard deviation (SD) values.</p>
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<p>Analysis of ascorbic acid metabolism in rapeseed leaves under rice growth substrate (RIS) and rapeseed growth substrate (RAS) in Zheyouza 1510 and Zheyou 51: (<b>A</b>) reduced ascorbic acid (AsA); (<b>B</b>) oxidized ascorbic acid (DHA); (<b>C</b>) dehydroascorbate reductase (DHAR); (<b>D</b>) monodehydroascorbate reductase (MDHAR); (<b>E</b>) ascorbic acid oxidase (AAO); (<b>F</b>) ascorbate peroxidase (APX); and (<b>G</b>) L-galactose-1,4-lactone dehydrogenase (Gal LDH). Different lowercase letters indicate significant difference among treatments using Duncan’s method (<span class="html-italic">p</span> &lt; 0.05). Error bars indicate standard deviation (SD) values.</p>
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<p>Analysis of ascorbic acid metabolism in rapeseed leaves under rice growth substrate (RIS) and rapeseed growth substrate (RAS) in Zheyouza 1510 and Zheyou 51: (<b>A</b>) reduced ascorbic acid (AsA); (<b>B</b>) oxidized ascorbic acid (DHA); (<b>C</b>) dehydroascorbate reductase (DHAR); (<b>D</b>) monodehydroascorbate reductase (MDHAR); (<b>E</b>) ascorbic acid oxidase (AAO); (<b>F</b>) ascorbate peroxidase (APX); and (<b>G</b>) L-galactose-1,4-lactone dehydrogenase (Gal LDH). Different lowercase letters indicate significant difference among treatments using Duncan’s method (<span class="html-italic">p</span> &lt; 0.05). Error bars indicate standard deviation (SD) values.</p>
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<p>Analysis of glutathione metabolism in rapeseed leaves under rice growth substrate (RIS) and rapeseed growth substrate (RAS) in Zheyouza 1510 and Zheyou 51: (<b>A</b>) reduced glutathione (GSH); (<b>B</b>) oxidized glutathione (GSSG); (<b>C</b>) glutathione peroxidase (GPX); (<b>D</b>) glutathione reductase (GR); (<b>E</b>) glutathione-S-transferase (GST); (<b>F</b>) thioredoxin peroxidase (TPX); (<b>G</b>) glutamate cysteine ligase (GCL); and (<b>H</b>) total sulfhydryls (TSHs). Different lowercase letters indicate significant difference among treatments using Duncan’s method (<span class="html-italic">p</span> &lt; 0.05). Error bars indicate standard deviation (SD) values.</p>
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<p>Analysis of glutathione metabolism in rapeseed leaves under rice growth substrate (RIS) and rapeseed growth substrate (RAS) in Zheyouza 1510 and Zheyou 51: (<b>A</b>) reduced glutathione (GSH); (<b>B</b>) oxidized glutathione (GSSG); (<b>C</b>) glutathione peroxidase (GPX); (<b>D</b>) glutathione reductase (GR); (<b>E</b>) glutathione-S-transferase (GST); (<b>F</b>) thioredoxin peroxidase (TPX); (<b>G</b>) glutamate cysteine ligase (GCL); and (<b>H</b>) total sulfhydryls (TSHs). Different lowercase letters indicate significant difference among treatments using Duncan’s method (<span class="html-italic">p</span> &lt; 0.05). Error bars indicate standard deviation (SD) values.</p>
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<p>RNA-seq analysis on effects of rice growth substrate (RIS) and rapeseed growth substrate (RAS) on seedling quality in Zheyouza 1510: (<b>A</b>) volcano plot of differentially expressed genes under two crop growth substrates; (<b>B</b>) KEGG analysis of differentially expressed genes under two crop growth substrates; (<b>C</b>) identified genes in enzymatic and non-enzymatic systems for ROS scavenging; and (<b>D</b>) correlation analysis between expression levels of selected genes via RNA-seq and qRT-PCR.</p>
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13 pages, 475 KiB  
Article
Paraoxonase I Activity and Its Relationship with Nutrition in Amyotrophic Lateral Sclerosis
by Belén Proaño, María Benlloch, Sandra Sancho-Castillo, Jesús Privado, Guillermo Bargues-Navarro, Claudia Emmanuela Sanchis-Sanchis, Palmira Martínez Bolós, Ana Belén Carriquí-Suárez, Laura Cubero-Plazas, Jose Luis Platero Armero, Dolores Escriva, Jose Joaquín Ceron, Asta Tvarijonaviciute and Jose Enrique de la Rubia Ortí
Antioxidants 2024, 13(8), 1021; https://doi.org/10.3390/antiox13081021 - 22 Aug 2024
Viewed by 1330
Abstract
Background: Amyotrophic lateral sclerosis (ALS) is characterized by progressive motor neuron degeneration, with oxidative stress playing a key role. Paraoxonase 1 (PON1) is an antioxidant enzyme that may influence ALS progression. This study aimed to establish a predictive model for the influence [...] Read more.
Background: Amyotrophic lateral sclerosis (ALS) is characterized by progressive motor neuron degeneration, with oxidative stress playing a key role. Paraoxonase 1 (PON1) is an antioxidant enzyme that may influence ALS progression. This study aimed to establish a predictive model for the influence of PON1 activity on functionality in ALS patients and explore its relationship with nutrition. Methods: In this observational cross-sectional study, 70 ALS patients underwent assessments of PON1 activity, lipid profile, functional capacity, respiratory function, and heart rate variability. A structural equation model was developed to determine the relationships between variables. Nutritional intake was analyzed in 65 patients. Results: The predictive model showed that PON1 activity and LDL levels positively influenced functionality, both directly and indirectly through respiratory capacity. Heart rate variability moderately predicted functionality independently. HDL levels were not significantly associated with functionality. Weak to moderate correlations were found between PON1 activity and intake of certain nutrients, with positive associations for monounsaturated fats and vitamin D, and negative associations for carbohydrates, proteins, and some micronutrients. Conclusions: PON1 activity appears to play an important role in ALS patient functionality, both directly and through effects on respiratory capacity. However, its relationship with nutritional intake was not strongly evident in this sample population. Full article
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<p>Predictive model of PON1 activity, lipoproteins (HDL and LDL), spirometry (FEV1), HRV, and functionality in ALS.</p>
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18 pages, 4596 KiB  
Article
Ribonuclease Inhibitor 1 (RNH1) Regulates Sperm tsRNA Generation for Paternal Inheritance through Interacting with Angiogenin in the Caput Epididymis
by Zhuoyao Ma, Ningyuan Tang, Ruiyan Zhang, Hanyu Deng, Kexin Chen, Yue Liu and Zhide Ding
Antioxidants 2024, 13(8), 1020; https://doi.org/10.3390/antiox13081020 - 22 Aug 2024
Viewed by 1067
Abstract
Environmental stressors can induce paternal epigenetic modifications that are a key determinant of the intergenerational inheritance of acquired phenotypes in mammals. Some of them can affect phenotypic expression through inducing changes in tRNA-derived small RNAs (tsRNAs), which modify paternal epigenetic regulation in sperm. [...] Read more.
Environmental stressors can induce paternal epigenetic modifications that are a key determinant of the intergenerational inheritance of acquired phenotypes in mammals. Some of them can affect phenotypic expression through inducing changes in tRNA-derived small RNAs (tsRNAs), which modify paternal epigenetic regulation in sperm. However, it is unclear how these stressors can affect changes in the expression levels of tsRNAs and their related endonucleases in the male reproductive organs. We found that Ribonuclease inhibitor 1 (RNH1), an oxidation responder, interacts with ANG to regulate sperm tsRNA generation in the mouse caput epididymis. On the other hand, inflammation and oxidative stress induced by either lipopolysaccharide (LPS) or palmitate (PA) treatments weakened the RNH1-ANG interaction in the epididymal epithelial cells (EEC). Accordingly, ANG translocation increased from the nucleus to the cytoplasm, which led to ANG upregulation and increases in cytoplasmic tsRNA expression levels. In conclusion, as an antioxidant, RNH1 regulates tsRNA generation through targeting ANG in the mouse caput epididymis. Moreover, the tsRNA is an epigenetic factor in sperm that modulates paternal inheritance in offspring via the fertilization process. Full article
(This article belongs to the Special Issue Oxidative and Nitrosative Stress in Male Reproduction)
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<p><b>High RNH1 expression in caput epididymis.</b> (<b>A</b>) Expression of RNH1 protein detected by Western blotting in various mouse tissues. The tissue samples (liver, spleen, lung, kidney, brain, intestine, testis, caput epididymis and cauda epididymis) were from 8-week-old male mice, and ovarian and uterine tissues were from 8-week-old female mice. (<b>B</b>) The expression of RNH1 in caput epididymis, corpus epididymis, and cauda epididymis. (<b>C</b>) Western blotting detection of RNH1 in caput epididymis at 1 w, 2 w, 3 w, 5 w, and 8 w, respectively. (<b>D</b>) Immunofluorescent staining was used to identify localization of RNH1 protein in EEC.</p>
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<p><b>Interaction between RNH1 and ANG in epididymal epithelial cells.</b> (<b>A</b>) Western blotting analysis validated interaction between RNH1 and ANG in EEC. (<b>B</b>) Immunofluorescent staining of ANG (green), RNH1 (red) and DAPI (blue) in EEC. (<b>C</b>) Western blotting analysis detected the interaction between the mutant RNH1 and ANG in 3T3 cells.</p>
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<p><b>Environmental stress-induced upregulation of both in vivo epididymal cytoplasmic ANG expression and in vitro.</b> (<b>A</b>) Western blot analysis of ANG in the cytoplasm of caput epididymis identified stress-induced ANG upregulation. (<b>B</b>) Western blot analysis of stress-induced upregulation of cytoplasmic ANG expression in EEC after 48 h. (<b>C</b>) Western blotting of ANG protein expression in the cytoplasm of PC1 cells after 48 h exposure to stress conditions. Average value of control group was identified as 1. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01.</p>
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<p><b>Inflammation and oxidative stress weaken the interaction between RNH1 and ANG.</b> (<b>A</b>,<b>B</b>) Western blot analysis of RNH1 in cytoplasm under environmental stresses in EEC (<b>A</b>) and PC1 cells (<b>B</b>). (<b>C</b>) Immunofluorescent staining reveals localization of RNH1 and ANG under LPS or PA treatment. (<b>D</b>,<b>E</b>) Western blot analyses documents interaction between RNH1 and ANG under LPS (<b>D</b>) and PA (<b>E</b>) treatments in EEC. (<b>F</b>,<b>G</b>) RT-qPCR analysis reveals ANG mRNA levels under LPS or PA treatment in EEC (<b>F</b>) and PC1 cells (<b>G</b>). (<b>H</b>,<b>I</b>) Western blot analysis reveals ANG levels in EEC (<b>H</b>) and PC1 (<b>I</b>), respectively, under LPS or PA treatment. (<b>J</b>,<b>K</b>) Western blot analysis reveals ANG cytoplasmic and nuclear expression levels in EEC (<b>J</b>) and PC1 cells (<b>K</b>), respectively, under environmental stresses. GAPDH was used as a cytoplasmic reference gene, whereas H3 was used as a nuclear reference gene. ns: no significance. ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p><b>LPS or PA-induced rises in tsRNAs in EEC and exosomes.</b> (<b>A</b>) The levels of three tsRNAs, 5′-tiRNA-Gly, 5′-tiRNA-Val, and 5′-tiRNA-Glu in EEC under stress conditions. (<b>B</b>) RT-qPCR analyses of three tsRNAs in PC1 in the presence of LPS or PA. (<b>C</b>) RT-qPCR analyses of three tsRNAs in exosomes isolated from EEC under LPS or PA treatment. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p><b>RNH1 blunts ANG-induced increases in tsRNA generation.</b> (<b>A</b>) RT-qPCR showed that overexpression of <span class="html-italic">Rnh1</span> significantly decreased the level of three tsRNAs, 5′-tiRNA-Gly, 5′-tiRNA-Val, and 5′-tiRNA-Glu, in EEC. (<b>B</b>) The levels of three tsRNAs increased in <span class="html-italic">Rnh1</span> knocked down EEC. (<b>C</b>) RT-qPCR analysis of three tsRNA levels in <span class="html-italic">Rnh1</span>-overexpressed EEC compared to those in the mutant <span class="html-italic">Rnh1</span> transfected EEC or in the control group (vector plasmid transfected EEC). * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p><b>Schematic representation of RNH1 function in epididymal epithelial cells.</b> Inflammation and oxidative stress impair RNH1 interaction with ANG in the EEC, which in turn induces ANG release and its translocation from the nucleus to the cytoplasm. Accordingly, rises in the cytoplasmic ANG expression level induce increases in the tsRNA expression levels in both the EEC cytoplasm and its exosomes (epididymosomes). Subsequently, exosomes deliver tsRNAs into the sperm.</p>
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23 pages, 4548 KiB  
Article
ARE/Nrf2 Transcription System Involved in Carotenoid, Polyphenol, and Estradiol Protection from Rotenone-Induced Mitochondrial Oxidative Stress in Dermal Fibroblasts
by Aya Darawsha, Aviram Trachtenberg and Yoav Sharoni
Antioxidants 2024, 13(8), 1019; https://doi.org/10.3390/antiox13081019 - 21 Aug 2024
Viewed by 1128
Abstract
Skin aging is associated with the increased production of mitochondrial reactive oxygen species (mtROS) due to mitochondrial dysfunction, and various phytonutrients and estrogens have been shown to improve skin health. Thus, the aim of the current study was to examine damage to dermal [...] Read more.
Skin aging is associated with the increased production of mitochondrial reactive oxygen species (mtROS) due to mitochondrial dysfunction, and various phytonutrients and estrogens have been shown to improve skin health. Thus, the aim of the current study was to examine damage to dermal fibroblasts by chemically induced mitochondrial dysfunction and to study the mechanism of the protective effects of carotenoids, polyphenols, and estradiol. Rotenone, a Complex I inhibitor, caused mitochondrial dysfunction in human dermal fibroblasts, substantially reducing respiration and ATP levels, followed by increased mitochondrial and cytosolic ROS, which resulted in apoptotic cell death, an increased number of senescent cells, increased matrix metalloproteinase-1 (MMP1) secretion, and decreased collagen secretion. Pre-treatment with carotenoid-rich tomato extracts, rosemary extract, and estradiol reversed these effects. These protective effects can be partially explained by a cooperative activation of antioxidant response element (ARE/Nrf2) transcriptional activity by the protective compounds and rotenone, which led to the upregulation of antioxidant proteins such as NQO1. To determine if ARE/Nrf2 activity is crucial for cell protection, we inhibited it using the Nrf2 inhibitors ML385 and ochratoxin A. This inhibition markedly reduced the protective effects of the test compounds by diminishing their effect to reduce cytosolic ROS. Our study results indicate that phytonutrients and estradiol protect skin cells from damage caused by mtROS, and thus may delay skin cell senescence and improve skin health. Full article
(This article belongs to the Special Issue Role of Mitochondria and ROS in Health and Disease)
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<p>Tomato and rosemary extracts and estradiol reduce rotenone-induced mitochondrial and cytosolic ROS. Cells were seeded in 96-well plates (5 × 10<sup>3</sup> cells/well) and pre-incubated for 24 h with rosemary extract (RE; 10 μM carnosic acid), red tomato extract (RTE; 10 μM lycopene), golden tomato extract (GTE; 40 μM phytoene), or estradiol (10 nM). Then, the medium was replaced with one containing the treatment compounds plus 1 µM rotenone and incubated for 90 min or 4 h to detect mitochondrial ROS (Mitosox fluorescence) or cytosolic ROS (DCF fluorescence), respectively. (<b>a</b>) Mitochondrial ROS. (<b>b</b>) Cytosolic ROS. Values are averaged geometric means of fluorescence intensities (MFI) of Mitosox or DCF, and are the means ± SEM of four experiments, each performed in triplicate. <span>$</span><span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.001, <span>$</span><span>$</span><span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.0001, significant difference between the control and rotenone. *, <span class="html-italic">p</span> &lt; 0.05, **, <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, ****, <span class="html-italic">p</span> &lt; 0.0001, significant difference between the vehicle and other treatments in the presence of rotenone.</p>
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<p>Rotenone triggers apoptotic cell death of dermal fibroblasts, which can be reduced by tomato and rosemary extracts and estradiol. (<b>a</b>–<b>d</b>) Cells were seeded in 96-well plates (5 × 10<sup>3</sup> cells/well) and pre-incubated for 24 h with the indicated concentrations of red (<b>a</b>) and golden (<b>b</b>) tomato extracts, rosemary extract (<b>c</b>), and estradiol (<b>d</b>). Then, the medium was replaced with one containing the treatment compounds plus 1 µM rotenone and incubated for an additional 48 h. Cell number was determined using the crystal violet method. Results are presented as percent of the control without rotenone and the treatment compounds. One hundred percent of the cell number was 22,080 ± 3153 cells/well. Values are the means ± SEM of four experiments, each performed in triplicate. (<b>e</b>,<b>f</b>) Cells were seeded in 96-well plates and treated as described above. The concentrations of the treatment compounds were estradiol (10 nM), rosemary extract (RE; 10 μM carnosic acid), red tomato extract (RTE; 10 μM lycopene), and golden tomato extract (GTE; 40 μM phytoene). At the end of the experiments, the media were removed for further analysis (see <a href="#sec3dot4-antioxidants-13-01019" class="html-sec">Section 3.4</a>), and cell number ((<b>e</b>), crystal violet) or viability ((<b>f</b>), ApoLive-GloTM Multiplex Viability Assay) were determined. (<b>g</b>,<b>h</b>) Cell apoptosis was determined using annexin V flow cytometry. Cells were seeded in 6-well plates (3 × 10<sup>5</sup> cells/well) and pre-incubated for 24 h with the treatment compounds at the concentrations shown for (<b>e</b>,<b>f</b>). Then, the medium was replaced with one containing the treatment compounds plus 1 µM rotenone and incubated for an additional 48 h. (<b>g</b>) Typical flow cytometric data of annexin V fluorescence. (<b>h</b>) Percent apoptotic cells. Results are the means ± SEM of five experiments, each performed in duplicate. (<b>i</b>) Apoptosis was analyzed by measuring caspase 3/7 activity using an ApoLive-Glo<sup>TM</sup> Multiplex Assay. Cells were seeded in 96-well plates (5 × 10<sup>3</sup> cells/well) and treated as described above. Results are presented as percent of the control without rotenone and the treatment compounds. Values are the means ± SEM of four experiments, each performed in triplicate. <span>$</span>, <span class="html-italic">p</span> &lt; 0.05, <span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.01, <span>$</span><span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.001, <span>$</span><span>$</span><span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.0001, significant difference from the control without rotenone and the treatment compounds. *, <span class="html-italic">p</span> &lt; 0.05, **, <span class="html-italic">p</span> &lt; 0.01, ***, <span class="html-italic">p</span> &lt; 0.001, ****, <span class="html-italic">p</span> &lt; 0.0001, significant difference between the vehicle and other treatments in the presence of rotenone.</p>
Full article ">Figure 2 Cont.
<p>Rotenone triggers apoptotic cell death of dermal fibroblasts, which can be reduced by tomato and rosemary extracts and estradiol. (<b>a</b>–<b>d</b>) Cells were seeded in 96-well plates (5 × 10<sup>3</sup> cells/well) and pre-incubated for 24 h with the indicated concentrations of red (<b>a</b>) and golden (<b>b</b>) tomato extracts, rosemary extract (<b>c</b>), and estradiol (<b>d</b>). Then, the medium was replaced with one containing the treatment compounds plus 1 µM rotenone and incubated for an additional 48 h. Cell number was determined using the crystal violet method. Results are presented as percent of the control without rotenone and the treatment compounds. One hundred percent of the cell number was 22,080 ± 3153 cells/well. Values are the means ± SEM of four experiments, each performed in triplicate. (<b>e</b>,<b>f</b>) Cells were seeded in 96-well plates and treated as described above. The concentrations of the treatment compounds were estradiol (10 nM), rosemary extract (RE; 10 μM carnosic acid), red tomato extract (RTE; 10 μM lycopene), and golden tomato extract (GTE; 40 μM phytoene). At the end of the experiments, the media were removed for further analysis (see <a href="#sec3dot4-antioxidants-13-01019" class="html-sec">Section 3.4</a>), and cell number ((<b>e</b>), crystal violet) or viability ((<b>f</b>), ApoLive-GloTM Multiplex Viability Assay) were determined. (<b>g</b>,<b>h</b>) Cell apoptosis was determined using annexin V flow cytometry. Cells were seeded in 6-well plates (3 × 10<sup>5</sup> cells/well) and pre-incubated for 24 h with the treatment compounds at the concentrations shown for (<b>e</b>,<b>f</b>). Then, the medium was replaced with one containing the treatment compounds plus 1 µM rotenone and incubated for an additional 48 h. (<b>g</b>) Typical flow cytometric data of annexin V fluorescence. (<b>h</b>) Percent apoptotic cells. Results are the means ± SEM of five experiments, each performed in duplicate. (<b>i</b>) Apoptosis was analyzed by measuring caspase 3/7 activity using an ApoLive-Glo<sup>TM</sup> Multiplex Assay. Cells were seeded in 96-well plates (5 × 10<sup>3</sup> cells/well) and treated as described above. Results are presented as percent of the control without rotenone and the treatment compounds. Values are the means ± SEM of four experiments, each performed in triplicate. <span>$</span>, <span class="html-italic">p</span> &lt; 0.05, <span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.01, <span>$</span><span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.001, <span>$</span><span>$</span><span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.0001, significant difference from the control without rotenone and the treatment compounds. *, <span class="html-italic">p</span> &lt; 0.05, **, <span class="html-italic">p</span> &lt; 0.01, ***, <span class="html-italic">p</span> &lt; 0.001, ****, <span class="html-italic">p</span> &lt; 0.0001, significant difference between the vehicle and other treatments in the presence of rotenone.</p>
Full article ">Figure 3
<p>An increase in SA-β-gal activity following exposure to rotenone was inhibited by dietary compounds and estradiol. Cells were seeded in 6-well plates (<b>a</b>,<b>b</b>) and 24-well plates (<b>c</b>,<b>d</b>) at a density of 2 × 10<sup>5</sup> cells/well and pre-incubated for 24 h with the treatment compounds at the concentrations shown in <a href="#antioxidants-13-01019-f002" class="html-fig">Figure 2</a>. Then, the medium was replaced with one containing the treatment compounds plus 1 µM rotenone and incubated for an additional 48 h. The cells were incubated with X-gal (<b>a</b>,<b>b</b>) or with the fluorogenic substrate (<b>c</b>,<b>d</b>) as described in the Methods section. Positive control cells were treated with 25 μM etoposide for 24 h. (<b>a</b>,<b>b</b>) Cells were imaged with a light microscope at 10× magnification (<b>a</b>), and the number of positive cells was counted in a 2.2 mm<sup>2</sup> area (<b>b</b>). Values are the average ± SEM of positive cells of five experiments each performed in triplicate. (<b>c</b>) Representative flow cytometry data. (<b>d</b>) SA-β-gal activity results are presented as a percent of the control without rotenone and the treatment compounds. Values are the means ± SEM of the averaged geometric mean of fluorescence intensities (MFI) of five experiments, each performed in triplicate. <span>$</span><span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.001, significant difference between the control and rotenone or etoposide. **, <span class="html-italic">p</span> &lt; 0.01, ***, 0.001, significant difference between the vehicle and other treatments in the presence of rotenone.</p>
Full article ">Figure 4
<p>Rotenone increases MMP1 secretion, and reduces collagen 1a1 secretion and mRNA expression, whereas pre-treatment with tomato (RTE, GTE) and rosemary (RE) extracts and estradiol reverse these effects. The secretion of MMP-1 and pro-collagen 1a1 was determined in the media collected in the experiments described in <a href="#antioxidants-13-01019-f002" class="html-fig">Figure 2</a>, using ELISA as described in the Methods section. (<b>a</b>) MMP-1 level. (<b>b</b>) Pro-collagen 1a1 level. Results are presented as percent of the control values. One hundred percent of the MMP-1 secretion was 42.7 ± 6.4 pmol/1000 cells. One hundred percent of the pro-collagen 1a1 secretion was 80.9 ± 14.6 pmol/1000 cells. (<b>c</b>) Collagen 1a1 mRNA expression was determined using RT-qPCR as described in the Methods section. Values are the means ± SEM of four experiments, each performed in triplicate. <span>$</span><span>$</span><span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.0001, significant difference between the control and rotenone. **, <span class="html-italic">p</span> &lt; 0.01, ***, <span class="html-italic">p</span> &lt; 0.001, ****, <span class="html-italic">p</span> &lt; 0.0001, significant difference between the vehicle and other treatments in the presence of rotenone.</p>
Full article ">Figure 5
<p>Mitochondrial dysfunction following exposure to rotenone and recovery by dietary compounds and estradiol. Cells were seeded in XF 96-well plates (1 × 10<sup>4</sup> cells/well) and treated with the treatment compounds as described in <a href="#antioxidants-13-01019-f001" class="html-fig">Figure 1</a>. Twenty-four hours after the addition of rotenone, the cells were washed for 1 h, and OCR and ECAR were measured using an Agilent SeahorseXFe96 analyzer according to the manufacturer’s protocol. OCR and ECAR were normalized to nuclei DAPI staining. (<b>a</b>) A representative OCR recording at basal conditions and after the injection of oligomycin (1.5 μM), FCCP (1 μM), and antimycin A plus rotenone (1 μM). (<b>b</b>) Basal OCR results are expressed as pmol/min×1000 cells. Results of the following parameters (<b>c</b>–<b>f</b>) are expressed in the percentage of control (<b>c</b>) ATP-linked OCR, 100% = 8.04 ± 0.46 pmol/min×1000 cells; (<b>d</b>) maximal respiration, 100% = 12.54 ± 0.76 pmol/min×1000 cells; (<b>e</b>) spare respiratory capacity, 100% = 4.53 ± 0.49 pmol/min×1000 cells; and (<b>f</b>) ATP cellular level measured using the CellTiter-Glo Luminescent Cell Viability Assay Kit. (<b>g</b>) ECAR; results are expressed as mpH/min×1000 cells. Results are means ± SEM of four experiments, each in six replicates. <span>$</span>, <span class="html-italic">p</span> &lt; 0.05, <span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.01, <span>$</span><span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.001, <span>$</span><span>$</span><span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.0001, significant difference from the control without rotenone and the treatment compounds. ****, <span class="html-italic">p</span> &lt; 0.0001, significant difference between the vehicle and other treatments in the presence of rotenone.</p>
Full article ">Figure 6
<p>Increased activation of ARE/Nrf2 by the combinations of rotenone with the phytonutrients or estradiol. (<b>a</b>,<b>b</b>) ARE/Nrf2 transcriptional activity. Cells were seeded in 24-well plates (10<sup>5</sup> cells/well), and 24 h later were transfected with the ARE/Nrf2 reporter gene. After transfection, the cells were incubated for 16 h with the dietary compounds and estradiol at the concentrations shown in <a href="#antioxidants-13-01019-f002" class="html-fig">Figure 2</a>, with or without 1 μM rotenone. ARE/Nrf2 transcriptional activity was determined using a luciferase reporter assay as described in the Methods section. Values (fold induction) are the means ± SEM of four experiments, each performed in triplicate. (<b>c</b>,<b>d</b>) NQO1 protein levels. Cells were seeded in 6-well plates (3 × 10<sup>5</sup> cells/well) and were treated as described in <a href="#antioxidants-13-01019-f002" class="html-fig">Figure 2</a>. The NQO-1 protein level was determined through Western blot. (<b>c</b>) Quantitation of NQO1 level. (<b>d</b>) Representative Western blot gel. Values (fold of control) are the means ± SEM of four experiments. <span>$</span><span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.001, <span>$</span><span>$</span><span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.0001, significant difference from the control without rotenone and the treatment compounds. **, <span class="html-italic">p</span> &lt; 0.01, ***, <span class="html-italic">p</span> &lt; 0.001, ****, <span class="html-italic">p</span> &lt; 0.0001, significant difference between the vehicle and other treatments in the presence of rotenone.</p>
Full article ">Figure 6 Cont.
<p>Increased activation of ARE/Nrf2 by the combinations of rotenone with the phytonutrients or estradiol. (<b>a</b>,<b>b</b>) ARE/Nrf2 transcriptional activity. Cells were seeded in 24-well plates (10<sup>5</sup> cells/well), and 24 h later were transfected with the ARE/Nrf2 reporter gene. After transfection, the cells were incubated for 16 h with the dietary compounds and estradiol at the concentrations shown in <a href="#antioxidants-13-01019-f002" class="html-fig">Figure 2</a>, with or without 1 μM rotenone. ARE/Nrf2 transcriptional activity was determined using a luciferase reporter assay as described in the Methods section. Values (fold induction) are the means ± SEM of four experiments, each performed in triplicate. (<b>c</b>,<b>d</b>) NQO1 protein levels. Cells were seeded in 6-well plates (3 × 10<sup>5</sup> cells/well) and were treated as described in <a href="#antioxidants-13-01019-f002" class="html-fig">Figure 2</a>. The NQO-1 protein level was determined through Western blot. (<b>c</b>) Quantitation of NQO1 level. (<b>d</b>) Representative Western blot gel. Values (fold of control) are the means ± SEM of four experiments. <span>$</span><span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.001, <span>$</span><span>$</span><span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.0001, significant difference from the control without rotenone and the treatment compounds. **, <span class="html-italic">p</span> &lt; 0.01, ***, <span class="html-italic">p</span> &lt; 0.001, ****, <span class="html-italic">p</span> &lt; 0.0001, significant difference between the vehicle and other treatments in the presence of rotenone.</p>
Full article ">Figure 7
<p>Protection from rotenone-induced damage by dietary compounds and estradiol is markedly reduced in the presence of ARE/Nrf2 inhibitors. Cells were seeded in 96-well plates (5 × 10<sup>3</sup> cells/well) and treated essentially as described in <a href="#antioxidants-13-01019-f001" class="html-fig">Figure 1</a>, <a href="#antioxidants-13-01019-f002" class="html-fig">Figure 2</a>, <a href="#antioxidants-13-01019-f004" class="html-fig">Figure 4</a> and <a href="#antioxidants-13-01019-f005" class="html-fig">Figure 5</a>, but incubation was either with or without OTA (25 μM) or ML385 (10 μM). (<b>a</b>,<b>b</b>) ARE/Nrf2 transcriptional activity. This activity was measured in the presence of rotenone and rosemary extract (RE) at the indicated inhibitor concentrations. (<b>c</b>,<b>d</b>) Cytosolic ROS. Values are the means ± SEM of four experiments of averaged geometric means of fluorescence intensities (MFI) of DCF. (<b>e</b>) mtROS. Values are the means ± SEM of four experiments of averaged geometric means of fluorescence intensities (MFI) of Mitosox. (<b>f</b>) ATP cellular level. Values (% of control) are the means ± SEM of four experiments. (<b>g</b>,<b>h</b>) Cell number. (<b>i</b>,<b>j</b>) MMP-1 level. (<b>k</b>,<b>l</b>) Pro-collagen 1a1 level. Values (% of control) are the means ± SEM of four experiments. <span>$</span><span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.001, <span>$</span><span>$</span><span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.0001, significant difference between the control and rotenone; *, <span class="html-italic">p</span> &lt; 0.05, **, <span class="html-italic">p</span> &lt; 0.01, ***, <span class="html-italic">p</span> &lt; 0.001, ****, <span class="html-italic">p</span> &lt; 0.0001, significant difference between the vehicle and other treatments in the presence of rotenone. ###, <span class="html-italic">p</span> &lt; 0.001, ####, <span class="html-italic">p</span> &lt; 0.0001, significant difference between the control and rotenone in the presence of the inhibitors. ^, <span class="html-italic">p</span> &lt; 0.05, significant difference between the vehicle and other treatments in the presence of rotenone and the inhibitors.</p>
Full article ">Figure 7 Cont.
<p>Protection from rotenone-induced damage by dietary compounds and estradiol is markedly reduced in the presence of ARE/Nrf2 inhibitors. Cells were seeded in 96-well plates (5 × 10<sup>3</sup> cells/well) and treated essentially as described in <a href="#antioxidants-13-01019-f001" class="html-fig">Figure 1</a>, <a href="#antioxidants-13-01019-f002" class="html-fig">Figure 2</a>, <a href="#antioxidants-13-01019-f004" class="html-fig">Figure 4</a> and <a href="#antioxidants-13-01019-f005" class="html-fig">Figure 5</a>, but incubation was either with or without OTA (25 μM) or ML385 (10 μM). (<b>a</b>,<b>b</b>) ARE/Nrf2 transcriptional activity. This activity was measured in the presence of rotenone and rosemary extract (RE) at the indicated inhibitor concentrations. (<b>c</b>,<b>d</b>) Cytosolic ROS. Values are the means ± SEM of four experiments of averaged geometric means of fluorescence intensities (MFI) of DCF. (<b>e</b>) mtROS. Values are the means ± SEM of four experiments of averaged geometric means of fluorescence intensities (MFI) of Mitosox. (<b>f</b>) ATP cellular level. Values (% of control) are the means ± SEM of four experiments. (<b>g</b>,<b>h</b>) Cell number. (<b>i</b>,<b>j</b>) MMP-1 level. (<b>k</b>,<b>l</b>) Pro-collagen 1a1 level. Values (% of control) are the means ± SEM of four experiments. <span>$</span><span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.001, <span>$</span><span>$</span><span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.0001, significant difference between the control and rotenone; *, <span class="html-italic">p</span> &lt; 0.05, **, <span class="html-italic">p</span> &lt; 0.01, ***, <span class="html-italic">p</span> &lt; 0.001, ****, <span class="html-italic">p</span> &lt; 0.0001, significant difference between the vehicle and other treatments in the presence of rotenone. ###, <span class="html-italic">p</span> &lt; 0.001, ####, <span class="html-italic">p</span> &lt; 0.0001, significant difference between the control and rotenone in the presence of the inhibitors. ^, <span class="html-italic">p</span> &lt; 0.05, significant difference between the vehicle and other treatments in the presence of rotenone and the inhibitors.</p>
Full article ">Scheme 1
<p>(<b>a</b>) Cell damage. Low mitochondrial dysfunction occurs in cellular aging, and high and almost complete dysfunction occurs due to rotenone treatment, with a significant reduction in mitochondrial respiration and ATP level (see red arrow). This dysfunction results in the production of superoxide radical and other ROS, which cause cell damage that is manifested as increased MMP secretion, reduced collagen secretion, apoptotic cell death, and cell senescence. (<b>b</b>) Cell protection. Carotenoids, polyphenols, and the hormone estradiol protect the cell from rotenone-induced damage by activating the ARE/Nrf2 transcription system, resulting in reduced ROS and the partial prevention of the harmful effects.</p>
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16 pages, 982 KiB  
Article
Effect of Supplementation of a Cryopreservation Extender with Pectoliv30 on Post-Thawing Semen Quality Parameters in Rooster Species
by Esther Díaz Ruiz, Juan Vicente Delgado Bermejo, José Manuel León Jurado, Francisco Javier Navas González, Ander Arando Arbulu, Juan Fernández-Bolaños Guzmán, Alejandra Bermúdez Oria and Antonio González Ariza
Antioxidants 2024, 13(8), 1018; https://doi.org/10.3390/antiox13081018 - 21 Aug 2024
Viewed by 818
Abstract
Sperm cryopreservation is a fundamental tool for the conservation of avian genetic resources; however, avian spermatozoa are susceptible to this process. To cope with the high production of reactive oxygen species (ROS), the addition of exogenous antioxidants is beneficial. Pectoliv30 is a substance [...] Read more.
Sperm cryopreservation is a fundamental tool for the conservation of avian genetic resources; however, avian spermatozoa are susceptible to this process. To cope with the high production of reactive oxygen species (ROS), the addition of exogenous antioxidants is beneficial. Pectoliv30 is a substance derived from alperujo, and in this study, its effect was analyzed on seminal quality after its addition to the cryopreservation extender of roosters at different concentrations. For this purpose, 16 Utrerana breed roosters were used, and seminal collection was performed in six replicates, creating a pool for each working day with ejaculates of quality. After cryopreservation, one sample per treatment and replicate was thawed, and several seminal quality parameters were evaluated. Statistical analysis revealed numerous correlations between these variables, both positive and negative according to the correlation matrix obtained. Furthermore, the chi-squared automatic interaction detection (CHAID) decision tree (DT) reported significant differences in the hypo-osmotic swelling test (HOST) variable between groups. Moreover, results for this parameter were more desirable at high concentrations of Pectoliv30. The application of this substance extracted from the by-product alperujo as an antioxidant allows the improvement of the post-thawing seminal quality in roosters and facilitates optimization of the cryopreservation process as a way to improve the conservation programs of different endangered poultry breeds. Full article
(This article belongs to the Special Issue Antioxidant Properties and Applications of Food By-Products)
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Figure 1

Figure 1
<p>Correlation matrix between the different semen quality traits evaluated in the frozen-thawed semen, taking into account all the cryopreservation treatments in this study (T1: extender without antioxidant; T2: 100 µg/mL of Pectoliv30; T3: 200 µg/mL of Pectoliv30; T4: 400 µg/mL of Pectoliv30).</p>
Full article ">Figure 2
<p>Graphic depiction of the data mining CHAID DT obtained from the chi-square dissimilarity matrix, considering the four freeze-thawing treatments as the clustering criterion (T1: extender without antioxidant; T2: 100 µg/mL of Pectoliv30; T3: 200 µg/mL of Pectoliv30; T4: 400 µg/mL of Pectoliv30). All sperm-quality-related parameters were used in this analysis. Six replicate compounds of all the ejaculates (from 16 roosters used) that met the minimum quality criteria were established.</p>
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12 pages, 710 KiB  
Review
The Role of Oxidative Stress in Hypomagnetic Field Effects
by Lanxiang Tian, Yukai Luo, Jie Ren and Chenchen Zhao
Antioxidants 2024, 13(8), 1017; https://doi.org/10.3390/antiox13081017 - 21 Aug 2024
Viewed by 1870
Abstract
The geomagnetic field (GMF) is crucial for the survival and evolution of life on Earth. The weakening of the GMF, known as the hypomagnetic field (HMF), significantly affects various aspects of life on Earth. HMF has become a potential health risk for future [...] Read more.
The geomagnetic field (GMF) is crucial for the survival and evolution of life on Earth. The weakening of the GMF, known as the hypomagnetic field (HMF), significantly affects various aspects of life on Earth. HMF has become a potential health risk for future deep space exploration. Oxidative stress is directly involved in the biological effects of HMF on animals or cells. Oxidative stress occurs when there is an imbalance favoring oxidants over antioxidants, resulting in cellular damage. Oxidative stress is a double-edged sword, depending on the degree of deviation from homeostasis. In this review, we summarize the important experimental findings from animal and cell studies on HMF exposure affecting intracellular reactive oxygen species (ROS), as well as the accompanying many physiological abnormalities, such as cognitive dysfunction, the imbalance of gut microbiota homeostasis, mood disorders, and osteoporosis. We discuss new insights into the molecular mechanisms underlying these HMF effects in the context of the signaling pathways related to ROS. Among them, mitochondria are considered to be the main organelles that respond to HMF-induced stress by regulating metabolism and ROS production in cells. In order to unravel the molecular mechanisms of HMF action, future studies need to consider the upstream and downstream pathways associated with ROS. Full article
(This article belongs to the Section Health Outcomes of Antioxidants and Oxidative Stress)
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Figure 1
<p>ROS-regulated signaling pathways. ROS act as second messengers to regulate four main signaling pathways, controlling a variety of biological processes in cells. Mitochondria and NOXs are the two main sources of ROS generation (black arrows: activation; black T-arrows: inhibition). Dark red font represents the transcription factors. TCA: tricarboxylic acid cycle, ETC: electron transport chain, AP-1: activator protein-1.</p>
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20 pages, 1378 KiB  
Article
Phytochemical Composition Antioxidant and Anti-Inflammatory Activity of Artemisia dracunculus and Artemisia abrotanum
by Mădălina Țicolea, Raluca Maria Pop, Marcel Pârvu, Lia-Oxana Usatiuc, Ana Uifălean, Floricuța Ranga and Alina Elena Pârvu
Antioxidants 2024, 13(8), 1016; https://doi.org/10.3390/antiox13081016 - 20 Aug 2024
Cited by 1 | Viewed by 1151
Abstract
This study aimed to investigate the antioxidant and anti-inflammatory activities mechanism of Artemisia dracunculus (A. dracunculus) and Artemisia abrotanum (A. abrotanum) ethanol extracts in acute rat inflammation induced in Wistar male rats with turpentine oil. The characterization of the [...] Read more.
This study aimed to investigate the antioxidant and anti-inflammatory activities mechanism of Artemisia dracunculus (A. dracunculus) and Artemisia abrotanum (A. abrotanum) ethanol extracts in acute rat inflammation induced in Wistar male rats with turpentine oil. The characterization of the polyphenolic compounds in the extracts was conducted using UV–Vis and Fourier-transform infrared spectroscopy and high-performance liquid chromatography coupled with mass spectrometry techniques. The antioxidant activity of the extracts was evaluated in vitro by DPPH, FRAP, H2O2, and NO scavenging tests and in vivo by measuring the total oxidative status (TOS), total antioxidant capacity (TAC), oxidative stress index (OSI), 8-hydroxy-deoxyguanosine (8-Oxo-dG), advanced oxidation protein products (AOPP), malondialdehyde (MDA), nitric oxide (NO), 3-nitrotyrosine (3NT), and total thiols (SH). Inflammation was evaluated by measuring nuclear factor-kB-p65 (NfkB-p65) and NLRP3 inflammasome activation with IL-1β, IL-18, and gasdermin D. Liver and renal toxicity was determined following transaminases (ALT and AST), creatinine, and urea. The experimental results indicated that A. dracunculus and A. abrotanum ethanol extracts have moderate in vitro antioxidant activity and had in vivo antioxidant activity and an anti-inflammatory effect by NfkB-p65, IL-1b, IL-18, and gasdermin D serum level reduction. The antioxidant activity correlated with the chemical composition of the extracts. These results bring evidence-based use of A. dracunculus and A. abrotanum’s in traditional and contemporary medicine. Full article
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Figure 1
<p>HPLC chromatogram of phenolic compounds from <span class="html-italic">A. abrotanum</span> and <span class="html-italic">A. dracunculus</span> ethanolic extracts. The peak identification is provided in <a href="#antioxidants-13-01016-t002" class="html-table">Table 2</a>.</p>
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<p>Comparative general FTIR spectra of <span class="html-italic">A. dracunculus</span> and <span class="html-italic">A. abrotanum</span> ethanol extracts (600–3500 cm<sup>−1</sup>).</p>
Full article ">Figure 3
<p>The PCA results of oxidative stress and inflammatory biomarkers based on the correlation matrix with PC1 and PC2 for <span class="html-italic">A. dracunculus</span> and <span class="html-italic">A. abrotanum</span> ethanol extracts: (<b>A</b>) PCA of AD 100%; (<b>B</b>) PCA of AD 50%; (<b>C</b>) PCA of AD 25%; (<b>D</b>) PCA of AA 100%; (<b>E</b>) PCA of AA 50%; (<b>F</b>) PCA of AA 25%. AD 100%—<span class="html-italic">A. dracunculus</span> 100%; AD 50%—<span class="html-italic">A. dracunculus</span> 50%; AD 25%—<span class="html-italic">A. dracunculus</span> 25%; AA 100%—<span class="html-italic">A. abrotanum</span> 100%; AA 50%—<span class="html-italic">A. abrotanum</span> 50%; AA 25%—<span class="html-italic">A. abrotanum</span> 25%.</p>
Full article ">Figure 3 Cont.
<p>The PCA results of oxidative stress and inflammatory biomarkers based on the correlation matrix with PC1 and PC2 for <span class="html-italic">A. dracunculus</span> and <span class="html-italic">A. abrotanum</span> ethanol extracts: (<b>A</b>) PCA of AD 100%; (<b>B</b>) PCA of AD 50%; (<b>C</b>) PCA of AD 25%; (<b>D</b>) PCA of AA 100%; (<b>E</b>) PCA of AA 50%; (<b>F</b>) PCA of AA 25%. AD 100%—<span class="html-italic">A. dracunculus</span> 100%; AD 50%—<span class="html-italic">A. dracunculus</span> 50%; AD 25%—<span class="html-italic">A. dracunculus</span> 25%; AA 100%—<span class="html-italic">A. abrotanum</span> 100%; AA 50%—<span class="html-italic">A. abrotanum</span> 50%; AA 25%—<span class="html-italic">A. abrotanum</span> 25%.</p>
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13 pages, 5296 KiB  
Article
Natural Product Auraptene Targets SLC7A11 for Degradation and Induces Hepatocellular Carcinoma Ferroptosis
by Donglin Li, Yingping Li, Liangjie Chen, Chengchang Gao, Bolei Dai, Wenjia Yu, Haoying Yang, Junxiang Pi and Xueli Bian
Antioxidants 2024, 13(8), 1015; https://doi.org/10.3390/antiox13081015 - 20 Aug 2024
Cited by 2 | Viewed by 1157
Abstract
The natural product auraptene can influence tumor cell proliferation and invasion, but its effect on hepatocellular carcinoma (HCC) cells is unknown. Here, we report that auraptene can exert anti-tumor effects in HCC cells via inhibition of cell proliferation and ferroptosis induction. Auraptene treatment [...] Read more.
The natural product auraptene can influence tumor cell proliferation and invasion, but its effect on hepatocellular carcinoma (HCC) cells is unknown. Here, we report that auraptene can exert anti-tumor effects in HCC cells via inhibition of cell proliferation and ferroptosis induction. Auraptene treatment induces total ROS and lipid ROS production in HCC cells to initiate ferroptosis. The cell death or cell growth inhibition of HCC cells induced by auraptene can be eliminated by the ROS scavenger NAC or GSH and ferroptosis inhibitor ferrostatin-1 or Deferoxamine Mesylate (DFO). Mechanistically, the key ferroptosis defense protein SLC7A11 is targeted for ubiquitin–proteasomal degradation by auraptene, resulting in ferroptosis of HCC cells. Importantly, low doses of auraptene can sensitize HCC cells to ferroptosis induced by RSL3 and cystine deprivation. These findings demonstrate a critical mechanism by which auraptene exhibits anti-HCC effects via ferroptosis induction and provides a possible therapeutic strategy for HCC by using auraptene or in combination with other ferroptosis inducers. Full article
(This article belongs to the Special Issue Antioxidant Capacity of Natural Products)
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Figure 1

Figure 1
<p>Auraptene exerts anti-tumor effects in HCC cells. (<b>A</b>) The molecular formula of auraptene with a molecular weight of 298.38. (<b>B</b>) HCCLM3 and HLE cells were plated into a 96-well plate at a density of 20,000 cells/well and treated with the indicated concentrations of auraptene for 24 h. Cell viability was detected with CCK-8 reagent and the IC50 was calculated. (<b>C</b>) HLE and HCCLM3 cells treated with the indicated concentrations of auraptene for 24 h were stained with crystal violet and photographed. (<b>D</b>) HLE and HCCLM3 cells treated with the indicated concentrations of auraptene for 16 h were photographed. Scale bar: 200 μm. (<b>E</b>) HLE and HCCLM3 cells treated with the indicated concentrations of auraptene for 16 h were stained with PI for flow cytometry analysis. Calculated cell death rate (Top) and representative pictures (Bottom) are shown. Aura: Auraptene. (<b>E</b>) Data are represented as the mean ± SD (n = 3), **** <span class="html-italic">p</span> &lt; 0.0001 (one-way ANOVA).</p>
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<p>ROS induction is responsible for auraptene-induced cell growth inhibition and cell death. (<b>A</b>) HCCLM3 and HLE cells treated with or without 100 μM auraptene and 5 mM NAC or 5 mM GSH were stained with DCFH-DA for 1 h, followed by flow cytometry analysis. The calculated relative cellular ROS levels (Top) and histogram of flow cytometric pictures are shown (Bottom). (<b>B</b>) The cell viability of HCCLM3 and HLE cells treated with or without 100 μM auraptene, 5 mM NAC, or 5 mM GSH for 24 h was analyzed with CCK-8. (<b>C</b>) HCCLM3 and HLE cells treated with or without 100 μM auraptene, 5 mM NAC or 5 mM GSH for 24 h were stained with crystal violet and the photographs are shown. (<b>D</b>) HCCLM3 and HLE cells treated with or without 100 μM auraptene, 5 mM NAC, or 5 mM GSH for 16 h were photographed and the representative images are shown. Scale bar: 200 μm. (<b>E</b>) HCCLM3 and HLE cells treated with or without 100 μM auraptene, 5 mM NAC, or 5 mM GSH for 16 h were harvested and stained with 10 μg/mL PI followed by flow cytometry analysis. Calculated cell death rate (left) and representative flow cytometric pictures (right) are shown. (<b>A</b>,<b>B</b>,<b>E</b>) Data are represented as the mean ± SD (n = 3); ** <span class="html-italic">p</span> &lt; 0.01, **** <span class="html-italic">p</span> &lt; 0.0001 (one-way ANOVA).</p>
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<p>Auraptene induces HCC cell ferroptosis. (<b>A</b>) HCCLM3 and HLE cells treated with or without 100 μM auraptene, 2 μM Fer-1, or 50 μM DFO for 4 h were incubated with the ROS probe DCFH-DA for 1 h followed by flow cytometry analysis. The calculated total ROS levels (Top) and histogram of flow cytometric pictures (Bottom) are shown. (<b>B</b>) HCCLM3 and HLE cells treated with or without auraptene (100 μM) for 10 h were incubated with lipid ROS probe C11-BODIPY 581/591 for 1 h followed by flow cytometry analysis. The calculated lipid ROS levels (Top) and histogram of flow cytometric pictures (Bottom) are shown. (<b>C</b>) HCCLM3 and HLE cells treated with or without 100 μM auraptene, 2 μM Fer-1, or 50 μM DFO for 24 h were stained with crystal violet and photographed; the images are shown. (<b>D</b>) HCCLM3 and HLE cells treated with or without 100 μM auraptene, 2 μM Fer-1 or 50 μM DFO for 24 h were incubated with CCK-8 followed by an analysis with a microplate reader. (<b>E</b>,<b>F</b>) HCCLM3 and HLE cells treated with or without 100 μM auraptene, 2 μM Fer-1 or 50 μM DFO for 16 h were photographed. Scale bar: 200 μm. (<b>E</b>) or stained with PI followed by analysis with flow cytometry. (<b>F</b>) The calculated cell death rate (<b>F</b>, <b>Top</b>) and the representative flow cytometric pictures (<b>F</b>, <b>Bottom</b>) are shown. (<b>A</b>,<b>B</b>,<b>D</b>,<b>F</b>) Data are represented as the mean ± SD (n = 3); *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001 (Unpaired Student’s <span class="html-italic">t</span> test for (<b>B</b>) and one-way ANOVA for (<b>A</b>,<b>D</b>,<b>F</b>)).</p>
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<p>A low dose of auraptene sensitizes HCC cells to ferroptosis. (<b>A</b>) HCCLM3 and HLE cells treated with or without the indicated concentrations of auraptene, RSL3 (2 μM) for 24 h, or cystine deprivation for 36 h were stained with crystal violet and photographed; the images are shown. (<b>B</b>) HCCLM3 and HLE cells treated with or without the indicated concentrations of auraptene, RSL3 (2 μM) for 24 h, or cystine deprivation for 36 h were photographed and the representative images are shown. Scale bar: 200 μm. (<b>C</b>) HCCLM3 and HLE cells treated with or without indicated concentration of auraptene or RSL3 (2 μM) for 24 h were stained with PI followed by flow cytometry analysis. The calculated cell death rate (left) and the representative flow cytometric pictures (right) are shown. (<b>D</b>) HCCLM3 and HLE cells treated with or without the indicated concentrations of auraptene or cystine deprivation for 36 h were stained with PI followed by flow cytometry analysis. The calculated cell death rate (left) and the representative flow cytometric pictures (right) are shown. (<b>C</b>,<b>D</b>) Data are represented as the mean ± SD (n = 3); ns: no significance, ** <span class="html-italic">p</span> &lt; 0.01, **** <span class="html-italic">p</span> &lt; 0.0001 (one-way ANOVA).</p>
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<p>Auraptene degrades SLC7A11. (<b>A</b>) HCCLM3 and HLE cells treated with indicated concentrations of auraptene for 10 h were harvested for WB analysis with indicated antibodies, with Vinculin as the loading control. (<b>B</b>) HLE cells treated with 100 μM auraptene at the indicated time points were harvested for WB analysis with indicated antibodies, with Vinculin as the loading control. (<b>C</b>) HLE cells were pretreated with 100 μM auraptene for 2 h and then treated with or without CHX (100 μg/mL) or MG132 (10 μM) for another 8 h, then cells were harvested for WB analysis with indicated antibodies. (<b>A</b>–<b>C</b>) The data of SLC7A11/Vinculin are represented as the mean ± SD (n = 3); ns: no significance, * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001 (one-way ANOVA). (<b>D</b>) HLE cells were transfected with the specified plasmids for 15 h and then treated with or without 100 μM auraptene for 10 h. Cells were lysed for immunoprecipitated with anti-flag antibodies followed by Western blotting with the specified antibodies. (<b>E</b>) HCCLM3 and HLE cells were treated with or without 100 μM auraptene for 10 h and the cellular GSH levels were determined by a microplate reader at 412 nm. Data are represented as the mean ± SD (n = 3); **** <span class="html-italic">p</span> &lt; 0.0001 (Unpaired Student’s <span class="html-italic">t</span> test).</p>
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<p>The working model of auraptene in HCC ferroptosis induction. Auraptene, the major coumarin of citrus plants, targets SLC7A11 for ubiquitin–proteasomal degradation, leading to lipid ROS production and ferroptosis of HCC. Ub: ubiquitin.</p>
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20 pages, 3737 KiB  
Article
A Sustainable Approach to Valuable Polyphenol and Iridoid Antioxidants from Medicinal Plant By-Products
by Filippo Marchetti, Irene Gugel, Stefania Costa, Anna Baldisserotto, Alberto Foletto, Ilenia Gugel, Erika Baldini, Stefano Manfredini and Silvia Vertuani
Antioxidants 2024, 13(8), 1014; https://doi.org/10.3390/antiox13081014 - 20 Aug 2024
Cited by 2 | Viewed by 1207
Abstract
Supply chain waste gives rise to significant challenges in terms of disposal, making upcycling a promising and sustainable alternative for the recovery of bioactive compounds from by-products. Lignocellulosic by-products like STF231, which are derived from the medicinal plant extract industry, offer valuable compounds [...] Read more.
Supply chain waste gives rise to significant challenges in terms of disposal, making upcycling a promising and sustainable alternative for the recovery of bioactive compounds from by-products. Lignocellulosic by-products like STF231, which are derived from the medicinal plant extract industry, offer valuable compounds such as polyphenols and iridoids that can be recovered through upcycling. In an unprecedented study, we explored and compared conventional hydroethanolic extraction, ultrasound hydroethanolic extraction, and natural deep eutectic solvents–ultrasound extraction methods on STF231 to obtain extracts with antioxidant activity. The extraction profile of total polyphenols (TPCs) was measured using the Folin–Ciocalteu test and the antioxidant capacity of the extracts was tested with FRAP and DPPH assays. HPLC-UV was employed to quantify the phenolic and iridoid markers in the extracts. Additionally, the sustainability profile of the process was assessed using the green analytical procedure index (GAPI), AGREEprep, and analytical GREEnness metric approach (AGREE) frameworks. Our findings indicate that a choline chloride and lactic acid mixture at a 1:5 ratio, under optimal extraction conditions, resulted in extracts with higher TPC and similar antioxidant activity compared with conventional hydroethanolic extracts. The innovative aspect of this study lies in the potential application of sustainable upcycling protocols to a previously unexamined matrix, resulting in extracts with potential health applications. Full article
(This article belongs to the Section Natural and Synthetic Antioxidants)
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Graphical abstract

Graphical abstract
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<p>Microscopy of STF 231 particulate (<b>a</b>) and surface morphological analysis (<b>b</b>).</p>
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<p>Effect of % ethanol in water (<b>a</b>) and temperature (<b>b</b>) on conventional hydroethanolic extraction.</p>
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<p>Effect on TPC of % ethanol (<b>a</b>), temperature (<b>b</b>), extraction time (<b>c</b>), and solid-to-solvent ratio (<b>d</b>) in ultrasound-assisted extraction at 37 kHz.</p>
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<p>Effect of water content, (<b>a</b>) temperature, and (<b>b</b>) solid-to-solvent ratio in ChCl:CA NADES.</p>
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<p>Effect of water content, (<b>a</b>) temperature, and (<b>b</b>) solid-to-solvent ratio in ChCl:LA NADES.</p>
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<p>Effect of water content, (<b>a</b>) temperature, and (<b>b</b>) solid-to-solvent ratio in Su:CA NADES.</p>
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<p>Comparison of TPC in original bitter tincture (BT) and TPC in best extraction conditions in conventional hydroethanolic extraction (CE), hydroethanolic ultrasound-assisted extraction (EtOH UAE), choline chloride/citric acid (ChCl:CA), choline chloride/lactic acid (ChCl:LA), and sucrose/citric acid (Su:CA).</p>
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<p>DPPH antioxidant activity (<b>a</b>) and FRAP reducing power (<b>b</b>) of original bitter tincture (BT), conventional hydroethanolic extraction (CE), hydroethanolic ultrasound-assisted extraction (EtOH UAE), choline chloride/citric acid ultrasound-assisted extraction (ChCl:CA UAE), choline chloride/lactic acid ultrasound-assisted extraction (ChCl:LA UAE), and sucrose/citric acid ultrasound-assisted extraction (Su:CA UAE).</p>
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<p>Green analytical procedure index evaluation of CE (<b>a</b>) and EtOH UAE, NADES UAE (<b>b</b>).</p>
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<p>AGREEprep assessment of CE (<b>a</b>) and EtOH UAE, NADES UAE (<b>b</b>). Numbers refer to: 1—sample preparation placement, 2—hazardous material, 3—sustainability, renewability, and reusability materials, 4—waste, 5—size economy of the sample, 6—sample throughput, 7—integration and automation, 8—energy consumption, 9—post-sample configuration for the analysis, 10—operator’s safety.</p>
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<p>AGREE assessment of CE (<b>a</b>) and EtOH UAE, NADES UAE (<b>b</b>). Numbers refer to: 1—avoid sample treatment, 2—minimal sample size, 3—in situ measurements, 4—save reagents, 5—automated and miniaturized methods, 6—derivatization avoided, 7—waste avoided, 8—multianalyte methods, 9—energy minimized, 10—reagents from renewable source, 11—toxic reagents eliminated, 12—operator safety.</p>
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15 pages, 2802 KiB  
Article
Mitogen-Activated Protein Kinase Kinase OsMEK2 Positively Regulates Ca2+ Influx and Ferroptotic Cell Death during Rice Immune Responses
by Juan Wang, Nam Khoa Nguyen, Dongping Liu and Nam-Soo Jwa
Antioxidants 2024, 13(8), 1013; https://doi.org/10.3390/antiox13081013 - 20 Aug 2024
Viewed by 1166
Abstract
Mitogen-activated protein (MAP) kinase (MAPK) signaling pathway is important in plant immune responses, involved in iron- and reactive oxygen species (ROS)-dependent ferroptotic cell death mediated by Ca2+. High Ca2+ influx triggered iron-dependent ROS accumulation, lipid peroxidation, and subsequent hypersensitive response [...] Read more.
Mitogen-activated protein (MAP) kinase (MAPK) signaling pathway is important in plant immune responses, involved in iron- and reactive oxygen species (ROS)-dependent ferroptotic cell death mediated by Ca2+. High Ca2+ influx triggered iron-dependent ROS accumulation, lipid peroxidation, and subsequent hypersensitive response (HR) cell death in rice (Oryza sativa). Apoplastic Ca2+ chelation by EGTA during avirulent Magnaporthe oryzae infection altered Ca2+, ROS, and Fe2+ accumulation, increasing rice susceptibility to infection. By contrast, acibenzolar-S-methyl (ASM), a plant defense activator, significantly enhanced Ca2+ influx, and H2O2 accumulation, triggering rice ferroptotic cell death during virulent Magnaporthe oryzae infection. Here, we report a novel role of the MAPK signaling pathway in regulating cytoplasmic Ca2+ increase during ferroptotic cell death in rice immunity, using the ΔOsmek2 knockout mutant rice. The knockout of rice OsMEK2 impaired the ROS accumulation, lipid peroxidation, and iron accumulation during avirulent M. oryzae infection. This study has shown that OsMEK2 could positively regulate iron- and ROS-dependent ferroptotic cell death in rice by modulating the expression of OsNADP-ME, OsRBOHB, OsPLC, and OsCNGC. This modulation indicates a possible mechanism for how OsMEK2 participates in Ca2+ regulation in rice ferroptotic cell death, suggesting its broader role in plant immune responses in response to M. oryzae infection. Full article
(This article belongs to the Special Issue Reactive Oxygen and Nitrogen Species in Plants―2nd Edition)
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Figure 1

Figure 1
<p>Time-course detection of cytoplasmic Ca<sup>2+</sup> and H<sub>2</sub>O<sub>2</sub> accumulation in WT rice DJ and <span class="html-italic">ΔOsmek2</span> mutant rice during avirulent <span class="html-italic">Magnaporthe oryzae</span> 007 infection. (<b>A</b>) Images of cytoplasmic Ca<sup>2+</sup> staining by Fluo-5F AM and H<sub>2</sub>O<sub>2</sub> staining by peroxy orange 1 (PO1) in wildtype (WT) rice DJ and <span class="html-italic">ΔOsmek2</span> mutant rice at 12, 24, 36, and 48 hpi, during avirulent <span class="html-italic">M. oryzae</span> 007 infection. Bars = 10 μm. (<b>B</b>) Quantification of cytoplasmic Ca<sup>2+</sup> accumulation in rice sheath cells infected with <span class="html-italic">M. oryzae</span> 007 at 12, 24, 36, and 48 hpi. (<b>C</b>) Quantification of cytoplasmic H<sub>2</sub>O<sub>2</sub> accumulations in rice sheath cells infected with <span class="html-italic">M. oryzae</span> 007 at 12, 24, 36, and 48 hpi. Values are means ± SD (<span class="html-italic">n</span> = 3 biological repeats). Asterisks above the bar indicated significantly different means, as determined by the ANOVA test (mixed-effects analysis) (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01; ns, not significant). SD, standard deviation.</p>
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<p>Effects of EGTA on HR cell death, cytoplasmic Ca<sup>2+</sup>, H<sub>2</sub>O<sub>2</sub> accumulation, and iron accumulation in WT rice DJ and <span class="html-italic">ΔOsmek2</span> mutant rice during avirulent <span class="html-italic">Magnaporthe oryzae</span> 007 infection. (<b>A</b>) Images of HR cell death, cytoplasmic Ca<sup>2+</sup> staining, H<sub>2</sub>O<sub>2</sub> staining, and iron staining in wildtype (WT) rice DJ and <span class="html-italic">ΔOsmek2</span> mutant rice. Bars = 10 μm. (<b>B</b>) Quantification of different infection types in rice sheath cells of wildtype (WT) rice DJ and <span class="html-italic">ΔOsmek2</span> mutant rice. IH, invasive hyphae; HR, hypersensitive response. (<b>C</b>) Quantification of cytoplasmic Ca<sup>2+</sup> accumulation in rice sheath cells of wildtype (WT) rice DJ and <span class="html-italic">ΔOsmek2</span> mutant rice. (<b>D</b>) Quantification of cytoplasmic H<sub>2</sub>O<sub>2</sub> accumulation in rice sheath cells of wildtype (WT) rice DJ and <span class="html-italic">ΔOsmek2</span> mutant rice. Values are means ± SD (<span class="html-italic">n</span> = 3 biological repeats). Different letters above the bar indicated significantly different means, as determined by one-way ANOVA test followed by Tukey’s HSD. SD, standard deviation.</p>
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<p>Time-course detection of cytoplasmic Ca<sup>2+</sup> and H<sub>2</sub>O<sub>2</sub> accumulation in WT rice DJ and <span class="html-italic">ΔOsmek2</span> mutant rice during virulent <span class="html-italic">Magnaporthe oryzae</span> PO6-6 infection. (<b>A</b>) Images of cytoplasmic Ca<sup>2+</sup> staining by Fluo-5F AM and H<sub>2</sub>O<sub>2</sub> staining by peroxy orange 1 (PO1) in wildtype (WT) rice DJ and <span class="html-italic">ΔOsmek2</span> mutant rice at 12, 24, 36, and 48 hpi, during virulent <span class="html-italic">M. oryzae</span> PO6-6 infection. Bars = 10 μm. (<b>B</b>) Quantification of cytoplasmic Ca<sup>2+</sup> accumulation in rice sheath cells infected with <span class="html-italic">M. oryzae</span> PO6-6 at 12, 24, 36, and 48 hpi. (<b>C</b>) Quantification of cytoplasmic H<sub>2</sub>O<sub>2</sub> accumulations in rice sheath cells infected with <span class="html-italic">M. oryzae</span> PO6-6 at 12, 24, 36, and 48 hpi. Values are means ± SD (<span class="html-italic">n</span> = 3 biological repeats). Data were analyzed by the ANOVA test (mixed-effects analysis) (ns, not significant). SD, standard deviation.</p>
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<p>Effects of ASM on HR cell death, cytoplasmic Ca<sup>2+</sup>, H<sub>2</sub>O<sub>2</sub> accumulation, and iron accumulation in WT rice DJ and <span class="html-italic">ΔOsmek2</span> mutant rice during virulent <span class="html-italic">Magnaporthe oryzae</span> PO6-6 infection. (<b>A</b>) Images of HR cell death, cytoplasmic Ca<sup>2+</sup> staining, H<sub>2</sub>O<sub>2</sub> staining, and iron staining in wildtype (WT) rice DJ and <span class="html-italic">ΔOsmek2</span> mutant rice. Bars = 10 μm. (<b>B</b>) Quantification of different infection types in rice sheath cells of wildtype (WT) rice DJ and <span class="html-italic">ΔOsmek2</span> mutant rice. IH, invasive hyphae; HR, hypersensitive response. (<b>C</b>) Quantification of cytoplasmic Ca<sup>2+</sup> accumulation in rice sheath cells of wildtype (WT) rice DJ and <span class="html-italic">ΔOsmek2</span> mutant rice. (<b>D</b>) Quantification of cytoplasmic H<sub>2</sub>O<sub>2</sub> accumulation in rice sheath cells of wildtype (WT) rice DJ and <span class="html-italic">ΔOsmek2</span> mutant rice. Values are means ± SD (<span class="html-italic">n</span> = 3 biological repeats). Different letters above the bar indicated significantly different means, as determined by one-way ANOVA test followed by Tukey’s HSD. SD, standard deviation.</p>
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<p>Comparisons of GSH depletion and lipid peroxidation in WT rice DJ and <span class="html-italic">ΔOsmek2</span> mutant rice during avirulent <span class="html-italic">Magnaporthe oryzae</span> 007 infection. (<b>A</b>) Quantification of reduced glutathione (GSH) in rice leaf sheaths in WT rice DJ and <span class="html-italic">ΔOsmek2</span> mutant rice at 48 hpi. Values are means ± SD (<span class="html-italic">n</span> = 3) of GSH contents. (<b>B</b>) Quantification of total glutathione (GSH + GSSG) in rice leaf sheaths in WT rice DJ and <span class="html-italic">ΔOsmek2</span> mutant rice at 48 hpi. Values are means ± SD (<span class="html-italic">n</span> = 3) of total glutathione contents. (<b>C</b>) Determination of lipid peroxidation by quantifying malondialdehyde (MDA) in rice leaf sheaths in WT rice DJ and <span class="html-italic">ΔOsmek2</span> mutant rice at 48 hpi. Values are means ± SD (<span class="html-italic">n</span> = 3) of MDA concentrations. Asterisks above the bar indicated significantly different means, as determined by the Student’s <span class="html-italic">t</span>-test (* <span class="html-italic">p</span> &lt; 0.05). SD, standard deviation.</p>
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<p>Real-time reverse transcription (qRT-PCR) analysis of time-course expressions of ROS- and calcium-related genes in leaf sheaths of rice DJ and <span class="html-italic">ΔOsmek2</span> during avirulent <span class="html-italic">Magnaporthe oryzae</span> 007 infection. Relative expression levels of <span class="html-italic">OsNADP-ME</span>, <span class="html-italic">OsRBOHB</span>, <span class="html-italic">OsPLC2</span>, <span class="html-italic">OsPLC4</span>, <span class="html-italic">OsCNGC2</span>, and <span class="html-italic">OsCNGC13</span> were normalized by the expression of <span class="html-italic">OsUbiquitin</span> (<span class="html-italic">OsUbi</span>). The data represent means ± SD of relative gene expression levels in rice leaf sheaths from three independent experiments. Asterisks above the bars indicate significant differences, as determined by the ANOVA test (mixed-effects analysis) (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01; ns, not significant). SD, standard deviation.</p>
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<p>Model of OsMEK2-mediated rice ferroptotic cell death and plant immune response. Membrane-bound PRRs recognize the <span class="html-italic">M. oryzae</span> effector and activate the mitogen-activated protein kinase (MAPK) pathway. Activated OsMEK2 triggers OsMPK1-OsWRKY90 pathway in the nucleus, upregulating the <span class="html-italic">NADP-malic enzyme</span> (<span class="html-italic">OsNADP-ME</span>), <span class="html-italic">NADP-oxidase</span> (<span class="html-italic">OsRBOHB</span>), <span class="html-italic">phospholipase C</span> (<span class="html-italic">OsPLC</span>), and <span class="html-italic">cyclic nucleotide-gated channels</span> (<span class="html-italic">OsCNGC</span>). OsNADP-ME and OsRBOHB regulate cellular ROS production, and OsPLC and OsCNGC facilitate internal (stores such as ER) and external (apoplast) Ca<sup>2+</sup> influx to the cytosol, playing important roles during rice ferroptotic cell death. Resistosomes form calcium-permeable channels which enable Ca<sup>2+</sup> influx to mediate cell death, while EGTA chelates apoplastic Ca<sup>2+</sup>, preventing Ca<sup>2+</sup> influx and subsequent cell death. The plant activator ASM stimulates cytoplasmic Ca<sup>2+</sup> increase through a yet undescribed mechanism, contributing to ferroptotic cell death. Solid arrows and solid T-shaped lines indicate positive and negative regulators, respectively. Dotted arrows indicate indirect or unverified connections.</p>
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30 pages, 1793 KiB  
Review
Mechanism of Action and Therapeutic Implications of Nrf2/HO-1 in Inflammatory Bowel Disease
by Lingling Yuan, Yingyi Wang, Na Li, Xuli Yang, Xuhui Sun, Huai’e Tian and Yi Zhang
Antioxidants 2024, 13(8), 1012; https://doi.org/10.3390/antiox13081012 - 20 Aug 2024
Cited by 1 | Viewed by 2483
Abstract
Oxidative stress (OS) is a key factor in the generation of various pathophysiological conditions. Nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) is a major transcriptional regulator of antioxidant reactions. Heme oxygenase-1 (HO-1), a gene regulated by Nrf2, is one of the most [...] Read more.
Oxidative stress (OS) is a key factor in the generation of various pathophysiological conditions. Nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) is a major transcriptional regulator of antioxidant reactions. Heme oxygenase-1 (HO-1), a gene regulated by Nrf2, is one of the most critical cytoprotective molecules. In recent years, Nrf2/HO-1 has received widespread attention as a major regulatory pathway for intracellular defense against oxidative stress. It is considered as a potential target for the treatment of inflammatory bowel disease (IBD). This review highlights the mechanism of action and therapeutic significance of Nrf2/HO-1 in IBD and IBD complications (intestinal fibrosis and colorectal cancer (CRC)), as well as the potential of phytochemicals targeting Nrf2/HO-1 in the treatment of IBD. The results suggest that the therapeutic effects of Nrf2/HO-1 on IBD mainly involve the following aspects: (1) Controlling of oxidative stress to reduce intestinal inflammation and injury; (2) Regulation of intestinal flora to repair the intestinal mucosal barrier; and (3) Prevention of ferroptosis in intestinal epithelial cells. However, due to the complex role of Nrf2/HO-1, a more nuanced understanding of the exact mechanisms involved in Nrf2/HO-1 is the way forward for the treatment of IBD in the future. Full article
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<p>The protein structure of Nrf2. The Nrf2 protein contains 7 domains, Neh1-7. The N-terminal Nrf2-ECH homology (Neh) 2 domain has ETGE and DLG motifs that bind to the Kelch domain of Kelch-like ECH-associated protein 1 (KEAP1). The Neh4 and Neh5 domains mediate the transactivation activity of Nrf2. The Neh7 domain inhibits the transcription of the Nrf2 target gene. The Neh6 domain has DSGIS and DSAPGS motifs. The sMaf element of the Neh1 domain helps Nrf2 dimerization with DNA binding to other transcription factors. The C-terminal Neh3 domain supports Nrf2 transcriptional activity. NH2, N-terminal; COOH, C-terminal; GSK3-β, glycogen synthase kinase-3 beta; β-TrCP, beta-transducin repeat containing E3 ubiquitin protein ligase; HRD1, E3 ubiquitin ligase; sMaf, small musculoaponeurotic fibrosarcoma protein.</p>
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<p>Nrf2/HO-1 axis activity in oxidative conditions. Heme is a pro-oxidant and generates reactive oxygen species (ROS). Under oxidative stress, Keap1 undergoes a conformational change that induces its ubiquitination and degradation, as well as the translocation of Nrf2 to the nucleus Nrf2, which translocates to the nucleus and binds to the ARE in the promoter region of antioxidant genes. Elevation of the target gene downstream of Nrf2, HO-1, helps to scavenge mROS, thus counteracting the deleterious effects of ROS on mitochondrial function. HO-1 degrades heme to bilirubin and carbon (CO). HO-1 degrades hemoglobin to biliverdin, unstable iron, and carbon monoxide (CO). Unstable iron can act as a pro-oxidant via the Fenton reaction, but it stimulates ferritin transcription, which stores it for antioxidant and anti-inflammatory activity. Carbon monoxide is involved in several downstream processes, ultimately acting as an antioxidant, anti-inflammatory, vasomodulator, and anti-apoptotic agent. Biliverdin is an antioxidant molecule that is rapidly converted to bilirubin by biliverdin reductase (BVR), which has antioxidant and anti-inflammatory properties. ARE, antioxidant response element; ROS, reactive oxygen species; GSH, glutathione; INOS, inductible nitric oxide synthase; IL, interleukin; MPO, myeloperoxidase; MDA, malondialdehyde; NO, nitric oxide; NQO1, NAD(P)H: quinone oxidoreductase; ROS, reactive oxygen species; SOD, superoxide dismutase; TNF-α, tumor necrosis factor α. The “↑” and “↓” in the picture indicate increase and decrease respectively.</p>
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<p>Mechanisms of targeting the Nrf2/HO-1 axis for the treatment of IBD. (<b>a</b>) Nrf2/HO-1 activates a series of signaling pathways targeting inflammation, such as the NF-κB pathway and the NLRP3 pathway, to reduce the levels of inflammatory factors, such as IL-6, TNF-α, and IL-1β, as well as inducing the production of antioxidant enzymes, such as SOD and GSH, to reduce the oxidative stress caused by the aggravation of ROS and to attenuate the pathologic inflammatory response of the intestine. (<b>b</b>) Activation of the Nrf2/HO-1 axis improves the intestinal microbiota, promotes the growth of beneficial bacteria, inhibits the colonization of pathogenic bacteria, increases the expression level of tight junction proteins, such as intestinal occludin, claudin-1, and ZO-1, and maintains the integrity of the intestinal epithelial barrier. (<b>c</b>) Activation of Nrf2/HO-1 upregulates GPX4 expression, inhibits oxidative stress and iron overload, and reduces ROS production, thereby inhibiting intracellular iron death and exerting anti-inflammatory effects.</p>
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28 pages, 4264 KiB  
Article
Antioxidant Marine Hydrolysates Isolated from Tuna Mixed Byproducts: An Example of Fishery Side Streams Upcycling
by Federica Grasso, María Mercedes Alonso Martínez, Federica Turrini, Diego Méndez Paz, Rebeca Vázquez Sobrado, Valentina Orlandi, Marte Jenssen, Kjersti Lian, Junio Rombi, Micaela Tiso, Elisabetta Razzuoli, Celina Costas and Raffaella Boggia
Antioxidants 2024, 13(8), 1011; https://doi.org/10.3390/antiox13081011 - 19 Aug 2024
Viewed by 1212
Abstract
The aim of this research is to propose simple and scalable processes to obtain bioactive peptides extensively hydrolyzed starting from a tuna mixed biomass. The upcycling of this powdered biomass is challenging since it comes from the unsorted industrial side streams of the [...] Read more.
The aim of this research is to propose simple and scalable processes to obtain bioactive peptides extensively hydrolyzed starting from a tuna mixed biomass. The upcycling of this powdered biomass is challenging since it comes from the unsorted industrial side streams of the tuna canning process (cooked residues from fillet trimming) after a patented mild dehydration useful for preventing its degradation until its exploitation. Two different protocols were proposed, with and without the inclusion of an exogenous enzyme (Enzymatic-Assisted Extraction, EAE), with no relevant differences in yields (24% vs. 22%) and a comparable amino acid composition. Nevertheless, the former protocol (with EAE) provided peptides with an average molecular weight of 1.3 kDa, and the second one (without EAE) provided peptides with an average molecular weight of 2.2 kDa. The two corresponding types of tuna protein hydrolysates (Enzymatic Hydrolysates (EH) and Non-Enzymatic Hydrolysates (NEH)) were characterized by proximate compositions, pH, color profile, amino acid analysis, FTIR spectra, and molecular weight distribution. In addition, several biological analyses were performed to assess their potential use as nutraceutical supplements: special attention has been paid to antioxidant activity using three different methods to quantify it. EH showed the most promising antioxidant activity which could be exploited also in other fields (e.g., biomaterials, cosmetics). Full article
(This article belongs to the Section Extraction and Industrial Applications of Antioxidants)
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<p>Cooked tuna side streams before (TFB) and after (TFP) the mild dehydration process.</p>
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<p>Tuna protein hydrolysates: EH (Enzymatic Hydrolysates) and NEH (Non-Enzymatic Hydrolysates).</p>
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<p>The amino acid profiles of EH (Enzymatic Hydrolysates) and NEH (Non-Enzymatic Hydrolysates).</p>
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<p>Spectra of EH (Enzymatic Hydrolysates) and NEH (Non-Enzymatic Hydrolysates).</p>
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<p>Determination of TEAC (Trolox Equivalent Antioxidant Capacity) and ORAC (Oxygen Radical Absorbance Capacity) for EH (Enzymatic Hydrolysates) and NEH (Non-Enzymatic Hydrolysates). (* Statistics between tests presented with different colors; same colors indicate that there is no significant difference, <span class="html-italic">p</span> ≤ 0.05).</p>
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<p>Cellular Antioxidant Activity (CAA) of the tuna protein hydrolysates EH (Enzymatic Hydrolysates); * significant differences (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Antihypertensive activity of EH (Enzymatic Hydrolysates) and NEH (Non-Enzymatic Hydrolysates).</p>
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<p>Cell viability in response to sample concentration (*) significant differences (<span class="html-italic">p</span> &lt; 0.05) of the protein hydrolysates EH (Enzymatic Hydrolysates) and NEH (Non-Enzymatic Hydrolysates). If cell viability is &lt; 70% (under blue line), the sample is considered to have cytotoxic potential.</p>
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<p>Inflammatory factor production of the hydrolysates from tuna byproduct EH (Enzymatic Hydrolysates) and NEH (Non-Enzymatic Hydrolysates) in response to sample concentration (* significant differences (<span class="html-italic">p</span> &lt; 0.05) with respect to LPS).</p>
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<p>Osteoblast proliferation and osteoclast inhibition of EH (Enzymatic Hydrolysates) and NEH (Non-Enzymatic Hydrolysates). (*) significant differences (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Comparison of osteoblast differentiation activity of EH (Enzymatic Hydrolysates) and NEH (Non-Enzymatic Hydrolysates); * significant difference (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Comparison of fatty acid accumulation, * significant difference (<span class="html-italic">p</span> &lt; 0.05) of EH (Enzymatic Hydrolysates) and NEH (Non-Enzymatic Hydrolysates).</p>
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19 pages, 1487 KiB  
Article
Modulation of the Hyperglycemia Condition in Diabetic Lab Rats with Extracts of the Creole Jamaica Flower (Hibiscus sabdariffa L.) from the Morelia Region (Mexico)
by Teodoro Suárez-Diéguez, Marta Palma-Morales, Gloria Isabel Camacho Bernal, Erick Noe Valdez López, Celia Rodríguez-Pérez, Nelly del Socorro Cruz-Cansino and Juan Antonio Nieto
Antioxidants 2024, 13(8), 1010; https://doi.org/10.3390/antiox13081010 - 19 Aug 2024
Viewed by 1145
Abstract
Extracts from Jamaica flowers (Hibiscus sabdariffa) from Morelia (Mexico) were evaluated as antidiabetic ingredients in a diabetic rat lab model for 80 days at doses of 200, 400, and 600 mg extract/kg rat weight. The hydroalcoholic extract (water:ethanol 80:20 (v [...] Read more.
Extracts from Jamaica flowers (Hibiscus sabdariffa) from Morelia (Mexico) were evaluated as antidiabetic ingredients in a diabetic rat lab model for 80 days at doses of 200, 400, and 600 mg extract/kg rat weight. The hydroalcoholic extract (water:ethanol 80:20 (v/v) at 50 °C) showed a TPC value of 403.28 ± 7.71 mg GAE/g extract, and an antioxidant activity of 0.219 ± 0.00003 mmol Trolox/g (ABTS) and 0.134 ± 0.00001 mmol Trolox/g (DPPH). The extract allowed reducing the diabetic glucose plasma levels under fasting conditions in a dose-dependent manner by 35.2%, 41.63%, and 50.1%. Additionally, the highest dose of the extract (600 mg/kg) slightly reduced the short-term postprandial glucose response while improving the long-term response, reducing hyperglycemia by 45.1%. The same dose also improved lipid metabolism by reducing total cholesterol, triglycerides, VLDL, and LDL, while the HDL level increased. The improvement in glucose and lipid management in the treated groups also led to reduced levels of glycosylated hemoglobin, as well as lower insulin resistance (TyG index), compared to the diabetic control group. The results of this study suggest that extracts from Hibiscus sabdariffa (Morelia) can be used as potential functional ingredients or nutraceuticals for managing the diabetic condition. Full article
(This article belongs to the Section Health Outcomes of Antioxidants and Oxidative Stress)
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<p>Whole experimental design for the extract effect evaluation with the animal model.</p>
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<p>Inhibitory capacity against the enzyme α-amylase activity of the obtained <span class="html-italic">Hibiscus</span> extracts (obtained with water:ethanol 80:20 <span class="html-italic">v</span>/<span class="html-italic">v</span>, at 50 °C).</p>
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<p>Postprandial response of the diverse experimental groups after the whole experimental period (80 days). (<b>a</b>) Short-term postprandial response; (<b>b</b>) Long-term postprandial response. * Indicate significant differences between treatment and diabetic control group.</p>
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<p>Postprandial response of the diverse experimental groups after the whole experimental period (80 days). (<b>a</b>) Short-term postprandial response; (<b>b</b>) Long-term postprandial response. * Indicate significant differences between treatment and diabetic control group.</p>
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18 pages, 868 KiB  
Article
Effects of Hyperbaric (Non-Thermal) Sanitization and the Method of Extracting Pomegranate Juice on Its Antioxidant and Antihypertensive Properties
by Gieraldin Campos-Lozada, Jonathan Hernández-Miranda, Leonardo del Valle-Mondragón, Araceli Ortiz-Polo, Gabriel Betanzos-Cabrera and Gabriel Aguirre-Álvarez
Antioxidants 2024, 13(8), 1009; https://doi.org/10.3390/antiox13081009 - 19 Aug 2024
Viewed by 993
Abstract
Pomegranate (Punica granatum L.) is considered a functional food due to its polyphenol content that benefits the body. The type of processing the fruit undergoes is important, as this also influences the concentrations of these compounds. The pomegranate juice was extracted by [...] Read more.
Pomegranate (Punica granatum L.) is considered a functional food due to its polyphenol content that benefits the body. The type of processing the fruit undergoes is important, as this also influences the concentrations of these compounds. The pomegranate juice was extracted by two methods: manual extraction using a manual juicer through heat treatment in a water bath (Man-P), and extraction through mechanical pressing using Good Nature X-1 equipment and hyperbaric sanitization (Mech-Hyp). Bromatological analyses showed significant differences (p ≤ 0.05) between the two treatments. When subjected to hyperbaric sanitization, the juice showed higher concentrations of moisture, soluble solids, protein, and carbohydrates. In an antioxidant analysis, the ABTS radical showed no significant difference in the treatments, with 96.99% inhibition. For the DPPH radical, the sample with the highest inhibition was Man-P with 98.48%. The determination of phenols showed that there was a higher concentration in juice that underwent pasteurization (104.566 mg GAE/mL). However, the Mech-Hyp treatment exhibited a minor concentration of phenols with 85.70 mg GAE/mL. FTIR spectra revealed that the functional groups were mainly associated with carbohydrates. Regarding ACE inhibition, it was observed that the Man-P and Mech-Hyp juices showed greater inhibition of enzyme in hypertensive patients compared to normotensive patients. This activity can be attributed to the mechanisms of action of antioxidant compounds. Both extraction methods manual and mechanical pressing resulted in increased antioxidant and antihypertensive activity. The antioxidant compounds accompanied by adequate sanitation were decisive in an antimicrobial analysis, since no pathogenic microorganisms were observed in the juices. Full article
(This article belongs to the Special Issue Bioactive Compounds and Antioxidants in Fruits and Vegetables)
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<p>This figure shows the spectra of the functional groups identified in the juices.</p>
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<p>Phenol content in the juices. Different letters represent significant differences (<span class="html-italic">p</span> ≤ 0.05). Error bars represent ±1 SD of three replicates.</p>
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<p>Flavonoid content in the pomegranate juices. Different letters represent significant differences (<span class="html-italic">p</span> ≤ 0.05). Error bars represent ± 1 SD of three replicates.</p>
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<p>Anthocyanin content in the pomegranate juices. Different letters represent significant differences (<span class="html-italic">p</span> ≤ 0.05). Error bars represent ±1 SD of three replicates.</p>
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<p>Radical inhibition in juice ABTS (<b>a</b>) and DPPH (<b>b</b>). Different letters represent significant differences (<span class="html-italic">p</span> ≤ 0.05). Error bars represent ±1 SD of three replicates.</p>
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<p>ACE inhibition in hypertensive and normotensive patients. Different letters represent significant differences (<span class="html-italic">p</span> ≤ 0.05). Error bars represent ±1 SD of three replicates.</p>
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<p>ACE inhibition in pomegranate juice. Different letters represent significant differences (<span class="html-italic">p</span> ≤ 0.05). Error bars represent ±1 SD of three replicates.</p>
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22 pages, 5582 KiB  
Article
Resolvin D5 Protects Female Hairless Mouse Skin from Pathological Alterations Caused by UVB Irradiation
by Priscila Saito, Ingrid C. Pinto, Camilla C. A. Rodrigues, Ricardo L. N. de Matos, David L. Vale, Cristina P. B. Melo, Victor Fattori, Telma Saraiva-Santos, Soraia Mendes-Pierotti, Mariana M. Bertozzi, Ana P. F. R. L. Bracarense, Josiane A. Vignoli, Marcela M. Baracat, Sandra R. Georgetti, Waldiceu A. Verri and Rubia Casagrande
Antioxidants 2024, 13(8), 1008; https://doi.org/10.3390/antiox13081008 - 19 Aug 2024
Cited by 1 | Viewed by 979
Abstract
Resolvin D5 (RvD5) is a lipid mediator that has been reported to present anti-inflammatory and pro-resolution properties. Evidence also supports its capability to enhance reactive oxygen species (ROS) production during bacterial infections, which would be detrimental in diseases driven by ROS. The biological [...] Read more.
Resolvin D5 (RvD5) is a lipid mediator that has been reported to present anti-inflammatory and pro-resolution properties. Evidence also supports its capability to enhance reactive oxygen species (ROS) production during bacterial infections, which would be detrimental in diseases driven by ROS. The biological activity of RvD5 and mechanisms against UVB irradiation skin pathology have not been investigated so far. Female hairless mice were treated intraperitoneally with RvD5 before UVB stimulus. RvD5 reduced skin edema in a dose-dependent manner as well as oxidative stress by increasing antioxidants (endogenous tissue antioxidant scavenging of cationic radical, iron reduction, catalase activity and reduced glutathione levels) and decreasing pro-oxidants (superoxide anion and lipid peroxidation). RvD5 antioxidant activity was accompanied by enhancement of Nrf2, HO-1 and NQO1 mRNA expression. RvD5 reduced the production of IL-1β, TNF-α, TGF-β, and IL-10. RvD5 also reduced the inflammatory cell counts, including mast cells and neutrophils/macrophages. The reduction of oxidative stress and inflammation resulted in diminished matrix metalloproteinase 9 activity, collagen degradation, epidermal thickening and sunburn cell development. Therefore, this study demonstrates, to our knowledge, the first body of evidence that RvD5 can be used to treat UVB skin pathology and unveils, at least in part, its mechanisms of action. Full article
(This article belongs to the Special Issue Antioxidants for Skin Health)
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<p>RvD5 reduces the development of skin edema induced by UVB irradiation. (<b>A</b>) Experimental protocol. (<b>B</b>) Results of edema are presented as tissue weight in milligrams of skin. Bars are representative of two separate experiments and represent means ± SEM of 6 mice per group per experiment. Statistical analysis was performed by one-way ANOVA followed by Tukey’s test [* <span class="html-italic">p</span> &lt; 0.05 compared to the non-irradiated control group; <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05 compared to the irradiated control group (vehicle)].</p>
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<p>RvD5 inhibited UVB irradiation-induced decrease of skin antioxidant capacity. Protocol was followed as depicted in <a href="#antioxidants-13-01008-f001" class="html-fig">Figure 1</a>A to investigate total antioxidant capacity FRAP (<b>A</b>), ABTS (<b>B</b>), and GSH levels (<b>C</b>). Results are presented as nmol of Trolox per milligrams of tissue for FRAP and ABTS assays and micromoles per milligrams of tissue for GSH assay. Bars are representative of two separate experiments and represent means ± SEM of 6 mice per group per experiment. Statistical analysis was performed by one-way ANOVA followed by Tukey’s test [* <span class="html-italic">p</span> &lt; 0.05 compared to the non-irradiated control group; <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05 compared to the irradiated control group (vehicle)].</p>
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<p>RvD5 inhibited UVB irradiation-induced decrease of skin catalase activity, and induction of superoxide anion production and lipid peroxidation. Protocol was followed as depicted in <a href="#antioxidants-13-01008-f001" class="html-fig">Figure 1</a>A to investigate (<b>A</b>) catalase activity, (<b>B</b>) superoxide anion production, and (<b>C</b>) lipid peroxidation end-product LOOH. Bars are representative of two separate experiments and represent means ± SEM of 6 mice per group per experiment. Statistical analysis was performed by one-way ANOVA followed by Tukey’s test [* <span class="html-italic">p</span> &lt; 0.05 compared to the non-irradiated control group; <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05 compared to the irradiated control group (vehicle)].</p>
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<p>RvD5 enhances Nrf2, NQO1 and HO-1 mRNA expression in UVB irradiation. Protocol was followed as depicted in <a href="#antioxidants-13-01008-f001" class="html-fig">Figure 1</a>A to investigate Nrf2 (<b>A</b>), NQO1 (<b>B</b>), and HO-1 (<b>C</b>) mRNA. Bars are representative of two separate experiments and represent means ± SEM of 6 mice per group per experiment. Statistical analysis was performed by one-way ANOVA followed by Tukey’s test. [* <span class="html-italic">p</span> &lt; 0.05 compared to the non-irradiated control group; <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05 compared to the irradiated control group (vehicle)].</p>
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<p>RvD5 inhibits UVB irradiation-induced cytokine production. Protocol was followed as depicted in <a href="#antioxidants-13-01008-f001" class="html-fig">Figure 1</a>A to investigate IL-1β (<b>A</b>), TNFα (<b>B</b>), IL-10 (<b>C</b>) and TGFβ (<b>D</b>) production. Bars are representative of two separate experiments and represent means ± SEM of 6 mice per group per experiment. Statistical analysis was performed by one-way ANOVA followed by Tukey’s test [* <span class="html-italic">p</span> &lt; 0.05 compared to the non-irradiated control group; <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05 compared to the irradiated control group (vehicle)].</p>
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<p>RvD5 reduced UVB irradiation-induced increase of mast cell count. Protocol was followed as depicted in <a href="#antioxidants-13-01008-f001" class="html-fig">Figure 1</a>A to determine mast cells counts in toluidine blue stained slices. Representative images of the groups: non-irradiated control (<b>A</b>), irradiated treated with vehicle (<b>B</b>), and irradiated treated with 30 pg/mouse of RvD5 (<b>C</b>). Mast cells count of experimental groups is presented per field (<b>D</b>). Original magnification 400×. Bars are representative of two separate experiments and represent means ± SEM of 6 mice per group per experiment. Statistical analysis was performed by one-way ANOVA followed by Tukey’s test. [* <span class="html-italic">p</span> &lt; 0.05 compared to the non-irradiated control group; <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05 compared to the irradiated control group (vehicle)]. Arrows indicate mast cells.</p>
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<p>RvD5 reduces the recruitment of LysM-eGFP<sup>+</sup> cells triggered by UVB irradiation. Protocol was followed as depicted in <a href="#antioxidants-13-01008-f001" class="html-fig">Figure 1</a>A to investigate by fluorescence the recruitment of LysM-eGFP<sup>+</sup> cells (neutrophils and macrophages) in the skin. Representative images of the groups: non-irradiated control treated with vehicle (salina) (<b>A</b>), irradiated treated with vehicle (saline) (<b>B</b>), and irradiated treated with 30 pg/mouse of RvD5 (<b>C</b>). Results are expressed in eGFP fluorescence intensity (<b>D</b>). Original magnification 20× (images <b>A</b>–<b>C</b>). Representative images from each group are presented with a 50 µm scale. Statistical analysis was performed by one-way ANOVA followed by Tukey’s test. [* <span class="html-italic">p</span> &lt; 0.05 compared to the non-irradiated control group; <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05 compared to the irradiated control group (vehicle)].</p>
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<p>RvD5 inhibited UVB irradiation-induced MMP-9 activity in the skin. Protocol was followed as depicted in <a href="#antioxidants-13-01008-f001" class="html-fig">Figure 1</a>A to investigate MMP-9 activity. (<b>A</b>) Representative image of gelatin zymography is presented. (<b>B</b>) Quantitation of skin MMP-9 activity. Results are presented as arbitrary units per sample for MMP-9 activity. Bars are representative of two separate experiments and represent means ± SEM of 6 mice per group per experiment. Statistical analysis was performed by one-way ANOVA followed by Tukey’s test. [* <span class="html-italic">p</span> &lt; 0.05 compared to the non-irradiated control group; <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05 compared to the irradiated control group (vehicle)].</p>
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<p>RvD5 inhibited UVB irradiation-induced collagen fiber degradation in the skin. Protocol was followed as depicted in <a href="#antioxidants-13-01008-f001" class="html-fig">Figure 1</a>A to investigate collagen degradation using Masson’s trichrome staining. Representative images of the groups: (<b>A</b>) non-irradiated control treated with vehicle, (<b>B</b>) UVB irradiated treated with vehicle, and (<b>C</b>) UVB irradiated treated with 30 pg/mouse of RvD5 (100× magnification). Quantitative analysis of collagen degradation of experimental groups is presented as percentage of staining in panel (<b>D</b>). Bars are representative of two separate experiments and represent means ± SEM of 6 mice per group per experiment. Statistical analysis was performed by one-way ANOVA followed by Tukey’s test [* <span class="html-italic">p</span> &lt; 0.05 compared to the non-irradiated control group; <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05 compared to the irradiated control group (vehicle)].</p>
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<p>RvD5 reduces UVB radiation-induced sunburn cells. Mice were treated intraperitoneally with 30 pg of RvD5 1 h before and 7 h after the beginning of UVB irradiation. Sunburn cells were evaluated using hematoxylin and eosin staining (H &amp; E) in skin samples collected 12 h after the end of irradiation. The sections stained with H &amp; E were examined using light microscopy at 1000× magnification. Representative images of the groups: non-irradiated control (<b>A</b>), irradiated treated with vehicle (<b>B</b>), irradiated treated with 30 pg/mouse of RvD5 (<b>C</b>). Sunburn cells count is presented in cells per field in panel (<b>D</b>). Bars are representative of two separate experiments and represent means ± SEM of 6 mice per group per experiment. Statistical analysis was performed by one-way ANOVA followed by Tukey’s test [* <span class="html-italic">p</span> &lt; 0.05 compared to the non-irradiated control group; <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05 compared to the irradiated control group (vehicle)]. Arrows indicate sunburn cells.</p>
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<p>RvD5 reduced UVB irradiation-induced increase of epidermal thickness. Mice were treated intraperitoneally with 30 pg of RvD5 1 h before and 7 h after the beginning of UVB irradiation. The epidermal thickness was determined in samples collected 12 h after the end of irradiation and stained with hematoxylin and eosin staining (H&amp;E). Representative images of the groups: non-irradiated control (<b>A</b>), irradiated treated with vehicle (<b>B</b>), irradiated treated with 30 pg/mouse of RvD5 (<b>C</b>). The epidermal thickness of experimental groups is presented in μm in panel (<b>D</b>). The sections stained with H &amp; E were examined using light microscopy at 400× magnification. Bars are representative of two separate experiments and represent means ± SEM of 6 mice per group per experiment. Statistical analysis was performed by one-way ANOVA followed by Tukey’s test [* <span class="html-italic">p</span> &lt; 0.05 compared to the non-irradiated control group; <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05 compared to the irradiated control group (vehicle)]. Arrows indicate the epidermal thickness.</p>
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19 pages, 27780 KiB  
Article
Lysophosphatidylcholine Impairs the Mitochondria Homeostasis Leading to Trophoblast Dysfunction in Gestational Diabetes Mellitus
by Shao-Chi Hung, Te-Fu Chan, Hsiu-Chuan Chan, Chia-Ying Wu, Mei-Lin Chan, Jie-Yang Jhuang, Ji-Qin Tan, Jia-Bin Mei, Shi-Hui Law, Vinoth Kumar Ponnusamy, Hua-Chen Chan and Liang-Yin Ke
Antioxidants 2024, 13(8), 1007; https://doi.org/10.3390/antiox13081007 - 19 Aug 2024
Viewed by 1208
Abstract
Gestational diabetes mellitus (GDM) is a common pregnancy disorder associated with an increased risk of pre-eclampsia and macrosomia. Recent research has shown that the buildup of excess lipids within the placental trophoblast impairs mitochondrial function. However, the exact lipids that impact the placental [...] Read more.
Gestational diabetes mellitus (GDM) is a common pregnancy disorder associated with an increased risk of pre-eclampsia and macrosomia. Recent research has shown that the buildup of excess lipids within the placental trophoblast impairs mitochondrial function. However, the exact lipids that impact the placental trophoblast and the underlying mechanism remain unclear. GDM cases and healthy controls were recruited at Kaohsiung Medical University Hospital. The placenta and cord blood were taken during birth. Confocal and electron microscopy were utilized to examine the morphology of the placenta and mitochondria. We determined the lipid composition using liquid chromatography-mass spectrometry in data-independent analysis mode (LC/MSE). In vitro studies were carried out on choriocarcinoma cells (JEG3) to investigate the mechanism of trophoblast mitochondrial dysfunction. Results showed that the GDM placenta was distinguished by increased syncytial knots, chorangiosis, lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) overexpression, and mitochondrial dysfunction. Lysophosphatidylcholine (LPC) 16:0 was significantly elevated in the cord blood LDL of GDM patients. In vitro, we demonstrated that LPC dose-dependently disrupts mitochondrial function by increasing reactive oxygen species (ROS) levels and HIF-1α signaling. In conclusion, highly elevated LPC in cord blood plays a pivotal role in GDM, contributing to trophoblast impairment and pregnancy complications. Full article
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Figure 1

Figure 1
<p>The GDM placenta had aberrant structures and LOX-1 overexpression. (<b>A</b>) H&amp;E staining of the placenta in GDM revealed more syncytial knots and chorangiosis than in healthy controls (<span class="html-italic">n</span> = 6, six placentas from six different donors for each group). Scale bar represents 50 µm. The red arrow represents placental lesion. (<b>B</b>) Representative immunohistochemical staining for LOX-1 expression in healthy and GDM patients (<span class="html-italic">n</span> = 6). Scale bar: 50 µm. (<b>C</b>) LOX-1 expression increased 3.4-fold in GDM compared to healthy controls (<span class="html-italic">n</span> = 6). ** <span class="html-italic">p</span> &lt; 0.01. Abbreviations: GDM: gestational diabetes mellitus; NGDM: non-gestational diabetes healthy controls; LOX-1: lectin-like oxidized LDL receptor-1.</p>
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<p>The trophoblast of the GDM placenta showed lower mitochondrial mass and enhanced mitochondrial fusion. (<b>A</b>) Confocal microscopy of the NGDM (<b>upper panel</b>) and GDM (<b>lower panel</b>) placenta. OPA1 controls mitochondrial fusion (green color), TOM20 depicts mitochondrial content (red color), and DAPI stains the background nuclei (blue color) (<span class="html-italic">n</span> = 6). Scale bar: 100 µm. (<b>B</b>) The fold change of TOM20 and (<b>C</b>) OPA1/TOM20 ratio was assessed according to the relative fluorescence signal intensity (<span class="html-italic">n</span> = 6). Data were quantified by ImageJ software version 1.54i. (<b>D</b>) Transmission electron microscopy (TEM) was used to examine mitochondrial structure and morphology in NGDM (<b>upper panel</b>) and GDM (<b>lower panel</b>) placenta. Scale bar in the representative Figure: 5 µm. The red arrow represents the location of mitochondria. (<b>E</b>) The fold change of circularity (4π × area/(perimeter))<sup>2</sup> and (<b>F</b>) aspect ratio (largest (R)/smallest (r)) of mitochondria in NGDM and GDM placenta (<span class="html-italic">n</span> = 10 fields under transmission electron microscopy). * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01. Abbreviations: GDM: gestational diabetes mellitus; NGDM: non-gestational diabetes healthy controls; OPA1: optic atrophy 1; TOM20: translocase of outer mitochondrial membrane 20; DAPI: 4′,6-diamidino-2-phenylindole; TEM: transmission electron microscopy.</p>
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<p>Lysophosphatidylcholine increased in GDM cord blood. (<b>A</b>) Representative lipid patterns in the cord blood of NGDM (<b>upper panel</b>) and GDM (<b>lower panel</b>) as determined by UPLC/MS<sup>E</sup> (<span class="html-italic">n</span> = 6 for the NGDM group, <span class="html-italic">n</span> = 6 for the GDM group; lipid extracts were from the cord blood of 12 Taiwanese women). Total lipids were separated using a CSH C18 column (Waters Corporation; Milford, MA, USA). Furthermore, the mass-to-charge signals were detected in the positive mode using XEVO G2 QTOF mass spectrometry (Waters Corporation; Milford, MA, USA). (<b>B</b>) We chose <span class="html-italic">m</span>/<span class="html-italic">z</span> with (1) abundance &gt; 5000, (2) <span class="html-italic">p</span> &lt; 0.0001, (3) fold change &gt; 5 as the criteria and selected the top 25 differentiative markers for GDM lipids. (<b>C</b>) By principal component analysis (PCA), PC1 and PC2 were determined. Furthermore, the most critical components in PC1 and PC2 were calculated, respectively. (<b>D</b>) According to the retention time and fragment daughter ions of lipid standards, we identified LPC signals, and (<b>E</b>) calculated the fold change of GDM in comparison to NGDM. ** <span class="html-italic">p</span> &lt; 0.01. Abbreviations: GDM: gestational diabetes mellitus; NGDM: non-gestational diabetes healthy controls; LPC: lysophosphatidylcholine; UPLC: ultra-pure liquid chromatography; MS<sup>E</sup>: mass spectrometry in data-independent analysis mode; CSH: charged surface hybrid.</p>
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<p>LPC-induced LOX-1 overexpression and excessive mitochondrial ROS production. (<b>A</b>) Human placental JEG3 cells were challenged with 25, 50, or 75 μM LPC. The glucose levels were 1.0 g/L for non-treat control and 4.5 g/L for vehicle control and other treatments. LOX-1 expression was evaluated using Western blot testing. (<b>B</b>) The fold changes of LOX-1 expression were calculated by chemiluminescence signal intensity (<span class="html-italic">n</span> = 5). Data were quantified using ImageJ software, and the differences were measured using one-way ANOVA. (<b>C</b>) 50 μM LPC was treated to human placental JEG3 cells with or without 2.5 μM CAY10585, the HIF-1α inhibitor, for 24 h. The mitochondria ROS production was detected by staining with MitoSOX<sup>TM</sup> (red color). The nuclei were stained with DAPI (blue). Signals were visualized by fluorescence microscopy. Scale bar, 20 µm. (<b>D</b>) The fold changes of the fluorescence signal were measured according to fluorescence signal intensity (<span class="html-italic">n</span> = 6). Data were quantified by ImageJ software. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, data were compared to control; <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, data were compared to 50 μM LPC treatment. Abbreviations: LPC: Lysophosphatidylcholine; LOX-1: lectin-like oxidized low-density lipoprotein (LDL) receptor-1; HIF-1α: Hypoxia-inducible factor 1-alpha; ROS: reactive oxygen species; MitoSOX: mitochondrial superoxide indicators for live cell imaging; DAPI: 4′,6-diamidino-2-phenylindole, a fluorescent stain that binds strongly to adenine–thymine-rich regions in DNA; ANOVA: analysis of variance, ns: no significant differences.</p>
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<p>Lysophosphatidylcholine disrupts mitochondrial homeostasis via the HIF-1α pathway. (<b>A</b>) Human placental JEG3 cells were treated with 25, 50, or 75 μM LPC to assess its effect on HIF-1α signaling. Besides, JEG3 cells were cotreated with 75 μM LPC and 2.5 μM CAY10585, the HIF-1α inhibitor, for 24 h. The glucose levels were 1.0 g/L for non-treat control and 4.5 g/L for vehicle control and other treatments. (<b>B</b>) Data were quantified by ImageJ software. The fold changes of signal intensity were tested (<span class="html-italic">n</span> = 6). (<b>C</b>) The mitochondrial mass of JEG3 cells was determined using the signal intensity of MitoTracker<sup>TM</sup> Green FM labeling. Scale bar, 20 µm. (<b>D</b>) Fluorescence signal intensity was measured (<span class="html-italic">n</span> = 6). (<b>E</b>) Finally, the expression of OPA1 was tested for mitochondrial fusion. (<b>F</b>) Data were quantified by ImageJ software (<span class="html-italic">n</span> = 5). ** <span class="html-italic">p</span> &lt; 0.01, data were compared to vehicle control; <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, data were compared to 75 or 50 μM LPC treatment (# for inhibitor effect). Abbreviations: LPC: Lysophosphatidylcholine; HIF-1α: Hypoxia-inducible factor 1-alpha; MitoTracker<sup>TM</sup> Green FM: enable mitochondria visualization with fluorescent imaging; DAPI: 4′,6-diamidino-2-phenylindole, a fluorescent stain that binds strongly to adenine–thymine-rich regions in DNA; ns: no significant differences.</p>
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<p>Lysophosphatidylcholine impaired the mitochondrial electron transport chain function. (<b>A</b>) Human placental JEG3 cells were treated with 50 μM LPC to evaluate the mitochondrial function. Besides, JEG3 cells were cotreated with 50 μM LPC and 2.5 μM CAY10585, the HIF-1α inhibitor, for 24 h (<span class="html-italic">n</span> = 6). The glucose levels were 1.0 g/L for non-treat control and 4.5 g/L for vehicle control and other treatments. Scale bar, 20 µm. (<b>B</b>) Fluorescence signal intensity was measured. Data were quantified by ImageJ software. The fold changes of signal intensity were tested (<span class="html-italic">n</span> = 5). * <span class="html-italic">p</span> &lt; 0.05, data were compared to vehicle control; <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05, data were compared to 50 μM LPC treatment. Abbreviations: LPC: Lysophosphatidylcholine; HIF-1α: Hypoxia-inducible factor 1-alpha; MitoTracker<sup>TM</sup> Red FM: fluorescent signal intensity was dependent on mitochondrial potential; DAPI: 4′,6-diamidino-2-phenylindole, a fluorescent stain that binds strongly to adenine–thymine-rich regions in DNA; ns: no significant differences.</p>
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<p>A schematic diagram showing the mechanism by which LPC triggers oxidative stress, mitochondria fusion, and dysfunction, which in turn causes trophoblast dysregulation and is positively connected with a greater incidence of chorangiosis and syncytial knots in GDM. The red arrow indicates the increasing levels of LPC, ROS, HIF-1α, and OPA-1. Figures created with BioRender.com. Abbreviations: GDM: gestational diabetes mellitus; LPC: lysophosphatidylcholine; oxLDL: oxidized low-density lipoprotein; LOX-1: lectin-like oxidized low-density lipoprotein (LDL) receptor-1; HIF-1α: hypoxia-inducible factor 1-alpha; ROS: reactive oxygen species; OPA1: optic atrophy 1.</p>
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21 pages, 14131 KiB  
Article
Activation of Nrf2 at Critical Windows of Development Alters Tissue-Specific Protein S-Glutathionylation in the Zebrafish (Danio rerio) Embryo
by Emily S. Marques, Emily G. Severance, Paige Arsenault, Sarah M. Zahn and Alicia R. Timme-Laragy
Antioxidants 2024, 13(8), 1006; https://doi.org/10.3390/antiox13081006 - 19 Aug 2024
Viewed by 959
Abstract
Activation of Nrf2—the master regulator of antioxidative response—at different stages of embryonic development has been shown to result in changes in gene expression, but the tissue-specific and downstream effects of Nrf2 activation during development remain unclear. This work seeks to elucidate the tissue-specific [...] Read more.
Activation of Nrf2—the master regulator of antioxidative response—at different stages of embryonic development has been shown to result in changes in gene expression, but the tissue-specific and downstream effects of Nrf2 activation during development remain unclear. This work seeks to elucidate the tissue-specific Nrf2 cellular localization and the downstream changes in protein S-glutathionylation during critical windows of zebrafish (Danio rerio) development. Wild-type and mutant zebrafish embryos with a loss-of-function mutation in Nrf2a were treated with two canonical activators, sulforaphane (SFN; 40 µM) or tert-butylhydroquinone (tBHQ; 1 µM), for 6 h at either pharyngula, hatching, or the protruding-mouth stage. Nrf2a protein and S-glutathionylation were visualized in situ using immunohistochemistry. At the hatching stage, Nrf2a protein levels were decreased with SFN, but not tBHQ, exposure. Exposure to both activators, however, decreased downstream S-glutathionylation. Stage- and tissue-specific differences in Nrf2a protein and S-glutathionylation were identified in the pancreatic islet and liver. Protein S-glutathionylation in Nrf2a mutant fish was increased in the liver by both activators, but not the islets, indicating a tissue-specific and Nrf2a-dependent dysregulation. This work demonstrates that critical windows of exposure and Nrf2a activity may influence redox homeostasis and highlights the importance of considering tissue-specific outcomes and sensitivity in developmental redox biology. Full article
(This article belongs to the Special Issue Antioxidant Defenses in Fish—2nd Edition)
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Graphical abstract
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<p>(<b>A</b>) Zebrafish were treated with 40 µM SFN or 1 µM tBHQ during the pharyngula, hatching, and protruding-mouth stages for 6 h and then fixed, and Nrf2a protein was labeled using immunohistochemistry (IHC). The mean fluorescence intensity (FI) of Nrf2a protein in the (<b>B</b>) body tissue, (<b>C</b>) brain, (<b>D</b>) heart, (<b>E</b>) gut, and (<b>F</b>) pancreas was measured; liver data are presented at higher magnification in Figure 3 below. Representative heatmaps were generated to visualize Nrf2a protein fluorescence differences at the (<b>G</b>) pharyngula, (<b>H</b>) hatching, and (<b>I</b>) protruding-mouth stages. The gut and pancreas are not present (n.p.) at the pharyngula stage. Statistics were performed with a two-way ANOVA and Fisher’s LSD post hoc test. <span class="html-italic">n</span> = 7–12 fish. Letters indicate significant differences (<span class="html-italic">p</span> ≤ 0.05) among groups. * <span class="html-italic">p</span> ≤ 0.05, *** <span class="html-italic">p</span> ≤ 0.001, **** <span class="html-italic">p</span> ≤ 0.0001. Scale bar = 100 µm.</p>
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<p>(<b>A</b>) Zebrafish were treated with 40 µM SFN or 1 µM tBHQ during the pharyngula, hatching, or protruding-mouth stage for 6 h and then fixed, and Nrf2a protein was labeled using immunohistochemistry (IHC). Z-stacks of the islet were acquired via confocal microscopy. (<b>A</b>) Islet volume and (<b>B</b>) islet mean fluorescence intensity (FI) of Nrf2a protein were measured with Nikon NIS elements software (available at Light Microscopy Core analysis workstations at the UMass Amherst Institute for Applied Life Sciences; <a href="https://www.microscope.healthcare.nikon.com/bioimaging-centers/nic-and-cofe/university-of-massachusetts-amherst" target="_blank">https://www.microscope.healthcare.nikon.com/bioimaging-centers/nic-and-cofe/university-of-massachusetts-amherst</a>). Representative images of each group at the (<b>C</b>) pharyngula, (<b>D</b>) hatching, and (<b>E</b>) protruding-mouth stage are shown. Images show max intensity projections of the islet Z-stack (circled in yellow) where FITC (green) represents β-cells, TRITC (red) represents Nrf2a protein, and DAPI (blue) represents nuclei. Statistics were performed with a two-way ANOVA and Fisher’s LSD post hoc test. <span class="html-italic">n</span> = 5–12 fish. Letters indicate significant differences (<span class="html-italic">p</span> ≤ 0.05) among groups. Scale bar = 25 µm.</p>
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<p>Zebrafish were treated with 40 µM SFN or 1 µM tBHQ during the protruding-mouth stage for 6 h and then fixed, and Nrf2a protein was labeled using immunohistochemistry (IHC). Liver images were acquired using confocal microscopy. (<b>A</b>) The mean fluorescence intensity (FI) of labeled Nrf2a protein in the liver was quantified via image analysis. (<b>B</b>) Representative images of the zebrafish liver are shown, where the liver is outlined in yellow, TRITC (red) represents Nrf2a, and DAPI (blue) represents nuclei. Statistics were performed using a one-way ANOVA followed by Fisher’s LSD post hoc test. <span class="html-italic">n</span> = 8–13 fish. Letters indicate significant differences (<span class="html-italic">p</span> ≤ 0.05). Scale bar = 50 µm.</p>
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<p>Zebrafish were treated with 40 µM SFN or 1 µM tBHQ during the pharyngula, hatching, and protruding-mouth stages for 6 h and then fixed, and Nrf2a protein was labeled using immunohistochemistry (IHC). Colocalization analysis was performed on 40× confocal images of the liver, and a representative image of the pancreatic islet was taken from the Z-stack. Pearson’s R coefficients were converted to normally distributed Z-scores, shown as means ± SEMs, where higher Z-scores indicate greater colocalization between Nrf2a protein and nuclear DAPI staining within the region of interest. Significance was assessed with a two-way or three-way ANOVA followed by Fisher’s LSD post hoc test. <span class="html-italic">n</span> = 5–13 fish. Different letters indicate significant differences (<span class="html-italic">p</span> ≤ 0.05) among tissues. **** <span class="html-italic">p</span> ≤ 0.0001 between islet and liver of the DMSO WT groups.</p>
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<p>(<b>A</b>) Zebrafish were treated with 40 µM SFN or 1 µM tBHQ during the pharyngula, hatching, and protruding-mouth stages for 6 h and incubated with BioGee for 2 h prior to fixation (24 h post-exposure). BioGee protein conjugates were labeled in situ using immunohistochemistry. (<b>B</b>) Mean fluorescence intensity (Fl) of BioGee protein conjugates. Representative heatmaps were generated to visualize BioGee-protein conjugate fluorescence at the (<b>C</b>) pharyngula/hatching, (<b>D</b>) hatching/protruding-mouth, and (<b>E</b>) protruding-mouth/larval stages. Statistics were performed using a two-way ANOVA followed by Fisher’s LSD post hoc test. <span class="html-italic">n</span> = 8–10 fish. Different letters indicate significant differences (<span class="html-italic">p</span> ≤ 0.05) among groups. Scale bar = 100 μm.</p>
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<p>Zebrafish were treated with 40 µM SFN or 1 µM tBHQ for 6 h during the pharyngula, hatching, and protruding-mouth stages and incubated with BioGee for 2 h before fixation 24 h after the start of the exposure. BioGee protein conjugates were labeled in situ using IHC. Confocal microscopy was used to acquire Z-stacks of the islet. (<b>A</b>) Islet volume and (<b>B</b>) islet mean fluorescence intensity (FI) of BioGee. Representative images at the (<b>C</b>) pharyngula (start of exposure)/hatching (BioGee labeling and fixation), (<b>D</b>) hatching/protruding-mouth, and (<b>E</b>) protruding-mouth/larval stages are shown. Images are max intensity projections of the Z-stack the islet (outlined in yellow), where FITC (green) represents β-cells, TRITC (purple) represents BioGee protein conjugates, and DAPI (blue) represents nuclei. Statistical significance was assessed via two-way ANOVA followed by Fisher’s LSD post hoc test. <span class="html-italic">n</span> = 7–9 fish. Letters indicate significant differences (<span class="html-italic">p</span> ≤ 0.05) among groups. Scale bar = 25 µm.</p>
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<p>Zebrafish were treated with 40 µM SFN or 1 µM tBHQ during the hatching and protruding-mouth stages for 6 h and incubated with BioGee for 2 h before fixation 24 h after treatment. BioGee protein conjugates were labeled in situ using IHC. Confocal microscopy was used to acquire images of the liver using a 40× objective. (<b>A</b>) Liver mean fluorescence intensity (FI) of BioGee protein conjugates was determined via image analysis. Representative images of the zebrafish liver treated during the (<b>B</b>) hatching/protruding-mouth and (<b>C</b>) protruding-mouth/larval stage are shown where the liver is outlined in yellow, TRITC (purple) represents BioGee, and DAPI (blue) represents nuclei. The liver is not present (n.p.) at the stage visualized. Statistics were performed using a two-way ANOVA followed by Fisher’s LSD post hoc test. <span class="html-italic">n</span> = 6–12 fish. Different letters indicate significant differences (<span class="html-italic">p</span> ≤ 0.05) among groups. Scale bar = 50 µm.</p>
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<p>Zebrafish were treated with 40 µM SFN or 1 µM tBHQ for 6 h during the pharyngula, hatching, and protruding-mouth stages and then collected for gene expression via quantitative real-time PCR. mRNA expression was measured for <span class="html-italic">glutathione S-transferase Pi</span> (<span class="html-italic">gstp</span>) along with the housekeeping gene <span class="html-italic">β2-Microglobulin</span> (<span class="html-italic">b2m).</span> The ΔΔCT method was used to calculate fold change. Values are mean fold change ± SEM. Statistical significance was assessed via two-way ANOVA followed by Fisher’s LSD post hoc test. <span class="html-italic">n</span> = 2–3 pools of 15–20 (hatching and protruding-mouth stages) or 20–40 (pharyngula) fish. Different letters indicate significant differences (<span class="html-italic">p</span> ≤ 0.05) among groups.</p>
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1 pages, 444 KiB  
Correction
Correction: Feng et al. Photobiomodulation Inhibits Ischemia-Induced Brain Endothelial Senescence via Endothelial Nitric Oxide Synthase. Antioxidants 2024, 13, 633
by Yu Feng, Zhihai Huang, Xiaohui Ma, Xuemei Zong, Vesna Tesic, Baojin Ding, Celeste Yin-Chieh Wu, Reggie Hui-Chao Lee and Quanguang Zhang
Antioxidants 2024, 13(8), 1005; https://doi.org/10.3390/antiox13081005 - 19 Aug 2024
Viewed by 573
Abstract
In the original publication [...] Full article
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Figure 7

Figure 7
<p>Pretreatment with L-NAME prevented a PBMT-induced decrease in histone H2AX phosphorylation in bEnd.3 cells. (<b>A</b>) Representative immunofluorescence images for γH2AX (purple). Nuclei were counterstained with DAPI (blue). (<b>B</b>) The ratio of γH2AX+ cell and total cell numbers was calculated and expressed as percentage changes relative to the control group. Scale bar = 50 µm (n = 6). * indicates <span class="html-italic">p</span> &lt; 0.05 vs. control group; # indicates <span class="html-italic">p</span> &lt; 0.05 vs. OGD group. &amp; indicates <span class="html-italic">p</span> &lt; 0.05 vs. OGD + PBMT group.</p>
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18 pages, 11060 KiB  
Article
Sodium Selenite Induces Autophagy and Apoptosis in Cervical Cancer Cells via Mitochondrial ROS-Activated AMPK/mTOR/FOXO3a Pathway
by Cunqi Lv, Qingyu Zeng, Lei Qi, Yuanyuan Wang, Jiacheng Li, Huixin Sun, Linlin Du, Shuxiu Hao, Guijin Li, Chen Feng, Yu Zhang, Cheng Wang, Xinshu Wang, Rong Ma, Tong Wang and Qi Li
Antioxidants 2024, 13(8), 1004; https://doi.org/10.3390/antiox13081004 - 19 Aug 2024
Cited by 1 | Viewed by 1650
Abstract
Selenium (Se) is an essential trace element known for its significant role in maintaining human health and mitigating disease progression. Selenium and its compounds exhibit high selective cytotoxicity against tumor cells. However, their anti-cervical cancer (CC) effects and underlying mechanisms have not been [...] Read more.
Selenium (Se) is an essential trace element known for its significant role in maintaining human health and mitigating disease progression. Selenium and its compounds exhibit high selective cytotoxicity against tumor cells. However, their anti-cervical cancer (CC) effects and underlying mechanisms have not been fully explored. This study found that sodium selenite (SS) inhibits the viability of HeLa and SiHa cells in a dose- and time-dependent manner. Intraperitoneal injection of 3 and 6 mg/kg SS for 14 days in female nude mice significantly inhibited the growth of HeLa cell xenografts without evident hepatotoxicity or nephrotoxicity. RNA sequencing results indicated that the AMP-activated protein kinase (AMPK), Forkhead box protein O (FOXO), and apoptosis signaling pathways are key regulatory pathways in SS’s anti-CC effects, and SS’s inhibition of HeLa cell proliferation may be related to autophagy and ROS-induced apoptosis. Further research has revealed that SS induces cell autophagy and apoptosis through the AMPK/mTOR/FOXO3a pathway, characterized by the upregulation of p-AMPK/AMPK, FOXO3a, LC3-II, cleaved-caspase3, and cleaved-PARP and the downregulation of p-mTOR/mTOR and p62. Additionally, SS impaired mitochondrial function, including decreased mitochondrial membrane potential, mitochondrial Ca2+ overload, and accumulation of mitochondrial reactive oxygen species (mtROS). Pretreatment with Mitoquinone mesylate (Mito Q) and compound C partially reversed SS-induced apoptosis, autophagy, and proliferation inhibition. Pretreatment with 3-methyladenine (3-MA) enhances SS-induced apoptosis and proliferation inhibition in HeLa cells but reverses these effects in SiHa cells. In summary, SS induces apoptosis, autophagy, and proliferation inhibition in HeLa and SiHa cells through the activation of the AMPK/mTOR/FOXO3a signaling pathway via mtROS. Autophagy activation may be a major risk factor for SS-induced apoptosis in SiHa cells but can protect HeLa cells from SS-induced apoptosis. These findings provide new evidence for understanding the molecular mechanisms underlying SS in potential new drug development for CC. Full article
(This article belongs to the Special Issue Role of Mitochondria and ROS in Health and Disease)
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<p>The inhibitory effects of sodium selenite on HeLa and SiHa cell proliferation, migration, and invasion. (<b>A</b>,<b>B</b>) Cell viability was assessed using the CCK-8 assay after treating cells with various concentrations of SS (0, 2.5, 5, 7.5, 10, 15, 20, and 40 µM) for 6, 12, and 24 h. (<b>C</b>) The proliferation ability of HeLa and SiHa cells was assessed using CFSE staining. (<b>D</b>) Migration and invasion abilities were evaluated using transwell assays in HeLa cells and quantitatively analyzed by image J. Scale bar, 100 µm. All experiments were repeated at least three times. * indicates statistical significance compared to the control group, * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001. SS: sodium selenite.</p>
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<p>Inhibitory effect of sodium selenite on the growth of HeLa xenografts. (<b>A</b>) Schematic diagram of the establishment of HeLa xenograft models and administration (n = 5/group). (<b>B</b>) Body weights of the mice were monitored during the experiments for toxicity. (<b>C</b>) Tumor volumes were measured by vernier calipers and calculated by the length and width every 2 days. (<b>D</b>) Tumor weight in different intervention groups (<b>E</b>) Representative histological images of liver and kidney H&amp;E staining in control and 6 mg/kg SS-treated groups, magnification ×400. (<b>F</b>) H&amp;E staining of xenografts from control, 3 mg/kg SS, and 6 mg/kg SS intervention groups, magnification ×400. All experimental results were repeated at least five times. * indicates compared to the control group, * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001. SS: sodium selenite.</p>
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<p>Sodium selenite induces autophagy and apoptosis in HeLa and SiHa cells. (<b>A</b>) Volcano plot displaying the distribution of DEGs, highlighting significant genes in AMPK, FOXO, and apoptosis signaling pathways. (<b>B</b>) KEGG enrichment analysis showing significant enrichment of DEGs in AMPK, FOXO, and apoptosis pathways. (<b>C</b>) Quantitative analysis of the change in the percentage of MDC-positive cells after CQ pretreatment was performed using flow cytometry. (<b>D</b>) Changes in LC3-II protein levels after CQ pretreatment. (<b>E</b>) Quantitative analysis of the percentage of MDC-positive cells after SS treatment at different times and doses. (<b>F</b>) The percentage of apoptotic cells was detected and quantified using flow cytometry. (<b>G</b>) Protein expression levels of autophagy markers (LC3-II and p62) and apoptosis markers (cleaved-caspase3 and cleaved-PARP). All experiments were repeated at least three times. Con: Control; SS: sodium selenite; CQ: Chloroquine diphosphate salt; c-PARP: cleaved-PARP; c-caspase3: cleaved-caspase3.</p>
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<p>Sodium selenite induces proliferation inhibition, autophagy, and apoptosis via the AMPK/mTOR/FOXO3a pathway in HeLa and SiHa cells. (<b>A</b>) Protein expression levels in the AMPK/mTOR/FOXO3a pathway in HeLa and SiHa cells. (<b>B</b>) Localization of FOXO3a in the nucleus observed by confocal microscopy in HeLa cells. (<b>C</b>) Evaluation of glucose uptake ability using the 2-NBDG fluorescent glucose analog by confocal microscopy in HeLa cells. (<b>D</b>) Changes in the ADP/ATP ratio in HeLa cells. Changes in cell proliferation (<b>E</b>), the percentage of apoptotic cells (<b>F</b>), and the percentage of MDC-positive cells (<b>I</b>) in HeLa and SiHa cells after compound C pretreatment. (<b>G</b>) Expression of AMPK/mTOR/FOXO3a pathway proteins after compound C pretreatment in HeLa cells. (<b>H</b>) Analysis of autophagy- and apoptosis-related protein expression levels in HeLa cells. All results were repeated at least three times. # indicates statistical significance compared to the control group, ns: <span class="html-italic">p</span> &gt; 0.05, #### <span class="html-italic">p</span> &lt; 0.0001; * indicates statistical significance compared to the SS treatment group, ** <span class="html-italic">p</span> &lt; 0.01, **** <span class="html-italic">p</span> &lt; 0.0001. Con: Control; SS: sodium selenite; Com C: Compound C, c-PARP: cleaved-PARP; c-caspase3: cleaved-caspase3.</p>
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<p>Effects of sodium selenite on Ca<sup>2+</sup> levels, mitochondrial ROS production, and membrane potential in HeLa and SiHa cells. (<b>A</b>,<b>B</b>) Observation of intracellular Ca<sup>2+</sup> (<b>A</b>, magnification ×400) and mtROS (<b>B</b>, magnification ×200) levels by confocal microscopy in HeLa cells. (<b>C</b>,<b>D</b>) Quantitative analysis of Ca<sup>2+</sup> (<b>C</b>) and mtROS (<b>D</b>) levels in HeLa and SiHa cells using flow cytometry. (<b>E</b>) The levels of MMP were evaluated using JC-1 staining, visualized with a confocal microscope, magnification ×200. All results were repeated at least three times. * indicates statistical significance compared to the control group, **** <span class="html-italic">p</span> &lt; 0.0001.</p>
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<p>Sodium selenite induces autophagy and apoptosis via mitochondrial ROS. (<b>A</b>,<b>B</b>) After Mito Q pretreatment, mtROS levels were detected by confocal microscopy (<b>A</b>, magnification ×200) and quantified by flow cytometry (<b>B</b>) in HeLa cells. Changes in cell proliferation (<b>C</b>), the percentage of apoptotic cells (<b>D</b>), and the percentage of MDC-positive cells (<b>E</b>) were measured in HeLa and SiHa cells. (<b>F</b>) Expression levels of AMPK/mTOR/FOXO3a pathway proteins, as well as autophagy and apoptosis proteins in HeLa cells. All results were repeated at least three times. # indicates statistical significance compared to the control group, ns: <span class="html-italic">p</span> &gt; 0.05, #### <span class="html-italic">p</span> &lt; 0.0001; * indicates statistical significance compared to the SS treatment group, **** <span class="html-italic">p</span> &lt; 0.0001. Con: Control; SS: sodium selenite; Mito Q: Mitoquinone mesylate; c-PARP: cleaved-PARP; c-caspase3: cleaved-caspase3.</p>
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<p>The effects of 3-MA pretreatment on sodium selenite-induced apoptosis and proliferation inhibition in HeLa and SiHa cells. After 3-MA pretreatment, changes in the percentage of MDC-positive cells (<b>A</b>), the percentage of apoptotic cells (<b>C</b>), and cell proliferation (<b>D</b>) in HeLa and SiHa cells. (<b>B</b>) Changes in autophagic flux in HeLa cells were detected using Ad-mCherry-GFP-LC3B, magnification ×400. (<b>E</b>) The expression of autophagy and apoptosis-related proteins was assessed by Western blot in HeLa cells. All results were repeated at least three times. # indicates statistical significance compared to the control group, ns: <span class="html-italic">p</span> &gt; 0.05, #### <span class="html-italic">p</span> &lt; 0.0001; * indicates statistical significance compared to the SS treatment group, **** <span class="html-italic">p</span> &lt; 0.0001. 3-MA: 3-methyladenine; SS: sodium selenite; c-PARP: cleaved-PARP; c-caspase3: cleaved-caspase3.</p>
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<p>Sodium selenite induces autophagy and apoptosis in vivo via the AMPK/mTOR/FOXO3a signaling pathway. (<b>A</b>) Protein expression levels of LC3-II, p62, cleaved-caspase3, and cleaved-PARP in tumors from different intervention groups; the expression of β-actin was used as a reference. (<b>B</b>,<b>C</b>) Immunofluorescence analysis of cleaved-caspase3 and LC3B in tumor sections, magnification ×200. (<b>D</b>) Expression levels and quantitative analysis of p-AMPK, p-mTOR, and FOXO3a in tumors from different intervention groups; the expression of β-actin was used as a reference. (<b>E</b>) Immunohistochemical analysis of FOXO3a in tumor sections, magnification ×400. (<b>F</b>) Evaluation of changes in the ADP/ATP ratio in tumors. All results were repeated at least five times. * indicates statistical significance compared to the control group, * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001. Con: Control; SS: sodium selenite; c-PARP: cleaved-PARP; c-caspase3: cleaved-caspase3.</p>
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17 pages, 7392 KiB  
Article
Photobiomodulation Mitigates PM2.5-Exacerbated Pathologies in a Mouse Model of Allergic Asthma
by Jisu Park, Bo-Young Kim, Eun Jung Park, Yong-Il Shin and Ji Hyeon Ryu
Antioxidants 2024, 13(8), 1003; https://doi.org/10.3390/antiox13081003 - 19 Aug 2024
Cited by 2 | Viewed by 1589
Abstract
Exposure to particulate matter (PM), especially PM2.5, is known to exacerbate asthma, posing a significant public health risk. This study investigated the asthma-reducing effects of photobiomodulation (PBM) in a mice model mimicking allergic airway inflammation exacerbated by PM2.5 exposure. The [...] Read more.
Exposure to particulate matter (PM), especially PM2.5, is known to exacerbate asthma, posing a significant public health risk. This study investigated the asthma-reducing effects of photobiomodulation (PBM) in a mice model mimicking allergic airway inflammation exacerbated by PM2.5 exposure. The mice received sensitization with ovalbumin (OVA) and were subsequently treated with PM2.5 at a dose of 0.1 mg/kg every 3 days, for 9 times over 3 weeks during the challenge. PBM, using a 610 nm wavelength LED, was applied at 1.7 mW/cm2 to the respiratory tract via direct skin contact for 20 min daily for 19 days. Results showed that PBM significantly reduced airway hyperresponsiveness, plasma immunoglobulin E (IgE) and OVA-specific IgE, airway inflammation, T-helper type 2 cytokine, histamine and tryptase in bronchoalveolar lavage fluid (BALF), and goblet cell hyperplasia in PM2.5-exposed asthmatic mice. Moreover, PBM alleviated subepithelial fibrosis by reducing collagen deposition, airway smooth muscle mass, and expression of fibrosis-related genes. It mitigated reactive oxygen species generation, oxidative stress, endoplasmic reticulum stress, apoptotic cell death, ferroptosis, and modulated autophagic signals in the asthmatic mice exposed to PM2.5. These findings suggest that PBM could be a promising intervention for PM2.5-induced respiratory complications in patients with allergic asthma. Full article
(This article belongs to the Special Issue Oxidative Stress Induced by Air Pollution, 2nd Edition)
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<p>Inhibitory effects of PBM on the induction of airway hyperresponsiveness (AHR) and plasma IgE in a PM<sub>2.5</sub>-exposed asthma exacerbation model. (<b>A</b>) Establishment of an allergic asthma exacerbation mouse model induced by PM<sub>2.5</sub> exposure. A timeline describing the asthma exacerbation model induction and PBM treatment. (<b>B</b>) Measurement of body weight, thymus-to-body-weight ratio, and spleen-to-body-weight ratio on the final day of the experiment. (<b>C</b>) Assessment of AHR to methacholine (MCh) at concentrations of 25 and 50 mg/mL. (<b>D</b>) Measurement of total immunoglobulin E (IgE) and ovalbumin (OVA)-specific IgE in plasma. Data are shown as the mean ± SEM (<span class="html-italic">n</span> = 8). <span class="html-italic">* p</span> &lt; 0.05 compared with control. <sup>†</sup> <span class="html-italic">p</span> &lt; 0.05 compared with PM + OVA. i.n., intranasal injection; i.p., intraperitoneal injection; BW, body weight; PM, particulate matter; OVA, ovalbumin; PBM, photobiomodulation; DEX, dexamethasone.</p>
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<p>Inhibitory effects of PBM on the elevation of allergic airway inflammation and goblet cell metaplasia in a PM<sub>2.5</sub>-exposed asthma exacerbation model. (<b>A</b>) Measurement of total and differential inflammatory cell counts (macrophage, neutrophils, lymphocytes, and eosinophils) in bronchoalveolar lavage fluid (BALF). (<b>B</b>) Evaluation of Th2 cytokines including interleukin (IL)-4, IL-5, and IL-13 in BALF. (<b>C</b>) Assessment of histamine and mast cell tryptase in BALF. (<b>D</b>) Representative images of H&amp;E staining revealed the infiltration of inflammatory cells in lung tissues. Scale bar represents 200 µm (up) and 100 µm (down). The bar graphs represent the summarized score of inflammation. (<b>E</b>) Goblet cells secreting mucus in lung tissues were identified using PAS staining. The bar graphs represent the number of PAS-reactive airway epithelial cells. Scale bar represents 50 µm. The bar graphs represent the summarized scores of PAS-positive mucus-producing cells. Data are shown as the mean ± SEM (<span class="html-italic">n</span> = 8). <span class="html-italic">* p</span> &lt; 0.05 compared with control. <sup>†</sup> <span class="html-italic">p</span> &lt; 0.05 compared with PM + OVA. <sup>‡</sup> <span class="html-italic">p</span> &lt; 0.05 compared with PM + OVA + PBM.</p>
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<p>Inhibitory effects of PBM on the elevation of subepithelial fibrosis in a PM<sub>2.5</sub>-exposed asthma exacerbation model. Representative histological images showing (<b>A</b>) lung collagen fiber (Masson’s trichrome staining) and (<b>B</b>) collagen deposition (Sirius Red staining) in the lung tissues. Scale bar represents 100 µm. The bar graphs represent the summarized scores of collagen fiber deposition (<span class="html-italic">n</span> = 8). (<b>C</b>) Representative images of α-smooth muscle actin (α-SMA) and FITC expression, as determined by immunohistochemistry, in bronchioles of similar size. Scale bar represents 20 µm. The bar graphs represent the area of α-SMA staining per micrometer length of the bronchiolar basement membrane (µm<sup>2</sup>/µm; <span class="html-italic">n</span> = 6). (<b>D</b>) Detection of the mRNA levels of <span class="html-italic">Acta2</span>, <span class="html-italic">Tgfb1</span>, <span class="html-italic">Col1a1</span>, and <span class="html-italic">Col3a1</span> in lung tissues using qRT-PCR (<span class="html-italic">n</span> = 4). Data are shown as the mean ± SEM. <span class="html-italic">* p</span> &lt; 0.05 compared with control. <sup>†</sup> <span class="html-italic">p</span> &lt; 0.05 compared with PM + OVA.</p>
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<p>Inhibitory effects of PBM on the ROS-mediated ER stress in a PM<sub>2.5</sub>-exposed asthma exacerbation model. (<b>A</b>) Representative lung sections were stained with antibody specific for 8-hydroxy-2′-deoxyguanosine (8-OHdG). Bar graphs represent the quantification of positive areas of 8-OHdG in each experimental group (<span class="html-italic">n</span> = 4). Scale bar represents 100 µm. (<b>B</b>) ROS levels in lung tissue were measured in relative fluorescence units (RFU). (<b>C</b>) Protein expression of superoxide dismutase 1 (SOD1) and peroxiredoxin 4 (PRDX4). (<b>D</b>) ER stress markers (PERK, eIF2α, ATF4, and CHOP) in lung tissues by Western blotting. β-actin was used as a loading control. Bar graphs represent the quantification protein expression (<span class="html-italic">n</span> = 3). Data are shown as the mean ± SEM. <span class="html-italic">* p</span> &lt; 0.05 compared with control. <sup>†</sup> <span class="html-italic">p</span> &lt; 0.05 compared with PM + OVA.</p>
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<p>Inhibitory effects of PBM on the cell death in a PM<sub>2.5</sub>-exposed asthma exacerbation model. (<b>A</b>) Representative immunofluorescence for TUNEL (green) and DAPI (blue) staining. Scale bar represents 50 µm. Bar graphs represent TUNEL (+)/DAPI (+) cells in the lung tissues (<span class="html-italic">n</span> = 3). (<b>B</b>) Apoptotic markers in lung tissues. Bar graphs represent the quantification protein expression (<span class="html-italic">n</span> = 3). Bar graphs represent the quantification protein expression (<span class="html-italic">n</span> = 3). Data are shown as the mean ± SEM. <span class="html-italic">* p</span> &lt; 0.05 compared with control. <sup>†</sup> <span class="html-italic">p</span> &lt; 0.05 compared with PM + OVA.</p>
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<p>Inhibitory effects of PBM on the ferroptosis and autophagic signals in a PM<sub>2.5</sub>-exposed asthma exacerbation model. (<b>A</b>) Deposition of iron in lung tissue using Perls Prussian blue staining in lung tissues (<span class="html-italic">n</span> = 6). Scale bar represents 20 µm. (<b>B</b>) Malondialdehyde (MDA) concentration in lung tissue (<span class="html-italic">n</span> = 6). (<b>C</b>) Glutathione (GSH) concentration in lung tissue (<span class="html-italic">n</span> = 6). (<b>D</b>) Ca<sup>2+</sup> levels in lung tissue (<span class="html-italic">n</span> = 5). (<b>E</b>) 4-Hydroxynonenal (4-HNE) levels in lung tissue (<span class="html-italic">n</span> = 6). (<b>F</b>) Ferroptosis markers in lung tissue (<span class="html-italic">n</span> = 3). (<b>G</b>) Autophagy markers in lung tissues. Bar graphs represent the quantification protein expression (<span class="html-italic">n</span> = 3). Data are shown as the mean ± SEM. <span class="html-italic">* p</span> &lt; 0.05 compared to control. <sup>†</sup> <span class="html-italic">p</span> &lt; 0.05 compared to PM + OVA.</p>
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<p>Schematic representation of the anti-asthmatic effects of photobiomodulation (PBM) therapy on PM<sub>2.5</sub> exposure-induced allergic asthma in a mouse model. PBM therapy reduces AHR, inflammation, Th2 cytokines, goblet cell hyperplasia, and subepithelial fibrosis in a PM<sub>2.5</sub>-exacerbated allergic asthma mouse model. PBM therapy also decreases oxidative and ER stress, apoptosis, and ferroptosis, while modulating autophagy in asthmatic mice exposed to PM<sub>2.5</sub>. These findings suggest PBM’s potential as an adjunct to asthma treatment in patients exposed to environmental pollutants. Abbreviations: DEX, dexamethasone; OVA, ovalbumin; PBM, photobiomodulation; PM, particulate matter, PM<sub>2.5</sub>, PM with a diameter &lt; 2.5 μm.</p>
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20 pages, 14344 KiB  
Article
Dermal Injection of Recombinant Filaggrin-2 Ameliorates UVB-Induced Epidermal Barrier Dysfunction and Photoaging
by Lu Li, Yuan Liu, Ruxue Chang, Tao Ye, Ziyi Li, Rufei Huang, Zhaoyang Wang, Jingxian Deng, Huan Xia, Yan Yang and Yadong Huang
Antioxidants 2024, 13(8), 1002; https://doi.org/10.3390/antiox13081002 - 19 Aug 2024
Viewed by 1453
Abstract
The epidermal barrier is vital for protecting the skin from environmental stressors and ultraviolet (UV) radiation. Filaggrin-2 (FLG2), a critical protein in the stratum corneum, plays a significant role in maintaining skin barrier homeostasis. However, the precise role of FLG2 in mitigating the [...] Read more.
The epidermal barrier is vital for protecting the skin from environmental stressors and ultraviolet (UV) radiation. Filaggrin-2 (FLG2), a critical protein in the stratum corneum, plays a significant role in maintaining skin barrier homeostasis. However, the precise role of FLG2 in mitigating the adverse effects of UV-induced barrier disruption and photoaging remains poorly understood. In this study, we revealed that UVB exposure resulted in a decreased expression of FLG2 in HaCaT keratinocytes, which correlated with a compromised barrier function. The administration of recombinant filaggrin-2 (rFLG2) enhanced keratinocyte differentiation, bolstered barrier integrity, and offered protection against apoptosis and oxidative stress induced by UVB irradiation. Furthermore, in a UV-induced photodamage murine model, the dermal injection of rFLG2 facilitated the enhanced restoration of the epidermal barrier, decreased oxidative stress and inflammation, and mitigated the collagen degradation that is typical of photoaging. Collectively, our findings suggested that targeting FLG2 could be a strategic approach to prevent and treat skin barrier dysfunction and combat the aging effects associated with photoaging. rFLG2 emerges as a potentially viable therapy for maintaining skin health and preventing skin aging processes amplified by photodamage. Full article
(This article belongs to the Section Natural and Synthetic Antioxidants)
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<p>UVB can induce photoaging in HaCaTs. (<b>a</b>) UVB-treated cells were measured for senescence-associated β-galactosidase activity and the percentage of cells positive for β-galactosidase staining. Scale bar represents 20 μm. (<b>b</b>) Determining early apoptosis after UVB exposure by flow cytometry Annexin V/PI assay. (<b>c</b>) Filaggrin-2 (FLG2) gene expression changes in HaCaT cells before and after UVB irradiation. (<b>d</b>,<b>e</b>) Changes in FLG2 protein expression in HaCaT cells before and after UVB irradiation; GAPDH was used as the internal reference. (<b>f</b>) Transmembrane resistance was measured in monolayer cells after UVB irradiation. The data are representative of at least three independent experiments. Values are shown as mean ± SD. ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p>Recombinant filaggrin-2 (rFLG2) promoted keratinocyte differentiation in vitro. (<b>a</b>) The purified protein was separated by SDS-PAGE. The arrow shows the target protein (rFLG2). (<b>b</b>) mRNA expression of involucrin, loricrin, and caspase14 in HaCaTs was measured by RT-qPCR and normalized to GAPDH levels. (<b>c</b>) mRNA expression of filaggrin (FLG) in HaCaTs was measured by RT-qPCR and normalized to GAPDH levels. (<b>d</b>–<b>h</b>) HaCaTs were treated with rFLG2 in combination with UVB, and the protein level of the FLG2, involucrin, loricrin, and caspase14 were detected by Western blot; GAPDH was used as the internal reference. (<b>i</b>–<b>k</b>) The protein levels of involucrin, loricrin, and caspase14 in HaCaTs treated with UVB and rFLG2 were detected by immunofluorescence. Scale bar represents 20 μm. The data are representative of at least three independent experiments. Values are shown as mean ± SD. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p>rFLG2 can alleviate UVB-induced damage to HaCaT barrier function. (<b>a</b>,<b>b</b>) Expression and distribution of zonula occludens-1 (ZO-1) and occludin proteins in HaCaTs were detected by immunofluorescence after being treated with rFLG2. Scale bar represents 20 μm. (<b>c</b>,<b>d</b>) HaCaTs were treated with rFLG2 in combination with UVB, and the expression level of ZO-1 was detected by Western blot; GAPDH was used as the internal reference. (<b>e</b>) Transmembrane resistance was measured in monolayer cells. The data are representative of at least three independent experiments. Values are shown as mean ± SD. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01.</p>
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<p>rFLG2 reduces UVB-induced oxidative stress. (<b>a</b>) Live cells were stained green, while dead cells were stained red with Calcein AM/PI. Scale bar represents 100 μm. (<b>b</b>) Fluorescence quantitative live and dead staining statistics. (<b>c</b>) Flow cytometry and analysis of ROS production in HaCaT cells after UVB and rFLG2 treatment. (<b>d</b>) Quantification of intracellular ROS levels. (<b>e</b>,<b>f</b>) Gene expression levels of inflammatory factors of IL-1β and IL-10 in HaCaT cells were measured by RT-qPCR and normalized to GAPDH levels. The data are representative of at least three independent experiments. Values are shown as mean ± SD. ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p>Dermal injection of rFLG2 mitigates UVB-induced epidermal hyperplasia in mice. (<b>a</b>) In vivo imaging of Cy5.5-labeled rFLG2 at different times after subcutaneous injection. (<b>b</b>) Schematic diagram of the animal experiment. (<b>c</b>) Representative images of the morphological changes from each group. (<b>d</b>) Representative micrographs of H&amp;E-stained skin tissue sections. Scale bar represents 200 μm. Arrows indicate epidermal thickness. (<b>e</b>) Statistics of epidermal thickness in mice (<span class="html-italic">n</span> = 7). Values are shown as mean ± SD. *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p>rFLG2 redistributes the expression of barrier proteins in vivo. (<b>a</b>,<b>b</b>) Immunohistochemical staining of involucrin, loricrin, caspase14, and ZO-1 in skin equivalents from mouse. Images are representative of groups. Scale bar represents 100 μm. (<b>c</b>,<b>d</b>) Protein level of ZO-1 in dorsal skin of UVB-irradiated mice was analyzed by Western blot (<span class="html-italic">n</span> = 3). GAPDH antibody was used as control. Values are shown as means ± SD. *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p>rFLG2 significantly inhibits UVB-induced oxidative stress and inflammatory responses in mice. (<b>a</b>) Lipid peroxidation in the dorsal skin in each group after rFLG2 treatment was measured by quantifying malondialdehyde (MDA) (<span class="html-italic">n</span> = 3). (<b>b</b>) GSH-PX activity in the dorsal skin after rFLG2 treatment (<span class="html-italic">n</span> = 3). (<b>c</b>) Superoxide dismutase (SOD) activity in the dorsal skin after rFLG2 treatment (<span class="html-italic">n</span> = 3). (<b>d</b>,<b>e</b>) mRNA expressions of <span class="html-italic">Il-10</span> and <span class="html-italic">Il-1β</span> in skin was measured by RT-qPCR and normalized to GAPDH levels (<span class="html-italic">n</span> = 3). Values are shown as means ± SD. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p>rFLG2 inhibits UVB-induced collagen depletion in mice. (<b>a</b>,<b>b</b>) Masson staining was used to detect the content of collagen fibers in the skin of each group of mice (<span class="html-italic">n</span> = 7). Scale bar represents 200 μm. (<b>c</b>,<b>d</b>) Picrosirius red staining of the dorsal skin (<span class="html-italic">n</span> = 7). Scale bar represents 200 μm. (<b>e</b>) HYP activity in the dorsal skin after rFLG2 treatment was quantified (<span class="html-italic">n</span> = 3). (<b>f</b>,<b>g</b>) Relative transcript levels of <span class="html-italic">Mmp-3</span> and <span class="html-italic">elastin</span>-related collagen genes were determined by RT-qPCR (<span class="html-italic">n</span> = 3). mRNA relative levels were normalized to GAPDH levels. Values are shown as means ± SD (<span class="html-italic">n</span> = 3). * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p>Mechanisms involved in protective effects of rFLG2. The up arrows represent increased protein expression, while the down arrows represent decreased protein expression.</p>
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17 pages, 5328 KiB  
Article
Involvement of KV3.4 Channel in Parkinson’s Disease: A Key Player in the Control of Midbrain and Striatum Differential Vulnerability during Disease Progression?
by Giorgia Magliocca, Emilia Esposito, Michele Tufano, Ilaria Piccialli, Valentina Rubino, Valentina Tedeschi, Maria Jose Sisalli, Flavia Carriero, Giuseppina Ruggiero, Agnese Secondo, Lucio Annunziato, Antonella Scorziello and Anna Pannaccione
Antioxidants 2024, 13(8), 999; https://doi.org/10.3390/antiox13080999 - 18 Aug 2024
Viewed by 1307
Abstract
Parkinson’s disease (PD), the second most common neurodegenerative disease in the elderly, is characterized by selective loss of dopaminergic neurons and accumulation of α-synuclein (α-syn), mitochondrial dysfunction, Ca2+ dyshomeostasis, and neuroinflammation. Since current treatments for PD merely address symptoms, there is an [...] Read more.
Parkinson’s disease (PD), the second most common neurodegenerative disease in the elderly, is characterized by selective loss of dopaminergic neurons and accumulation of α-synuclein (α-syn), mitochondrial dysfunction, Ca2+ dyshomeostasis, and neuroinflammation. Since current treatments for PD merely address symptoms, there is an urgent need to identify the PD pathophysiological mechanisms to develop better therapies. Increasing evidence has identified KV3.4, a ROS-sensitive KV channel carrying fast-inactivating currents, as a potential therapeutic target against neurodegeneration. In fact, it has been hypothesized that KV3.4 channels could play a role in PD etiopathogenesis, controlling astrocytic activation and detrimental pathways in A53T mice, a well-known model of familial PD. Here, we showed that the A53T midbrain, primarily involved in the initial phase of PD pathogenesis, displayed an early upregulation of the KV3.4 channel at 4 months, followed by its reduction at 12 months, compared with age-matched WT. On the other hand, in the A53T striatum, the expression of KV3.4 remained high at 12 months, decreasing thereafter, in 16-month-old mice. The proteomic profile highlighted a different detrimental phenotype in A53T brain areas. In fact, the A53T striatum and midbrain differently expressed neuroprotective/detrimental pathways, with the variation of astrocytic p27kip1, XIAP, and Smac/DIABLO expression. Of note, a switch from protective to detrimental phenotype was characterized by the upregulation of Smac/DIABLO and downregulation of p27kip1 and XIAP. This occurred earlier in the A53T midbrain, at 12 months, compared with the striatum proteomic profile. In accordance, an upregulation of Smac/DIABLO and a downregulation of p27kip1 occurred in the A53T striatum only at 16 months, showing the slowest involvement of this brain area. Of interest, HIF-1α overexpression was associated with the detrimental profile in midbrain and its major vulnerability. At the cellular level, patch-clamp recordings revealed that primary A53T striatum astrocytes showed hyperpolarized resting membrane potentials and lower firing frequency associated with KV3.4 ROS-dependent hyperactivity, whereas primary A53T midbrain astrocytes displayed a depolarized resting membrane potential accompanied by a slight increase of KV3.4 currents. Accordingly, intracellular Ca2+ homeostasis was significantly altered in A53T midbrain astrocytes, in which the ER Ca2+ level was lower than in A53T striatum astrocytes and the respective littermate controls. Collectively, these results suggest that the early KV3.4 overexpression and ROS-dependent hyperactivation in astrocytes could take part in the different vulnerabilities of midbrain and striatum, highlighting astrocytic KV3.4 as a possible new therapeutic target in PD. Full article
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<p>K<sub>V</sub>3.4, p27<sup>kip1</sup>, and XIAP protein expression in midbrain and striatum obtained from 4- and 12-month-old A53T and WT mice. (<b>A</b>) Representative Western blotting (upper) and densitometric quantification (lower) of K<sub>V</sub>3.4 protein expression in A53T and WT midbrain at 4 and 12 months. (<b>B</b>) Representative Western blotting (upper) and densitometric quantification (lower) of K<sub>V</sub>3.4 protein expression in A53T and WT striatum at 4 and 12 months. (<b>C</b>) Representative Western blotting (upper) and densitometric quantification (lower) of p27<sup>kip1</sup> protein expression in A53T and WT midbrain at 4 and 12 months. (<b>D</b>) Representative Western blotting (upper) and densitometric quantification (lower) of p27<sup>kip1</sup> protein expression in A53T and WT striatum at 4 and 12 months. (<b>E</b>) Representative Western blotting (upper) and densitometric quantification (lower) of XIAP protein expression in A53T and WT midbrain at 4 and 12 months. (<b>F</b>) Representative Western blotting (upper) and densitometric quantification (lower) of XIAP protein expression in A53T and WT striatum at 4 and 12 months. Number of animals at 4 months, WT #7 and A53T #5, and at 12 months, WT #5 and A53T #5. Each bar represents the mean% ± S.E.M. of different experimental values obtained in 3 independent experimental sessions. * <span class="html-italic">p</span> &lt; 0.05 compared to age-matched WT midbrain and striatum; ** <span class="html-italic">p</span> &lt; 0.05 compared to age-matched A53T midbrain and striatum.</p>
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<p>HIF-1α and Smac/DIABLO protein expression in midbrain and striatum obtained from 4- and 12-month-old A53T and WT mice. (<b>A</b>) Representative Western blotting (upper) and densitometric quantification (lower) of HIF-1α protein expression in A53T and WT midbrain at 4 and 12 months. (<b>B</b>) Representative Western blotting (upper) and densitometric quantification (lower) of HIF-1α protein expression in A53T and WT striatum at 4 and 12 months. (<b>C</b>) Representative Western blotting (upper) and densitometric quantification (lower) of Smac/DIABLO protein expression in A53T and WT midbrain at 4 and 12 months. (<b>D</b>) Representative Western blotting (upper) and densitometric quantification (lower) of Smac/DIABLO protein expression in A53T and WT striatum at 4 and 12 months. Number of animals at 4 months, WT #7 and A53T #5, and at 12 months, WT #5 and A53T #5. Each bar represents the mean% ± S.E.M. of different experimental values obtained in 3 independent experimental sessions. * <span class="html-italic">p</span> &lt; 0.05 compared to age-matched WT midbrain and striatum; ** <span class="html-italic">p</span> &lt; 0.05 compared to age-matched A53T midbrain and striatum.</p>
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<p>Proteomic immunoassay of p27<sup>kip1</sup>, XIAP, HIF-1α, and Smac/DIABLO protein expression in the midbrain and striatum obtained from 4- and 12-month-old A53T and WT mice. (<b>A</b>) Representative blots of proteomic immunoassay (upper) and densitometric quantification (lower) of (4) p27<sup>kip1</sup>, (3) XIAP (blue circles), (1) HIF-1α, and (2) Smac/DIABLO (red circles) protein expression in A53T and WT midbrain at 4 months. (<b>B</b>) Representative blots of proteomic immunoassay (upper) and densitometric quantification (lower) of p27<sup>kip1</sup>, XIAP, HIF-1α, and Smac/DIABLO protein expression in A53T and WT striatum at 4 months. (<b>C</b>) Representative blots of proteomic immunoassay (upper) and densitometric quantification (lower) of p27<sup>kip1</sup>, XIAP, HIF-1α, and Smac/DIABLO protein expression in A53T and WT midbrain at 12 months. (<b>D</b>) Representative blots of proteomic immunoassay (upper) and densitometric quantification (lower) of p27<sup>kip1</sup>, XIAP, HIF-1α, and Smac/DIABLO protein expression in A53T and WT striatum at 12 months. Number of animals at 4 months, WT #7 and A53T #5, and at 12 months, WT #5 and A53T #5. Each bar represents the mean% ± S.E.M. of different experimental values obtained in 3 independent experimental sessions. Anti-apoptotic proteins in blue circle and pro-apoptotic proteins in red circle * <span class="html-italic">p</span> &lt; 0.05 compared to age-matched WT midbrain and striatum.</p>
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<p>K<sub>V</sub>3.4, p27<sup>kip1</sup>, XIAP, and Smac/DIABLO protein expression in striatum obtained from 16-month-old A53T and WT mice. (<b>A</b>) Representative Western blotting (upper) and densitometry quantification (lower) of K<sub>V</sub>3.4 protein expression in A53T and WT striatum at 16 months. (<b>B</b>) Representative Western blotting (upper) and densitometry quantification (lower) of p27<sup>kip1</sup> protein expression in A53T and WT striatum at 16 months (<b>C</b>) Representative Western blotting (upper) and densitometry quantification (lower) of XIAP protein expression in A53T and WT striatum at 16 months. (<b>D</b>) Representative Western blotting (upper) and densitometry quantification (lower) of Smac/DIABLO protein expression in A53T and WT striatum at 16 months. Number of animals at 16 months, WT #5 and A53T #5. Each bar represents the mean% ± S.E.M. of different experimental values obtained in 3 independent experimental sessions. * <span class="html-italic">p</span> &lt; 0.05 compared to age-matched WT midbrain and striatum.</p>
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<p>Different modulation of ROS-dependent K<sub>V</sub>3.4 channels in primary A53T midbrain and striatum astrocytes. (<b>A</b>) Representative Western blotting and densitometry quantification of K<sub>V</sub>3.4 protein expression in primary A53T and WT astrocytes from midbrain. (<b>B</b>) Representative Western blotting and densitometry quantification of K<sub>V</sub>3.4 protein expression in primary A53T and WT astrocytes from striatum. Each bar represents the mean ± S.E.M. of the percentage of different experimental values obtained in three independent experimental sessions. (<b>C</b>) Representative dot plots of DCF density in primary A53T (magenta) and WT astrocytes (black) from striatum (left) and midbrain (right). (<b>D</b>) Mean Fluorescence Intensity of ROS production in C panel. Each bar represents the mean ± S.E.M. of the DCF fluorescence of different experimental values obtained in three independent experimental sessions. (<b>E</b>) Representative Western blotting and densitometry quantification of HIF-1α protein expression in primary A53T and WT astrocytes from midbrain and striatum. Each bar represents the mean% ± S.E.M. of different experimental values obtained in three independent experimental sessions. * <span class="html-italic">p</span> &lt; 0.05 compared to midbrain and striatum WT astrocytes.</p>
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<p>Effect of the activity of K<sub>V</sub>3.4 channels on resting membrane potentials and [Ca<sup>2+</sup>]<sub>i</sub> transients in primary A53T and WT astrocytes from midbrain and striatum. (<b>A</b>) Representative traces (left) and quantification of K<sub>V</sub>3.4-mediated fast inactivating K<sup>+</sup> currents (I<sub>A</sub>; right) recorded from primary A53T and WT astrocytes from striatum. (<b>B</b>) Representative traces (left) and quantification of K<sub>V</sub>3.4-mediated I<sub>A</sub> currents (right) recorded from primary A53T and WT astrocytes from midbrain. The peak values of I<sub>A</sub> currents, measured at the beginning of the +40 mV depolarizing pulse, are expressed as mean% ± SEM of 3 independent experiments performed on 3 different preparations (for both (<b>A</b>,<b>B</b>): n = 12 cells in each cell culture and for each group). (<b>C</b>) Representative traces recorded in the gap-free mode (left) and quantification of membrane resting potential (right) in A53T and WT astrocytes from striatum. (<b>D</b>) Representative traces recorded in the gap-free mode (left) and its quantification (right) in A53T and WT astrocytes from midbrain. Vertical scale bar below the trace represents 1 pA. The values are expressed as mV and represent the mean ± SEM of 3 independent experiments performed on 3 different preparations (for both (<b>C</b>,<b>D</b>): n = 12 cells for each group). (<b>E</b>) Quantification of Ca<sup>2+</sup><sub>i</sub> in A53T and WT astrocytes from striatum and midbrain loaded with Fluo-4AM; * <span class="html-italic">p</span> &lt; 0.05 vs. its respective WT. (<b>F</b>) Quantification of ER Ca<sup>2+</sup> in A53T and WT astrocytes from striatum and midbrain loaded with Fluo-4AM; * <span class="html-italic">p</span> &lt; 0.05 vs. WT astrocytes from midbrain. ** <span class="html-italic">p</span>&lt; 0.05 A53T astrocytes from midbrain vs. A53T astrocytes from striatum</p>
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9 pages, 1763 KiB  
Communication
Oxidative Stress in Transthyretin-Mediated Amyloidosis: An Exploratory Study
by Marco Fiore, Chiara Cambieri, Laura Libonati, Federica Moret, Edoardo D’Andrea, Maria Grazia Di Certo, Claudio Passananti, Francesca Gabanella, Nicoletta Corbi, Matteo Garibaldi, Cristina Chimenti, Maria Alfarano, Giampiero Ferraguti, Silvia Francati, Maurizio Inghilleri and Marco Ceccanti
Antioxidants 2024, 13(8), 998; https://doi.org/10.3390/antiox13080998 - 18 Aug 2024
Cited by 1 | Viewed by 950
Abstract
Transthyretin-mediated amyloidosis (ATTR) is a systemic disease with protein precipitation in many tissues, mainly the peripheral nerve and heart. Both genetic (ATTRv, “v” for variant) and wild-type (ATTRwt) forms are known. Beyond the steric encumbrance, precipitated transthyretin seems to have a toxic effect. [...] Read more.
Transthyretin-mediated amyloidosis (ATTR) is a systemic disease with protein precipitation in many tissues, mainly the peripheral nerve and heart. Both genetic (ATTRv, “v” for variant) and wild-type (ATTRwt) forms are known. Beyond the steric encumbrance, precipitated transthyretin seems to have a toxic effect. In this study carried out in men, we recruited 15 ATTRv patients, 7 ATTRv asymptomatic carriers, 14 ATTRwt patients and 10 young and 13 old healthy controls to evaluate the oxidative stress using FORD (Free Oxygen Radicals Defense) and FORT (Free Oxygen Radicals Test) analyses. ATTRv patients showed reduced FORD compared to ATTRwt and ATTRv asymptomatic carriers. FORD independently predicted the disease stage, with the early stages characterized by the highest consumption. These findings suggest a role for oxidative stress in the early stages of ATTRv. Full article
(This article belongs to the Section Health Outcomes of Antioxidants and Oxidative Stress)
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<p>FORD levels in the different groups. * <span class="html-italic">p</span> &lt; 0.05. Boxes indicate upper and lower quartiles, and whiskers indicate the minimum to the maximum value.</p>
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<p>FORD levels in the different FAP stages. * <span class="html-italic">p</span> &lt; 0.05. Boxes indicate upper and lower quartiles, and whiskers indicate the minimum to the maximum value.</p>
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59 pages, 3076 KiB  
Review
The Potential Health Benefits of Gallic Acid: Therapeutic and Food Applications
by Milad Hadidi, Rafael Liñán-Atero, Mohammad Tarahi, Marios C. Christodoulou and Fatemeh Aghababaei
Antioxidants 2024, 13(8), 1001; https://doi.org/10.3390/antiox13081001 - 18 Aug 2024
Cited by 9 | Viewed by 5883
Abstract
Gallic acid (GA), a phenolic acid found in fruits and vegetables, has been consumed by humans for centuries. Its extensive health benefits, such as antimicrobial, antioxidant, anticancer, anti-inflammatory, and antiviral properties, have been well-documented. GA’s potent antioxidant capabilities enable it to neutralize free [...] Read more.
Gallic acid (GA), a phenolic acid found in fruits and vegetables, has been consumed by humans for centuries. Its extensive health benefits, such as antimicrobial, antioxidant, anticancer, anti-inflammatory, and antiviral properties, have been well-documented. GA’s potent antioxidant capabilities enable it to neutralize free radicals, reduce oxidative stress, and protect cells from damage. Additionally, GA exerts anti-inflammatory effects by inhibiting inflammatory cytokines and enzymes, making it a potential therapeutic agent for inflammatory diseases. It also demonstrates anticancer properties by inhibiting cancer cell growth and promoting apoptosis. Furthermore, GA offers cardiovascular benefits, such as lowering blood pressure, decreasing cholesterol, and enhancing endothelial function, which may aid in the prevention and management of cardiovascular diseases. This review covers the chemical structure, sources, identification and quantification methods, and biological and therapeutic properties of GA, along with its applications in food. As research progresses, the future for GA appears promising, with potential uses in functional foods, pharmaceuticals, and nutraceuticals aimed at improving overall health and preventing disease. However, ongoing research and innovation are necessary to fully understand its functional benefits, address current challenges, and establish GA as a mainstay in therapeutic and nutritional interventions. Full article
(This article belongs to the Section Extraction and Industrial Applications of Antioxidants)
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<p>General classification and main natural sources of GA and its derivatives.</p>
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<p>Production of GA from some of its derivatives by tannase-catalyzed reactions: propyl gallate (<b>A</b>), epigallocatechin gallate (<b>B</b>), and tannic acid (<b>C</b>).</p>
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<p>Advantages and drawbacks of the techniques used in GA analysis.</p>
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<p>Main mechanisms of antimicrobial action of polyphenols on bacteria.</p>
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18 pages, 2427 KiB  
Article
Controlled Cultivation Confers Rhodiola rosea Synergistic Activity on Muscle Cell Homeostasis, Metabolism and Antioxidant Defense in Primary Human Myoblasts
by Fortuna Iannuzzo, Elisabetta Schiano, Arianna Pastore, Fabrizia Guerra, Gian Carlo Tenore, Ettore Novellino and Mariano Stornaiuolo
Antioxidants 2024, 13(8), 1000; https://doi.org/10.3390/antiox13081000 - 18 Aug 2024
Viewed by 1281
Abstract
Rhodiola rosea L. is recognized for its adaptogenic properties and ability to promote muscle health, function and recovery from exercise. The plethora of biological effects of this plant is ascribed to the synergism existing among the molecules composing its phytocomplex. In this manuscript, [...] Read more.
Rhodiola rosea L. is recognized for its adaptogenic properties and ability to promote muscle health, function and recovery from exercise. The plethora of biological effects of this plant is ascribed to the synergism existing among the molecules composing its phytocomplex. In this manuscript, we analyze the activity of a bioactive fraction extracted from Rhodiola rosea L. controlled cultivation. Biological assays were performed on human skeletal myoblasts and revealed that the extract is able to modulate in vitro expression of transcription factors, namely Pax7 and myoD, involved in muscle differentiation and recovery. The extract also promotes ROS scavenging, ATP production and mitochondrial respiration. Untargeted metabolomics further reveals that the mechanism underpinning the plant involves the synergistic interconnection between antioxidant enzymes and the folic/acid polyamine pathway. Finally, by examining the phytochemical profiles of the extract, we identify the specific combination of secondary plant metabolites contributing to muscle repair, recovery from stress and regeneration. Full article
(This article belongs to the Special Issue Antioxidant Response in Skeletal Muscle)
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<p>RRcc modulates undifferentiated muscle cells and promotes defense from oxidative stress. Growth (<b>A</b>), qPCR analysis of differentiation-related muscle cell transcription factors (<b>B</b>), antioxidant enzymes (<b>C</b>) and total ROS content (<b>D</b>) in primary muscle myoblasts treated for 48 h in the presence of RRcc (30 μg/mL), RRwh (30 μg/mL), an equal amount of vehicle (veh) or left untreated (untr). Data are shown as mean ± SD of five independent experiments. Statistical analysis was performed by ANOVA test comparing each mean with that of untreated cells. <span class="html-italic">p</span> value = * &lt;0.05, otherwise differences were not statistically significant.</p>
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<p>RRcc promotes ATP production and mitochondrial activity in cultured human myoblasts. Intracellular ATP content (<b>A</b>), <span class="html-italic">Pgc1α</span> mRNA levels (<b>B</b>) and mitochondrial activity(<b>C</b>) in primary muscle myoblasts treated for 48 h in the presence of RRcc (30 μg/mL), RRwh (30 μg/mL), an equal amount of vehicle (veh) or left untreated (untr). Data are shown as mean ± SD of five independent experiments. Statistical analysis was performed by ANOVA test comparing each mean with that of untreated cells. <span class="html-italic">p</span> value = * &lt;0.05, *** &lt; 0.001, otherwise differences were not statistically significant.</p>
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<p>RRcc presents folic acid-cycle and polyamine pathway modulatory activity in human myoblasts. Metabolic profile of primary muscle myoblasts treated for 48 h with RRcc (30 μg/mL), RRwh (30 μg/mL), an equal amount of vehicle (veh) or left untreated (untr). (<b>A</b>) Content (fold variation vs. untr) of the indicated metabolites as measured in the four different experimental conditions; (<b>B</b>) primary component analysis (PCA) score plot showing that the metabolic profiles of RRcc and RRwh are different from veh and untr cells; (<b>C</b>) content (fold variation vs. untr) of the folic acid-cycle and polyamine pathway metabolites as measured in the four different experimental conditions (<span class="html-italic">n</span> = 3 independent experiments); (<b>D</b>) PCA score plot showing the metabolic profile of RRcc being different from RRwh. In A and C, values are represented as mean ± SD. Two-way ANOVA and Bonferroni post-test analysis were performed; * = <span class="html-italic">p</span> &lt; 0.05; ** = <span class="html-italic">p</span> &lt; 0.01; *** = <span class="html-italic">p</span> &lt; 0.001; **** = <span class="html-italic">p</span> &lt; 0.0001; otherwise differences were not statistically significant.</p>
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<p>RRcc activity relies on the synergism between the folic acid pathway and antioxidant activity and on the presence of quercetin-glycosides. (<b>A</b>,<b>B</b>) mRNA levels of muscle cell differentiation markers in primary muscle myoblasts treated for 48 h in the presence of RRcc (30 μg/mL), RRwh (30 μg/mL), an equal amount of vehicle (veh) or left untreated (untr). When indicated, folic acid (FA, 5μM), cystine (Cys2, 1 mM), quercetin (Q, 1 μM), quercetin 3-glycoside (QG, 1 μM), rutin (Q2G, 1 μM) and quercetin 3-rhamnoglucoside 7-glucoside (Q3G, 1 μM) were included in the treatment. Data are shown as mean ± SD of five independent experiments. Statistical analysis was performed by ANOVA test comparing each mean with that of untreated cells. <span class="html-italic">p</span> value = * &lt;0.05, *** &lt;0.001, otherwise differences not statistically significant.</p>
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<p>Overlapped UHPL-HRMS base peak chromatogram of <span class="html-italic">Rhodiola rosea</span> L. wild harvest (RRwh, black chromatogram) and <span class="html-italic">Rhodiola rosea</span> L. controlled cultivation (RRcc, red chromatogram) with peak annotation.</p>
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14 pages, 2847 KiB  
Article
Associations between Brain Alpha-Tocopherol Stereoisomer Profile and Hallmarks of Brain Aging in Centenarians
by Jia Pei Chan, Jirayu Tanprasertsuk, Elizabeth J. Johnson, Priyankar Dey, Richard S. Bruno, Mary Ann Johnson, Leonard W. Poon, Adam Davey, John L. Woodard and Matthew J. Kuchan
Antioxidants 2024, 13(8), 997; https://doi.org/10.3390/antiox13080997 - 17 Aug 2024
Viewed by 1361
Abstract
Brain alpha-tocopherol (αT) concentration was previously reported to be inversely associated with neurofibrillary tangle (NFT) counts in specific brain structures from centenarians. However, the contribution of natural or synthetic αT stereoisomers to this relationship is unknown. In this study, αT stereoisomers were quantified [...] Read more.
Brain alpha-tocopherol (αT) concentration was previously reported to be inversely associated with neurofibrillary tangle (NFT) counts in specific brain structures from centenarians. However, the contribution of natural or synthetic αT stereoisomers to this relationship is unknown. In this study, αT stereoisomers were quantified in the temporal cortex (TC) of 47 centenarians in the Georgia Centenarian Study (age: 102.2 ± 2.5 years, BMI: 22.1 ± 3.9 kg/m2) and then correlated with amyloid plaques (diffuse and neuritic plaques; DPs, NPs) and NFTs in seven brain regions. The natural stereoisomer, RRR-αT, was the primary stereoisomer in all subjects, accounting for >50% of total αT in all but five subjects. %RRR was inversely correlated with DPs in the frontal cortex (FC) (ρ = −0.35, p = 0.032) and TC (ρ = −0.34, p = 0.038). %RSS (a synthetic αT stereoisomer) was positively correlated with DPs in the TC (ρ = 0.39, p = 0.017) and with NFTs in the FC (ρ = 0.37, p = 0.024), TC (ρ = 0.42, p = 0.009), and amygdala (ρ = 0.43, p = 0.008) after controlling for covariates. Neither RRR- nor RSS-αT were associated with premortem global cognition. Even with the narrow and normal range of BMIs, BMI was correlated with %RRR-αT (ρ = 0.34, p = 0.021) and %RSS-αT (ρ = −0.45, p = 0.002). These results providing the first characterization of TC αT stereoisomer profiles in centenarians suggest that DP and NFT counts, but not premortem global cognition, are influenced by the brain accumulation of specific αT stereoisomers. Further study is needed to confirm these findings and to determine the potential role of BMI in mediating this relationship. Full article
(This article belongs to the Section Health Outcomes of Antioxidants and Oxidative Stress)
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<p>(<b>A</b>) α-tocopherol (αT) stereoisomer concentrations (pmol/mg tissue) in the brains of 47 subjects. Each bar represents a subject and subjects are ordered from the lowest to the highest total α-TP concentration. (<b>B</b>) αT stereoisomer relative concentrations in the brains of 47 subjects. Each bar represents a subject and subjects are ordered from the lowest to the highest %RRR, and 2S includes <span class="html-italic">SSS</span>, <span class="html-italic">SSR</span>, <span class="html-italic">SRS</span>, and <span class="html-italic">SRR</span>.</p>
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<p>Correlation between %<span class="html-italic">RRR</span> or %<span class="html-italic">RSS</span> and diffuse plaque (DP), neuritic plaque (NP), or neurofibrillary tangle (NFT) counts in different brain regions (<span class="html-italic">n</span> = 43, excluding one double amputee and three without pathology assessment data). Partial correlation adjusting for sex, race, education, ApoE genotype, diabetes, and hypertension (<b>upper row</b>). Partial correlation adjusting for body mass index in addition to the variables in the first model (<b>lower row</b>). * <span class="html-italic">p</span> &lt; 0.10 and ** <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>The relationship between brain tocopherol concentrations (% of total α-tocopherol) and body mass index (<span class="html-italic">n</span> = 46, excluding one double amputee). <span class="html-italic">RRR</span> (<b>left</b>) and <span class="html-italic">RSS</span> (<b>right</b>).</p>
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19 pages, 1974 KiB  
Review
Antioxidant Functions of Vitamin D and CYP11A1-Derived Vitamin D, Tachysterol, and Lumisterol Metabolites: Mechanisms, Clinical Implications, and Future Directions
by Héctor Vázquez-Lorente, Lourdes Herrera-Quintana, Laura Jiménez-Sánchez, Beatriz Fernández-Perea and Julio Plaza-Diaz
Antioxidants 2024, 13(8), 996; https://doi.org/10.3390/antiox13080996 - 17 Aug 2024
Cited by 1 | Viewed by 1333
Abstract
Evidence is increasing that vitamin D and CYP11A1-derived vitamin D, tachysterol, and lumisterol metabolites play a significant antioxidant role beyond its classical functions in bone health and calcium metabolism. Several recent studies have linked these elements to reduced oxidative stress as well as [...] Read more.
Evidence is increasing that vitamin D and CYP11A1-derived vitamin D, tachysterol, and lumisterol metabolites play a significant antioxidant role beyond its classical functions in bone health and calcium metabolism. Several recent studies have linked these elements to reduced oxidative stress as well as improved immune, cardiovascular, and neurological functions as a result of chronic kidney disease and cancer. Additionally, supplementation with this vitamin has been shown to be one of the most cost-effective micronutrient interventions worldwide, highlighting its potential as a therapeutic approach. The underlying mechanisms and implications of this antioxidant function of vitamin D or CYP11A1-derived vitamin D, tachysterol, and lumisterol metabolites are not well understood. This comprehensive and narrative review is aimed at summarizing the current evidence regarding the molecular mechanisms implicated in this antioxidant function of vitamin D, as well as to provide a general overview and to identify key research areas for the future, offering an extensive perspective that can guide both researchers and clinicians in the management of diseases associated with oxidative stress and/or insufficient vitamin D status. Full article
(This article belongs to the Section Health Outcomes of Antioxidants and Oxidative Stress)
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<p>Vitamin D sources, synthesis, metabolism, and antioxidant mechanisms. Abbreviations. UV, ultraviolet.</p>
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<p>Antioxidant functions of vitamin D. Abbreviations: MDA, Malondialdehyde; Nrf2, nuclear factor erythroid 2-related factor 2; <sup>•</sup>OH, hydroxyl radicals; <sup>•</sup>O<sub>2</sub><sup>−</sup>, superoxide radicals; <sup>•</sup>O<sub>2</sub><sup>2−</sup>, hydrogen peroxide; ROS, reactive oxygen species.</p>
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<p>Main future directions for vitamin D.</p>
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