Ginsenoside Rg1 Acts as a Selective Glucocorticoid Receptor Agonist with Anti-Inflammatory Action without Affecting Tissue Regeneration in Zebrafish Larvae
<p>Inhibitory effects of Rg1 and beclomethasone on wounding-induced leukocyte migration. (<b>A</b>) The chemical structures of the classical GC beclomethasone, and the ginsenoside Rg1, which has a steroid-like skeleton consisting of four trans-rings, (three six-member cyclohexane rings and one five-member cyclopentane ring). (<b>B</b>) Schematic drawing of a zebrafish larva at 3 dpf, indicating the site of tail fin amputation, the region of microscopic imaging, and the area for quantification of neutrophils and macrophages (i.e., the counting area: posterior to the tail vein). (<b>C</b>) Dose-dependent anti-inflammatory effect of Rg1. Numbers of neutrophils that have migrated towards the amputated tail fin at 4 hpa and 2 h pre- and 4 h post-treatment with Rg1 or beclomethasone at the indicated concentrations. The graph shows data from an experiment with 25 larvae per group, representative of three experimental repeats. No mortality was observed at Rg1 concentrations up to 120 μM, while 20% and 40% of embryos on average died in the 150 μM and 180 μM groups. (<b>D</b>) Representative fluorescence microscopy images of wound-induced migration of neutrophils (in green fluorescence channel) and macrophages (in red fluorescence channel), in combination with vehicle, beclomethasone (Beclo), and Rg1 treatments. Scale bar: 100 μm. (<b>E</b>,<b>F</b>) Numbers of neutrophils (<b>E</b>) and macrophages (<b>F</b>) that have migrated towards the amputation site, after treatment of 3 dpf larvae with vehicle, Beclo, or Rg1. The graphs show data from an experiment with 20 larvae per group (each indicated as individual data points), representative of three experimental repeats. (<b>G</b>,<b>H</b>) The total number of neutrophils (<b>G</b>) and macrophages (<b>H</b>) present in the entire tail region (posterior to the yolk sac extension). ns—not significant. * <span class="html-italic">p</span> < 0.05.</p> "> Figure 2
<p>Glucocorticoid receptor (GR)-dependency of the anti-inflammatory effects of Rg1 and beclomethasone. Quantification of neutrophil migration towards the amputated site in the tail fin at 3 dpf in Gr mutant (<span class="html-italic">gr<sup>−/−</sup></span>) and wild type (<span class="html-italic">gr<sup>+/+</sup></span>) larvae, after treatment with vehicle, beclomethasone (Beclo), or Rg1. The graphs show data from an experiment with 25 larvae per group (each indicated as individual data points), representative of three experimental repeats. ns—not significant. * <span class="html-italic">p</span> < 0.05.</p> "> Figure 3
<p>Differential effects of Rg1 and beclomethasone on inflammatory gene expression. Expression analysis by qPCR using total RNA from 3 dpf larvae with (Amp) or without (Non-Amp) amputation and treated with vehicle, beclomethasone (Beclo), or Rg1. (<b>A</b>) Genes encoding proteins in general inflammatory pathways (Toll-like receptors and NF-κB inhibitor α-like protein (<b>A</b>). (<b>B</b>) Genes encoding proinflammatory cytokines (Interleukins and Tnfα). (<b>C</b>) Genes encoding Matrix metalloproteinases (Mmps), implicated in inflammation and tissue regeneration. (<b>D</b>,<b>E</b>) Genes encoding neutrophil- or macrophage-related chemokines (<b>D</b>) or chemokine receptors (<b>E</b>). The relative expression values were normalized to those of <span class="html-italic">ppial</span> and are shown on a log2 scale. Bars represent the mean ± SEM of three independent experiments (each with technical duplicates). * <span class="html-italic">p</span> < 0.05 compared with the nonamputated vehicle group. <sup>#</sup> <span class="html-italic">p</span> < 0.01 compared with the amputated vehicle group. + <span class="html-italic">p</span> < 0.05 compared with the amputated Beclo group.</p> "> Figure 4
<p>Differential effects of Rg1 and beclomethasone on the expression of endogenous Gr target genes. Expression analysis by qPCR using total RNA from 3 dpf larvae with (Amp) or without (Non-Amp) amputation and treated with vehicle, beclomethasone (Beclo), or Rg1. (<b>A</b>) Gene encoding Phosphoenolpyruvate Carboxykinase (Pck1). (<b>B</b>) Gene encoding FK506 binding protein 5 (Fkbp5). The relative expression values were normalized to those of <span class="html-italic">ppial</span> and are shown on a log2 scale. Bars represent the mean ± SEM of three independent experiments (each with technical duplicates). <sup>#</sup> <span class="html-italic">p</span> < 0.01 compared with the amputated vehicle group. + <span class="html-italic">p</span> < 0.05 compared with the amputated Beclo group.</p> "> Figure 5
<p>Effects of beclomethasone and Rg1 on the regeneration of the tail fin. (<b>A</b>–<b>D</b>) Representative images of the tail fins of zebrafish larvae at three days after amputation (Amp) or without amputation (Non-Amp) and treated with vehicle, beclomethasone (Beclo), or Rg1. The approximate location of the amputation site is indicated with arrowheads on the dorsal and ventral side of the tail fin. The images show full regeneration of the tail fin in the vehicle and Rg1 treated larvae, and a total lack of regeneration in the Beclo group. (<b>E</b>) Quantification of the tail fin regeneration in zebrafish larvae treated with Vehicle, Beclo, or Rg1 (<span class="html-italic">n</span> = 90 larvae per group, accumulated from three independent experiments. (<b>F</b>) Quantification of the tail fin regeneration in Gr mutant (<span class="html-italic">gr<sup>−/−</sup></span>) and control (<span class="html-italic">gr</span><sup>+/+</sup>) larvae treated with vehicle, Beclo, or Rg1 (<span class="html-italic">n</span> = 90 larvae per group, accumulated from three independent experiments).</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Zebrafish Lines and Maintenance
2.2. Fish Embryo Acute Toxicity Test (FET) for Rg1
2.3. Tail Fin Amputation and Drug Treatments
2.4. Visualization and Quantification of Macrophages and Neutrophils
2.5. Regeneration of Amputated Larvae Tail Fin
2.6. Quantitative PCR (qPCR) Analysis
2.7. Statistical Analysis
3. Results
3.1. Ginsenoside Rg1 Has Anti-Inflammatory Effects that Are Mediated by the Gr
3.2. Rg1 And Beclomethasone Differentially Regulate Gene Expression
3.3. Rg1 Does Not Inhibit Tissue Regeneration, Unlike Beclomethasone
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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He, M.; Halima, M.; Xie, Y.; Schaaf, M.J.M.; Meijer, A.H.; Wang, M. Ginsenoside Rg1 Acts as a Selective Glucocorticoid Receptor Agonist with Anti-Inflammatory Action without Affecting Tissue Regeneration in Zebrafish Larvae. Cells 2020, 9, 1107. https://doi.org/10.3390/cells9051107
He M, Halima M, Xie Y, Schaaf MJM, Meijer AH, Wang M. Ginsenoside Rg1 Acts as a Selective Glucocorticoid Receptor Agonist with Anti-Inflammatory Action without Affecting Tissue Regeneration in Zebrafish Larvae. Cells. 2020; 9(5):1107. https://doi.org/10.3390/cells9051107
Chicago/Turabian StyleHe, Min, Mahmoud Halima, Yufei Xie, Marcel J. M. Schaaf, Annemarie H. Meijer, and Mei Wang. 2020. "Ginsenoside Rg1 Acts as a Selective Glucocorticoid Receptor Agonist with Anti-Inflammatory Action without Affecting Tissue Regeneration in Zebrafish Larvae" Cells 9, no. 5: 1107. https://doi.org/10.3390/cells9051107
APA StyleHe, M., Halima, M., Xie, Y., Schaaf, M. J. M., Meijer, A. H., & Wang, M. (2020). Ginsenoside Rg1 Acts as a Selective Glucocorticoid Receptor Agonist with Anti-Inflammatory Action without Affecting Tissue Regeneration in Zebrafish Larvae. Cells, 9(5), 1107. https://doi.org/10.3390/cells9051107