Methyl Ferulic Acid Attenuates Human Cardiac Fibroblasts Differentiation and Myocardial Fibrosis by Suppressing pRB-E2F1/CCNE2 and RhoA/ROCK2 Pathway

Figures

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  FIGURE 1. MFA reduced fibrosis-related genes expression in TGF-β1–stimulated HCFs. (A) Chemical structural formula of MFA. (B–C) Cell viabilities when HCFs were treated by different concentrations of TGF-β1 (B) and MFA (C) (n = 6). (D–F) Protein (D. E) and mRNA (F) expression levels of fibrosis-related genes (n = 4). (*compared with negative control: *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. #Compared with the TGF-β1 group: # p < 0.05, ## p < 0.01, ### p < 0.005).

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  FIGURE 2. MFA suppressed proliferation and migration ability in TGF-β1–stimulated HCFs. (A–B) EDU assay (A) and CCK-8 assay (B) were performed to evaluate the proliferation ability of HCFs (n = 6). (C–D) Wound healing assay (C) and Transwell assay (D) were performed to evaluate the migration ability of HCFs treated as below (n = 6). (*compared with negative control: ***p < 0.005, ****p < 0.001. #Compared with the TGF-β1 group: # p < 0.05, ## p < 0.01).

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  FIGURE 3. MFA reduced fibrosis level in MI mice. (A–C) Protein (A. B) and mRNA (C) expression levels of fibrosis-related genes in different mice groups (n = 6). (D–E) Tissue staining (D), and immunofluorescence (E) were performed to compare the fibrosis area and fibrosis-related genes expression. (*compared with negative control: **p < 0.01, ***p < 0.005, ****p < 0.001. #Compared with the MI group: # p < 0.05, ## p < 0.01, ### p < 0.005).

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  FIGURE 4. MFA improved the survival and cardiac function in MI mice. (A) General indicator among four groups of mice (n = 6). (B) K-M analysis among four groups of mice (n = 15). (C–D) Echocardiography images (in both B and M mode) (C), and cardiac functions among four groups of mice (n = 6). (*compared with negative control: *p < 0.05, **p < 0.01, ****p < 0.001. #Compared with the MI group: # p < 0.05, ## p < 0.01, ### p < 0.005, #### p < 0.005).

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  FIGURE 5. MFA suppressed the expression of pRB, E2F1, CCNE2, RhoA, and ROCK2, regulated the cell cycle, and conjunction of RB and E2F1. (A–C) Protein expression levels of proliferation and migration-related genes in HCFs (A), mice LVs (B), and mice tissue (C) (n = 3). (D) Cell cycle analysis in HCFs (n = 6). (E) Immunoprecipitation of anti-pRB to E2F1 in HCFs lysates. (F) Sketch map of our assumptions. (*compared with negative control: ***p < 0.005. #Compared with the TGF-β1+MFA group: ## p < 0.01).

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  FIGURE 6. MFA suppressed the expression of pRB, E2F1, CCNE2, RhoA, and ROCK2, regulated the cell cycle, and conjunction of RB and E2F1. (A–D) Rescue test for cell proliferation ability and ECM formation by Western blot (A) (n = 3), cell cycle analysis (B), EDU assay (C), and CCK-8 assay (D) (n = 6). (E–G) Rescue test for cell migration ability and ECM formation by Western blot (E) (n = 3), Wound healing assay (F), and TRANSWELL assay (n = 6) (G). (*compared with negative control: *p < 0.05, **p < 0.01, ***p < 0.005. #Compared with the TGF-β1 group: # p < 0.05, ### p < 0.005. &Compared with the TGF-β1 + MFA group: & p < 0.05, && p < 0.01).