C188‐9 reduces TGF‐β1‐induced fibroblast activation and alleviates ISO‐induced cardiac fibrosis in mice

Fig. 1. C188‐9 reduces TGF‐β1‐induced fibroblast activation. CFs were isolated, pretreated with C188‐9 (10 μm) for 12 h, and then stimulated with C188‐9 and TGF‐β1 for another 48 h. Total RNA or protein extracts were isolated. (A–D) Quantitative RT‐PCR (qRT‐PCR) of Col1a1 (A), Col1a2 (B), Col3a1 (C), and α‐SMA (D) transcript levels in CFs. Results are normalized to 1, n = 6, *P < 0.05, TGF‐β1‐DMSO vs. vehicle DMSO. # P < 0.05, TGF‐β1‐C188‐9 vs. TGFβ1‐DMSO; two‐way ANOVA, followed by Tukey's post hoc test. (E) Western blots of collagen Ⅰ, α‐SMA, p‐STAT3, and total STAT3 levels. GAPDH was used as loading control. (F) Quantification of relative collagen I and α‐SMA levels. n = 6. *P < 0.05, TGF‐β1‐DMSO vs. vehicle DMSO. # P < 0.05, TGF‐β1‐C188‐9 vs. TGFβ1‐DMSO; two‐way ANOVA, followed by Tukey's post hoc test. Data are presented as mean ± SEM.

Fig. 2. C188‐9 treatment prevents ISO‐induced left ventricular dilatation and dysfunction in mice. C57BL/6 mice aged 10 weeks were administered ISO perfusion with or without C188‐9 treatment. (A) Scheme of C188‐9 treatment in the ISO‐induced mouse model. Mice received ISO perfusion, echocardiography was performed, and heart tissues were collected on day 21. (B) Representative M‐mode echocardiography of the left ventricular chamber. (C, D) Statistical analysis of ejection fraction (EF) (C) and fractional shortening (FS) (D). Saline group, n = 5; ISO group, n = 7; *P < 0.05, ISO‐vehicle vs. saline‐vehicle. # P < 0.05, ISO‐C188‐9 vs. ISO‐vehicle. Two‐way ANOVA, followed by Tukey's post hoc test. (E, F) Heart weight‐to‐body weight ratio (HW/BW) (E) and heart weight‐to‐tibia length ratio (HW/TL) (F) were assessed. Saline group, n = 5; ISO group, n = 7; *P < 0.05, ISO‐vehicle vs. saline‐vehicle. # P < 0.05, ISO‐C188‐9 vs ISO‐vehicle. Two‐way ANOVA, followed by Tukey's post hoc test. (G, H) qRT‐PCR of ANF (G) and BNP (H) mRNA levels in ISO or/and C188‐9‐treated mouse heart tissues. n = 6. *P < 0.05, ISO‐vehicle vs. saline‐vehicle. # P < 0.05, ISO‐C188‐9 vs. ISO‐vehicle. Two‐way ANOVA, followed…

Fig. 3. C188‐9 treatment suppresses ISO‐induced fibrosis and hypertrophy in mice. (A, B) Representative images (A) and quantification (B) of Masson's trichrome staining in heart sections from saline‐ or ISO‐treated mice. Saline group, n = 4; ISO group, n = 6; *P < 0.05, ISO‐vehicle vs. saline‐vehicle. # P < 0.05, ISO‐C188‐9 vs. ISO‐vehicle. Scale bar: 50 μm. (C–F) qRT‐PCR of Col1a1 (C), Col1a2 (D), Col3a1 (E), and α‐SMA (F) in mouse heart tissues. n = 6. *P < 0.05, ISO‐vehicle vs saline‐vehicle. # P < 0.05, ISO‐C188‐9 vs. ISO‐vehicle. (G, H) Representative images (G) and quantification (H) of WGA staining in heart cross sections from saline‐ or ISO‐treated mice. Saline group, n = 4; ISO group, n = 6; *P < 0.05, ISO‐vehicle vs. saline‐vehicle. # P < 0.05, ISO‐C188‐9 vs. ISO‐vehicle. Scale bar: 50 μm. Two‐way ANOVA, followed by Tukey's post hoc test. Data are presented as mean ± SEM.

Fig. 4. C188‐9 treatment inhibits fibrotic protein expression and STAT3 phosphorylation in cardiac tissues of mice. (A) Western blots of collagen Ⅰ and α‐SMA levels in ISO‐ or saline‐treated mouse heart tissues with or without C188‐9 treatment. GAPDH was used as loading control. (B) Quantification of relative collagen I and α‐SMA levels. n = 6 *P < 0.05, ISO‐vehicle vs. saline‐vehicle. # P < 0.05, ISO‐C188‐9 vs. ISO‐vehicle. Two‐way ANOVA, followed by Tukey's post hoc test. (C) Western blots of p‐STAT3 and total STAT3 levels in ISO‐ or saline‐treated mouse heart tissues with or without C188‐9 treatment (n = 6). (D). Quantification of relative p‐STAT3/STAT3 levels. n = 6 *P < 0.05, ISO‐vehicle vs. saline‐vehicle. # P < 0.05, ISO‐C188‐9 vs. ISO‐vehicle. Two‐way ANOVA, followed by Tukey's post hoc test. Data are presented as mean ± SEM. (E) Proposed model for C188‐9 regulation of fibrosis‐related gene expression in CFs through STAT3 phosphorylation. Data are presented as mean ± SEM.