Proteomic Analysis Reveals the Protective Effects of Yiqi Fumai Lyophilized Injection on Chronic Heart Failure by Improving Myocardial Energy Metabolism

Figures

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  FIGURE 1. Comparison of the liquid chromatogram of the mixed reference substance and YQFM sample. (A) Standard solution and (B) Sample solution: 1: ginsenoside Rg1, 2: ginsenoside Rf, 3: ginsenoside Rb1, 4: ginsenoside Rc, 5: ginsenoside Rb2, 6: ginsenoside Rb3, 7: ginsenoside Re, 8: ginsenoside Rd, 9: schizandrol A, 10:20(S)-ginsenoside F2, 11: schizandrol B, 12: ginsenoside Rg2, 13: schisandrin A, 14: schisandrin B, 15: ginsenoside Rh1.

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  FIGURE 2. The effect of YQFM on cardiac function in CHF rat after being treated for 28 days. (A) Representative echocardiograph from the various groups. (B) Left ventricle ejection fraction (LVEF) (n = 6 animals/group). (C) Left ventricle fractional shortening (LVFS) (n = 6 animals/group). Data are means ± SD. # p < 0.05, ## p < 0.01, ### p < 0.001 vs the Control group, *p < 0.05, **p < 0.01, ***p < 0.001 vs the Model group.

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  FIGURE 3. The effect of YQFM on Cardiac Morphology in CHF rat after being treated for 28 d. (A) HE, Masson and PAS pathology section (B–D) staining of the myocardial. (n = 6 animals/group). Data are means ± SD. # p < 0.05, ## p < 0.01, ### p < 0.001 vs the Control group, *p < 0.05, **p < 0.01, ***p < 0.001 vs the Model group.

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  FIGURE 4. The effect of YQFM on Cardiac Morphology in CHF rat after being treated for 28 days. (A,B) Immunohistochemistry analysis of cTnt expression in heart sections of different groups. The diseased areas in the heart are magnified in the panel below (n = 3 per group). Concentration of cTn t (C), CK-MB (D) and LDH (E) in CHF rat after being treated for 28 days (n = 6 animals/group). Data are means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs the Model group, # p < 0.05, ## p < 0.01, ### p < 0.001 vs the Control group.

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  FIGURE 5. Quality control of protein data validation. (A) Protein information. The x-axis in the figure represents the identified protein information, from left to right: the total number of secondary spectra, the number of matched spectra, the number of identified peptides, the number of identified proteins, and the number of identified protein groups. (B) Protein mass distribution of all identified proteins. (C) Distribution of protein’s sequences coverage. (D) Protein length distribution of all identified peptides.

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  FIGURE 6. The differentially abundant proteins (DEPs) in serum between Model:Control and Treatment: Model. (A) Volcano Plots of DEPs of Model:Control. (B) Volcano Plots of DEPs of Treatment:Model. (C) Venn-diagram showing the overlap of all DEPs. (D) DEPs involved in Model:Control. (E) DEPs involved in Treatment:Model. Red or Green represents the numbers of upregulated and downregulated proteins, respectively.

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  FIGURE 7. Cluster analysis of differentially expressed proteins among the three groups. Color changes with protein expression, blue indicates negative correlation, red indicates positive correlation, the stronger the color, the strongest correlation.

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  FIGURE 8. Functional annotation and categories of DEPs. (A) GO annotations analisis of Model:Control. (B) GO annotations analisis of Treatment:Model. (C) KEGG analyses of protein functions. (D) Enrichment analysis of KEGG energy metabolism pathway.

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  FIGURE 9. YQFM ameliorated the mitochondrial dysfunction induced by H2O2 in vitro. (A) TEM images of the ultrastructural morphology of H9C2 cells mitochondria. (B) Oxygen consumption rates (OCR) in H9C2 cells treated with YQFM-L, YQFM-M& YQFM-H. (C) H9C2 cells were subjected to Mitotracker red staining. The model group refers to H9C2 cells induced by H2O2.

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  FIGURE 10. YQFM suppressed cardiomyocyte apoptosis caused by H2O2. (A,B) Flow cytometric measurement of H9C2 cells apoptosis caused by H2O2. (C) The amount of Bax and Bcl-2 from H9C2 cells lysate was examined by Western blot analysis. (D,E) Relative protein levels were measured by densitometry for Bax and Bcl-2 (n = 3 per group). Data are means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs the Model group, # p < 0.05, ## p < 0.01, ### p < 0.001 vs the Control group.

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  FIGURE 11. YQFM upregulated mitochondrial biogenesis-related genes with PGC-1α activation. (A) The amount of PGC-1α,PPAR-α and RXR-α from H9C2 cells lysate was examined by Western blot analysis. (B–D) Relative protein levels were measured by densitometry for PGC-1α,PPAR-α and RXR-α. (E) The amount of NRF-1 and Tfam from H9C2 cells lysate was examined by Western blot analysis. (F,G) Relative protein levels were measured by densitometry for NRF-1 and Tfam (n = 3 per group). Data are means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs the Model group, # p < 0.05, ## p < 0.01, ### p < 0.001 vs the Control group.