Ferruginol Restores SIRT1-PGC-1α-Mediated Mitochondrial Biogenesis and Fatty Acid Oxidation for the Treatment of DOX-Induced Cardiotoxicity

Figures

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  FIGURE 1. FGL relieved DOX-induced cardiac structural and functional lesion. (A) A schematic diagram of animal model generation and drug administration. (B) Body weight changes in various groups (n = 10). (C) Representative images of echocardiograms. (D) Representative images of morphological and structural changes in the myocardium. Scale bar: 50 μm. (E) Myocardial fibrosis detection by Masson staining. (F) The effects of FGL and ENA on DOX-induced cardiac EF and FS (n = 6). (G) The effects of FGL and ENA on cardiac serum CK-MB and LDH (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 vs DOX group.

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  FIGURE 2. The results of mRNA sequencing and analysis. (A) Hierarchical clustering of proteins and samples. (B, C) GO and KEGG enrichment analysis between the control and DOX groups. (D, E) Representative gene heatmap and PPI diagram associated with MB&FAO. (F) Quantitative analysis of representative mRNA associated with MB&FAO by RT-qPCR. (n = 3–5). *p < 0.05, **p < 0.01, ***p < 0.001 vs DOX group.

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  FIGURE 3. FGL inhibited DOX-induced apoptosis and oxidative injury in vivo and in vitro. (A, B) Representative images and quantitative analysis of apoptosis by TUNEL in the mouse myocardium (n = 6). Scale bar: 20 μm. (C) Effective concentration of FGL in H9C2 cells (n = 6). (D, E) Representative images and quantitative analysis of apoptosis by Hoechst staining in H9C2 cells (n = 5). Scale bar: 100 μm. (F, G) Representative images and quantitative analysis of mitochondrial membrane potential measured by JC-1 staining in H9C2 cells (n = 3). Scale bar: 25 μm. (H, I) Representative images and quantitative analysis of ROS by DCFH-DA staining in H9C2 cells (n = 6). Scale bar: 100 μm *p < 0.05, ***p < 0.001 vs DOX group. #p < 0.05 vs. (DOX + FGL) group.

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  FIGURE 4. FGL promoted mitochondrial biogenesis and fatty acid oxidization in vivo. (A, B) Representative images and quantitative analysis of electron micrographs of the heart (n = 6). Scale bar: 5.0 μm. (C–F) Expression levels of mitochondrial biogenesis and fatty acid oxidization-associated proteins were quantified and shown as relative protein expression levels after normalization to GAPDH (n = 3–5). *p < 0.05, **p < 0.01, ***p < 0.001 vs DOX group.

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  FIGURE 5. FGL promoted mitochondrial biogenesis and fatty acid oxidization in vitro. (A, B) Representative images and quantitative analysis of the number of mitochondria by mitotracker staining in H9C2 cells (n = 6). Scale bar: 25 μm. (C) The assessment of the mtDNA copy number in H9C2 cells by RT-qPCR (n = 4). (D) The content of ATP in H9C2 cells (n = 5). (E, F) The expression level of mitochondrial biogenesis and fatty acid oxidization-associated mRNA in H9C2 cells quantified and shown as relative RNA levels after normalization to GAPDH (n = 5). (G–J) The expression level of mitochondrial biogenesis and fatty acid oxidization-associated proteins in H9C2 cells quantified and shown as relative protein expression levels after normalization to GAPDH (n = 3–5). *p < 0.05, **p < 0.01, ***p < 0.001 vs DOX group. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. (DOX + FGL) group.

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  FIGURE 6. FGL promoted the deacetylation of SIRT1 to PGC-1α in vitro. (A, B) Representative images and quantitative analysis of SIRT1 and PGC-1α and their colocalization in the nucleus (n = 6). Scale bar: 10 μm. (C, D) Representative bands and the ratio of acetylated PGC-1α to total PGC-1α detected by CO-IP of PGC-1α and acetylated lysine (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001 vs DOX group. #p < 0.05 vs. (DOX + FGL) group.

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  FIGURE 7. FGL did not interfere with the antitumor effect of DOX. (A, B) The influence of FGL on the anticancer effect of DOX on MCF-7 and HepG2 cells was determined by CCK-8 assay (n = 6). ***p < 0.001 vs DOX group.