Sorbus commixta Fruit Extract Suppresses Lipopolysaccharide-Induced Neuroinflammation in BV-2 Microglia Cells via the MAPK and NF-κB Signaling Pathways
<p>Effects of SFW and SFE on NO production (<b>A</b>) and cell viabilities (<b>B</b>) in LPS-induced BV-2 cells. BV-2 cells (2 × 10<sup>5</sup> cells/mL) were treated with various concentrations of SFW (12.5–200 μg/mL) for 1 h and treated with LPS (200 ng/mL) for 24 h. Controls were samples without LPS or SFW treatment. (<b>A</b>) NO production in the culture medium was determined using the Griess reagent and a standard curve using NaNO<sub>2</sub>. (<b>B</b>) Cell viability was assessed by the MTT assay, and the results are expressed as the percentage of control cells. Data are presented as mean ± SD from three independent experiments. <sup>###</sup> <span class="html-italic">p</span> < 0.001 versus control. *** <span class="html-italic">p</span> < 0.001 versus LPS.</p> "> Figure 2
<p>Effects of pro-inflammatory cytokines IL-6 and TNF-<span class="html-italic">α</span> in LPS-induced BV-2 cells (<b>A</b>,<b>B</b>). BV-2 cells were pretreated with SFW for 1 h and stimulated with LPS (200 ng/mL) for 18 h (<b>A</b>) or 6 h (<b>B</b>), and then IL-6 and TNF-<span class="html-italic">α</span> levels were determined using ELISA. Data are presented as mean ± SD from three independent experiments. <sup>###</sup> <span class="html-italic">p</span> < 0.001 versus control. *** <span class="html-italic">p</span> < 0.001 versus LPS.</p> "> Figure 3
<p>Effect of SFW on LPS-induced iNOS and COX-2 protein expressions in BV-2 cells (<b>A</b>,<b>B</b>). BV-2 cells were pretreated with SFW for 1 h, stimulated with LPS (200 ng/mL) for 18 h, and examined by Western blotting. Data are presented as mean ± SD from three independent experiments. β-actin was used as an internal control. <sup>###</sup> <span class="html-italic">p</span> < 0.001 versus control. *** <span class="html-italic">p</span> < 0.001 versus LPS.</p> "> Figure 4
<p>Effect of SFW on LPS-induced MAPK activation in BV-2 cells (<b>A</b>–<b>D</b>). BV-2 cells were pretreated with SFW (50–100 μg/mL) for 1 h prior to stimulation with LPS (200 ng/mL) for 30 min. Total protein (15 μg) was subjected to 12% SDS-PAGE, followed by Western blotting using anti-ERK, anti-p38, and anti-JNK. Results are representative of those obtained from three independent experiments. <sup>###</sup> <span class="html-italic">p</span> < 0.001 versus control. *** <span class="html-italic">p</span> < 0.001 versus LPS.</p> "> Figure 5
<p>Effects of SFW on nuclear translocation of NF-<span class="html-italic">κ</span>B p65 through suppression of I<span class="html-italic">κ</span>Bα phosphorylation in LPS-stimulated BV-2 microglia. BV-2 cells were pretreated with SFW (200 μg/mL) for 1 h prior to stimulation with LPS (200 ng/mL) for 30 min. Cell lysates were prepared and analyzed by Western blotting with anti-phospho-I<span class="html-italic">κ</span>Bα antibody (<b>A</b>,<b>B</b>). Results are representative of those obtained from three independent experiments. <sup>###</sup> <span class="html-italic">p</span> < 0.001 versus control. *** <span class="html-italic">p</span> < 0.001 versus LPS. Localization of NF-<span class="html-italic">κ</span>B p65 was visualized with fluorescence microscopy after immunofluorescence staining with NF-<span class="html-italic">κ</span>B p65 antibody (red fluorescence) (<b>C</b>). Cells were stained with DAPI for visualization of nuclei (blue fluorescence). Scale bar: 50 μm. Arrows: highlight areas of cells with a translocation of NF-<span class="html-italic">κ</span>B p65.</p> "> Figure 6
<p>Chemical structure of gallic acid (<b>1</b>), chlorogenic acid (<b>2</b>), caffeic acid (<b>3</b>), <span class="html-italic">p</span>-coumaric acid (<b>4</b>), and rutin (<b>5</b>).</p> "> Figure 7
<p>HPLC chromatogram of caffeic acid (<b>A</b>) and SFW (<b>B</b>). <span class="html-italic">X</span>-axis, retention time; <span class="html-italic">Y</span>-axis, absorbance unit. The monitoring wavelength was set at 320 nm.</p> "> Figure 8
<p>Effect of caffeic acid on NO production (<b>A</b>), cell viability (<b>B</b>) and iNOS and COX-2 protein expression (<b>C</b>,<b>D</b>) in LPS-induced BV-2 microglia. The BV-2 cells were pretreated with caffeic acid for 1 h, stimulated with LPS (200 ng/mL) for 18 h, and examined by Western blotting. Data are presented as mean ± SD from three independent experiments. β-actin was used as an internal control. <sup>###</sup> <span class="html-italic">p</span> < 0.001 versus control. *** <span class="html-italic">p</span> < 0.001 versus LPS.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Effects of S. commixta Fruit Water Extract (SFW) and Ethanol Extract (SFE) on NO Production and Cytotoxicity
2.2. Effects of SFW on Pro-Inflammatory Cytokines
2.3. Effects of SFW on iNOS and COX-2 Protein Expression
2.4. Effects of SFW on MAPK Protein Expression
2.5. Effects of SFW on Phosphorylation of IĸBα and Nuclear Translocation of NF-ĸB
2.6. HPLC Analysis of Caffeic Acid
2.7. Anti-Inflammatory Effect of Caffeic Acid
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Plant Materials and Sample Preparation
4.3. Cell Culture and Cell Viability
4.4. Nitric Oxide Assay
4.5. Enzyme-Linked Immunosorbent Assay (ELISA) for the Determination of Cytokine Levels
4.6. Western Blot Analysis
4.7. Immunocytochemistry Staining
4.8. HPLC Analysis
4.9. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Compound | Retention Time (min) | Formular | Molecular Weight | CAS Number | |
---|---|---|---|---|---|
1 | Gallic acid | 9.60 | C7H6O5 | 170.12 | 149-91-7 |
2 | Chlorogenic acid | 18.45 | C16H18O9 | 354.31 | 327-97-9 |
3 | Caffeic acid | 20.58 | C9H8O4 | 180.16 | 331-39-5 |
4 | p-Coumaric acid | 23.03 | C9H8O3 | 164.16 | 501-98-4 |
5 | Rutin hydrate | 24.17 | C27H30O16 × H2O | 610.52 | 207671-50-9 |
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Kim, Y.-S.; Jung, J.-H.; Kim, K.-T. Sorbus commixta Fruit Extract Suppresses Lipopolysaccharide-Induced Neuroinflammation in BV-2 Microglia Cells via the MAPK and NF-κB Signaling Pathways. Molecules 2024, 29, 5592. https://doi.org/10.3390/molecules29235592
Kim Y-S, Jung J-H, Kim K-T. Sorbus commixta Fruit Extract Suppresses Lipopolysaccharide-Induced Neuroinflammation in BV-2 Microglia Cells via the MAPK and NF-κB Signaling Pathways. Molecules. 2024; 29(23):5592. https://doi.org/10.3390/molecules29235592
Chicago/Turabian StyleKim, Yon-Suk, Jin-Hwa Jung, and Ki-Tae Kim. 2024. "Sorbus commixta Fruit Extract Suppresses Lipopolysaccharide-Induced Neuroinflammation in BV-2 Microglia Cells via the MAPK and NF-κB Signaling Pathways" Molecules 29, no. 23: 5592. https://doi.org/10.3390/molecules29235592
APA StyleKim, Y. -S., Jung, J. -H., & Kim, K. -T. (2024). Sorbus commixta Fruit Extract Suppresses Lipopolysaccharide-Induced Neuroinflammation in BV-2 Microglia Cells via the MAPK and NF-κB Signaling Pathways. Molecules, 29(23), 5592. https://doi.org/10.3390/molecules29235592