Pirfenidone suppresses TGF-β1-induced human intestinal fibroblasts activities by regulating proliferation and apoptosis via the inhibition of the Smad and PI3K/AKT signaling pathway

Abstract

Intestinal fibroblasts, the main effector cells of intestinal fibrosis, are considered to be a good target for anti-fibrotic therapy. The aim of the present study was to examine the effects of pirfenidone (PFD) on human intestinal fibroblasts (HIFs) stimulated by transforming growth factor (TGF)-β1 and to explore the potential mechanism. Prior to stimulation with TGF-β1 (10 ng/ml), HIFs were treated with or without PFD (1 mg/ml). Cell proliferation was determined by Cell Counting Kit (CCK)-8 and colony formation assays, and cell apoptosis was assessed using flow cytometry and a TUNEL assay. Reverse transcription-quantitative polymerase chain reaction and western blotting were performed to evaluate the mRNA and protein expressions of α-smooth muscle actin (α-SMA), collagen I and fibronectin. The protein expression of TGF-β1/mothers against decapentaplegic homolog (Smad) and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathways was evaluated by western blotting. CCK-8 and colony formation assays demonstrated that PFD significantly inhibited cell proliferation in HIFs stimulated with TGF-β1. Flow cytometry and TUNEL assays revealed that PFD treatment significantly enhanced apoptosis in TGF-β1-stimulated HIFs. In addition, PFD markedly reduced TGF-β1-induced HIF activities, such as myofibroblast differentiation (α-SMA), and collagen production (collagen I and fibronectin). These effects of PFD were mediated by the inhibition of the TGF-β1/Smad and PI3K/AKT signaling pathways. Therefore, the present study demonstrated that PFD reduced TGF-β1-induced fibrogenic activities of HIFs, suggesting that PFD may be a potential therapeutic agent for intestinal fibrosis.