Mirodenafil ameliorates skin fibrosis in bleomycin-induced mouse model of systemic sclerosis

Abstract

Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by fibrosis of the skin and internal organs. Despite the recent advances in the pathogenesis and treatment of SSc, effective therapies for fibrosis caused by SSc have not yet been established. In this study, we investigated the potential role of mirodenafil, a potent phosphodiesterase 5 (PDE5) inhibitor, in the treatment of fibrosis in SSc. We used a bleomycin (BLM)-induced SSc mouse model to mimic the typical features of fibrosis in human SSc and examined the dermal thickness to assess the degree of skin fibrosis after staining with hematoxylin and eosin or Masson's trichrome stains. The effect of mirodenafil on the expression of profibrotic genes was also analyzed by treating fibroblasts with transforming growth factor (TGF)-β and mirodenafil. We showed that mirodenafil ameliorated dermal fibrosis and downregulated the protein levels of fibrosis markers including COL1A1 and α-SMA in the BLM-induced SSc mouse model. Further, using mouse embryonic fibroblasts and human lung fibroblasts, we demonstrated that the expression of collagen and profibrotic genes was reduced by treatment with mirodenafil. Finally, we showed that mirodenafil inhibited TGF-β-induced phosphorylation of Smad2/3 in fibroblasts, which suggested that this drug may ameliorate fibrosis by suppressing the TGF-β/Smad signaling pathway. Our findings suggest that mirodenafil possesses a therapeutic potential for treating fibrosis in SSc.

Keywords: Fibrosis; cyclic guanosine monophosphate; mirodenafil; systemic sclerosis.

Figure 1.

The summary of the systemic sclerosis experimental model. (A) Eight weeks-old male BALB/c mice were subcutaneously injected with either bleomycin (BLM) (100 µg) or phosphate-buffered saline (PBS) on their shaved back on alternate days for 3 weeks. Mirodenafil (5 or 10 mg/kg/day) or distilled water was orally administered every day for 3 weeks. (B) A visible change in the inner surface of the mouse dorsal skin is shown.

Figure 2.

Mirodenafil ameliorated skin fibrosis in BLM-induced systemic sclerosis (SSc) mouse model. Hematoxylin and eosin staining (A) and Masson’s trichrome staining (B) in normal control (NC), BLM, and mirodenafil groups (The original magnification 40×). (C) The dermal thickness of each group (n=8∼10). Data are presented as mean ± standard deviation (SD). *P < 0.05. ***P < 0.001.

Figure 3.

Mirodenafil downregulated the expression of fibrosis markers in BLM-induced SSc mouse model. The protein levels of COL1A1 and α-SMA in the lesioned skin from each group were measured by western blot. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. (B) Data from western blot were quantified by densitometry. Data are presented as mean ± SD. *P < 0.05.

Figure 4.

The anti-fibrotic effect of mirodenafil in mouse embryonic fibroblasts. NIH3T3 mouse embryonic fibroblasts were treated with the indicated concentration of mirodenafil and mouse transforming growth factor (TGF)-β1 (10 ng/ml) for 24 hr. The protein levels of COL1A1 and α-SMA were analyzed by western blot (A), and mRNA levels of Col1a1, α-SMA, and Ctgf were assessed by quantitative real-time polymerase chain reaction (qPCR) (B). GAPDH was used as loading control for western blot and normalization control for qPCR. Data are presented as mean ± SD. **P < 0.01, ***P < 0.001.

Figure 5.

The anti-fibrotic effect of mirodenafil in human lung fibroblasts. (A) MRC-5 human fetal lung fibroblasts were treated with the indicated concentration of mirodenafil and human TGF-β1 (10 ng/ml) for 24 hr. The protein levels of COL1A1 and α-SMA were analyzed by western blot. (B) Data from western blots were quantified by densitometry. Data are presented as mean ± SD. *P < 0.05, **P < 0.01.

Figure 6.

Mirodenafil inhibited the mRNA expression of the fibrosis marker in human lung fibroblasts. MRC-5 fibroblasts were treated with the indicated concentration of mirodenafil and human TGF-β1 (10 ng/ml) for 24 hr. The mRNA levels of COL1A1, α-SMA, and CTGF were assessed by qPCR. Expression data were normalized to GAPDH and presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7.

Mirodenafil inhibited TGF-β signaling. (A) NIH3T3 fibroblasts were treated with the indicated concentration of mirodenafil and mouse TGF-β1 (10 ng/ml) for 24 hr. The protein levels of p-Smad2/3 and Smad2/3 were analyzed by western blot. Beta-actin was used as loading control. (B) Data from western blots were quantified by densitometry. Data are presented as mean ± SD. *P < 0.05, **P < 0.01.