Cordycepin Ameliorates Renal Interstitial Fibrosis by Inhibiting Drp1-Mediated Mitochondrial Fission

Figures

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  Figure 1. Cordycepin effectively alleviated renal injury and fibrosis. (A) Flow chart of in vivo study; (B) Images of representative mice kidneys; (C) Representative images of Masson’s trichrome staining (scale bar 100 μm), n = 6; Representative images of PAS staining (scale bar 100 μm), n = 6; Representative immunohistochemical staining for α-SMA(scale bar 100 μm), n = 6; (D) Serum creatinine and kidney index of mice, n = 6; (E) Western blotting analysis of FN and α-SMA expression, n = 6; (F) Co-immunostaining for E-cadherin and α-SMA (scale bar 100 μm), n=6; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by One-way ANOVA, \documentclass[12pt]{minimal} \usepackage{wasysym} \usepackage[substack]{amsmath} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage[mathscr]{eucal} \usepackage{mathrsfs} \DeclareFontFamily{T1}{linotext}{} \DeclareFontShape{T1}{linotext}{m}{n} {linotext }{} \DeclareSymbolFont{linotext}{T1}{linotext}{m}{n} \DeclareSymbolFontAlphabet{\mathLINOTEXT}{linotext} \begin{document}$\bar x\pm SEM$\end{document}.

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  Figure 2. Transcriptome Analysis of Kidney. (A) PCA-plot of gene expression changes. The dots indicate samples. (B) Bar plot of DEGs in groups. (C) Clustered heatmap of groups. (D) Box plots of selected genes of interest. (E) DEGs expression (normalized counts) of fibrosis markers, inflammation factors and mitochondrial dynamics genes. (F) GO analysis of DEGs (enriched for pathways involved in mitochondrial processes). (G) KEGG analysis of DEGs.

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  Figure 3. Cordycepin inhibited Drp1-mediated aberrant mitochondrial fission. (A) Representative electron micrographs of mitochondrial morphology in renal tubular epithelial cells. (B) Western blotting analysis of Drp1, MFF, Fis1, OPA1 and Mfn2 expression of kidneys, n = 6; (C) Western blotting analysis of Drp1, MFF, Fis1, OPA1 and Mfn2 expression in NRK-52E, n = 3; (D) Real-time qPCR analysis of Drp1, MFF, Fis1, OPA1 and Mfn2 mRNA levels, n = 3; (E) Relative ROS levels of NRK-52E, n = 6; (F) Co-localization of the Drp1 and Mitotracker (scale bar = 100 μm). (G) Representative images and quantitative analysis of JC-1 assay determined by flow cytometry. (H) JC-1 assay determined by microplate reader; n = 6. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by One-way ANOVA, \documentclass[12pt]{minimal} \usepackage{wasysym} \usepackage[substack]{amsmath} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage[mathscr]{eucal} \usepackage{mathrsfs} \DeclareFontFamily{T1}{linotext}{} \DeclareFontShape{T1}{linotext}{m}{n} {linotext }{} \DeclareSymbolFont{linotext}{T1}{linotext}{m}{n} \DeclareSymbolFontAlphabet{\mathLINOTEXT}{linotext} \begin{document}$\bar x\pm…

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  Figure 4. Cordycepin reduced IL-6 secretion in tubular epithelial cells by Inhibiting mitochondrial fission. (A) Co-immunostaining for IL-6 and LTL (scale bar 100 μm); (B) Western blotting analysis of IL-6 in kidneys, n = 6; (C) ELISA analysis of blood serum IL-6, n = 6; (D) Western blotting analysis of α-SMA and FN in kidneys, n = 6; (E) Western blotting analysis of IL-6 in NRK-52E, n = 3; (F) Real-time qPCR analysis of IL-6 mRNA level, n = 6; (G) ELISA analysis of IL-6 secretion in cell supernatant, n = 6. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by One-way ANOVA, \documentclass[12pt]{minimal} \usepackage{wasysym} \usepackage[substack]{amsmath} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage[mathscr]{eucal} \usepackage{mathrsfs} \DeclareFontFamily{T1}{linotext}{} \DeclareFontShape{T1}{linotext}{m}{n} {linotext }{} \DeclareSymbolFont{linotext}{T1}{linotext}{m}{n} \DeclareSymbolFontAlphabet{\mathLINOTEXT}{linotext} \begin{document}$\bar x\pm SEM$\end{document}.

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  Figure 5. Cordycepin inhibited fibroblast proliferation and activation by reducing IL-6 secretion of adjacent PTECs. (A) Co-immunostaining for IL-6 and α-SMA. White boxes: IL-6 secretion from injured renal tubular epithelial cells and adjacent α-SMA-positive fibroblast accumulation (scale bar 100 μm); (B) Western blotting analysis of FN, α-SMA, p-STAT3 and STAT3 expression in NRK-49F, n = 3; (C) Real-time qPCR analysis of FN and α-SMA mRNA levels, n = 3; (D) Cell viability was measured by the CCK-8 on different conditions. n = 6. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by One-way ANOVA, \documentclass[12pt]{minimal} \usepackage{wasysym} \usepackage[substack]{amsmath} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage[mathscr]{eucal} \usepackage{mathrsfs} \DeclareFontFamily{T1}{linotext}{} \DeclareFontShape{T1}{linotext}{m}{n} {linotext }{} \DeclareSymbolFont{linotext}{T1}{linotext}{m}{n} \DeclareSymbolFontAlphabet{\mathLINOTEXT}{linotext} \begin{document}$\bar x\pm SEM$\end{document}.

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  Figure 6. (A) Molecular dynamics simulation of Cordycepin with Drp1; (B and C): RMSD and SASA analyzation of MD simulation; (D-F): SPR analysis of cordycepin binding to Drp1; (G) Molecular mechanism of inhibiting renal fibrosis by inhibiting mitochondrial division with Cordycepin.