Pirfenidone restores metabolic hormones and cardiac autophagy via p-AMPK in MASH

Figures

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  Fig. 1. Effects of PFD on body weight, caloric intake, glucose levels, and serum lipid profile. A) Body weight during the experimental period (n = 7 mice per group); the arrow black marks the initiation of standard PFD treatment at week 8. Evaluation of definitive weight changes and statistical significance is presented in Table 1. B-C) Daily calorie intake between groups (Student’s t-test with Welch’s correction: n = 24 per group, 1–8 weeks; and one-way ANOVA followed by Tukey’s post hoc test: n = 24 per group, 9–16 weeks). D) Fasting blood glucose levels. E) Glucose tolerance test (GTT) curves. F) Area under the curve (AUC). G) Serum triglycerides. H) Total cholesterol. I) LDL cholesterol. J) VLDL cholesterol levels. Data are expressed as mean ± standard error of the mean (SEM). D-J) One-way ANOVA followed by Tukey’s post hoc test, n = 6 per group. *P < 0.05, ***P < 0.001 vs. ND; #P < 0.05, ###P < 0.001 vs. HFHC; +++P < 0.001 vs. ND

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  Fig. 2. PFD improves cardiac hypertrophy and cardiac inflammatory markers in obese mice. (A) Micrographs of the ventricular chamber tissue sections. (B) Relative mRNA expression levels of hypertrophy markers (Nppa, Nppb). (C) Number of inflammatory foci (Student’s t-test, n = 3 per group). (D) Relative mRNA expression levels of inflammatory markers (Tnf, Il6, Adgre1, Ccl2), n = 4 per group. One-way ANOVA followed by Tukey’s post hoc test. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. ND; #P < 0.05, ##P < 0.01 vs. HFHC; ++P < 0.01 vs. ND

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  Fig. 3. Cardiac fibrosis is prevented by PFD. (A) Cardiac fibrosis was assessed with Masson’s trichrome staining (n = 6 per group). (B) Relative mRNA expression levels of fibrotic markers: Tgfb1, Col3a1, and Col1a1 (n = 4 per group). Data are expressed as mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001 vs. ND; #P < 0.05, ###P < 0.001 vs. HFHC

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  Fig. 4. Evaluation of cardiac autophagy-related markers in mice. (A) Western blotting image and relative protein expression levels of SQSTM1, BECN1, LC3B-II/LC3B-I ratio, and phosphorylated p-AMPKα (Thr172) normalized to total AMPKα and GAPDH (n = 4 per group). (B) Relative mRNA expression levels: Becn1, Atg7, and Sqstm1 (n = 4 per group). Data are expressed as mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test. *P < 0.05, ***P < 0.001 vs. ND; #P < 0.05, ##P < 0.01 vs. HFHC

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  Fig. 5. Cell viability by MTT assay in H9c2 cardiomyoblasts. (A) Concentrations of compounds chosen for experiments in H9c2 cells. (B) Representative micrographs showing intracellular lipid droplets (ORO). Scale bar = 200 μm. (C) Quantification of Oil Red O-positive stained area percentage (n = 4 fields per condition. (D-F) Relative mRNA expression levels of Nppa, Nppb, Tnf, Il6, Tgfb1, Col3a1, and Col1a1 normalized to Gapdh in H9c2 cells treated with bovine serum albumin (BSA), palmitic acid (PA), glucose (Glc), or pirfenidone (PFD), compound C (CC), and bafilomycin A1 (BafA1) (n = 4). Data are expressed as mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test (n = 6 per group). *P < 0.05, ***P < 0.001 vs. control; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. BSA/NaOH; +++P < 0.001 vs. DMSO. °P < 0.05, °°°P < 0.001 vs. BSA; qP < 0.05, qqP < 0.01 vs. PA 150 µM; &P < 0.05, &&P < 0.01, &&&P < 0.001 vs. PA 150 µM + Glc 15 mM

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  Fig. 6. Evaluation of autophagy-related markers in H9c2 cardiomyoblasts. A-B) Western blotting image and relative protein expression levels of SQSTM1, BECLIN-1, LC3B-II/LC3B-I ratio, and p-AMPKα (Thr172) normalized to total AMPKα and GAPDH (n = 4 per group). C-D) Relative mRNA expression levels: Becn1, Atg7, and Sqstm1 (n = 4 per group). Data are expressed as mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test. °P < 0.05, °°P < 0.01, °°°P < 0.001 vs. control; +P < 0.05, +++P < 0.001 vs. PA 200 µM; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. Glc 30 mM; &P < 0.05, &&&P < 0.001 vs. PA 150 µM + Glc 15 mM; TTP < 0.01, TTTP < 0.001 vs. PA 150 µM + Glc 15 mM + CC 10 µM; $P < 0.05, $$P < 0.01, $$$P < 0.001 vs. PA 150 µM + Glc 15 mM + CC 10 µM; %P < 0.05, %%P < 0.01, %%%P < 0.001 vs. PA 150 µM + Glc 15 mM + BafA1 50 nM

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  Fig. 7. Differentially expressed genes in ventricular tissue induced by HFHC diet and PFD. (A) Venn diagram analysis comparing gene expression identified among the mouse groups. (B) Biological processes-structured visualization of differentially expressed genes. The color intensity corresponds to Z-score magnitude. (C) Kernel density plots showing the Z-score distribution of gene expression. Panels A-C include genes with Z-score ≥ + 1.5 (up-regulated) or Z-score ≤ -1.5 (down-regulated). (D) Heatmaps displaying gene-specific Z-scores (Z-score ≥ + 1.5 or Z-score ≤ -1.5), where rows represent comparisons and columns represent individual genes

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  Fig. 8. Schematic summary of the cardioprotective mechanisms of pirfenidone in obesity-associated MASH. The left panel represents the dysregulation of systemic hormone levels, alongside the induction of cardiac inflammation, hypertrophy, and fibrosis markers, including the ventricular microarray signature. Conversely, the right panel illustrates the therapeutic efficacy of PFD in reversing these pathological processes and normalizing the ventricular microarray signature profile