Pirfenidone Inhibits Cell Proliferation and Collagen I Production of Primary Human Intestinal Fibroblasts

Abstract

Intestinal fibrosis is a common complication of inflammatory bowel disease. So far, there is no safe and effective drug for intestinal fibrosis. Pirfenidone is an anti-fibrotic compound available for the treatment of idiopathic pulmonary fibrosis. Here, we explored the anti-proliferative and anti-fibrotic properties of pirfenidone on primary human intestinal fibroblasts (p-hIFs). p-hIFs were cultured in the absence and presence of pirfenidone. Cell proliferation was measured by a real-time cell analyzer (xCELLigence) and BrdU incorporation. Cell motility was monitored by live cell imaging. Cytotoxicity and cell viability were analyzed by Sytox green, Caspase-3 and Water Soluble Tetrazolium Salt-1 (WST-1) assays. Gene expression of fibrosis markers was determined by quantitative reverse transcription PCR (RT-qPCR). The mammalian target of rapamycin (mTOR) signaling was analyzed by Western blotting and type I collagen protein expression additionally by immunofluorescence microscopy. Pirfenidone dose-dependently inhibited p-hIF proliferation and motility, without inducing cell death. Pirfenidone suppressed mRNA levels of genes that contribute to extracellular matrix production, as well as basal and TGF-β1-induced collagen I protein production, which was associated with inhibition of the rapamycin-sensitive mTOR/p70S6K pathway in p-hIFs. Thus, pirfenidone inhibits the proliferation of intestinal fibroblasts and suppresses collagen I production through the TGF-β1/mTOR/p70S6K signaling pathway, which might be a novel and safe anti-fibrotic strategy to treat intestinal fibrosis.

A Human Cellular Model for Colorectal Anastomotic Repair: The Effect of Localization and Transforming Growth Factor-β1 Treatment on Collagen Deposition and Biomarkers

“…We applied an explant procedure to obtain primary fibroblasts [ 40 ]. This method has been used previously to isolate colonic and rectal fibroblasts [ 31 , 41 , 42 ].…”

“…Fibroblasts are key cells in the production of type I, III, and VI collagens [ 25 ]. Growth factors, and transforming growth factor (TGF)-β1 in particular, are important mediators of ECM formation and wound healing [ 5 , 15 , 25 , 26 , 27 , 28 , 29 , 30 , 31 ]. Notably, increased TGF-β1 expression coincides with COL1A1 mRNA levels in an experimental model of anastomotic wound healing [ 32 ].…”

Expert Perspectives On Clinical Challenges: Expert Perspectives: Challenges in Scleroderma

“…Pirfenidone. Pirfenidone is an antifibrotic agent approved by the FDA for the treatment of IPF, and although its mechanism of action is not completely understood, it appears to target transforming growth factor β signaling, a key feature in the pathogenesis of fibrosis (67,68). An open-label phase II study of pirfenidone (69) showed an acceptable tolerability profile, especially with a longer titration schedule, despite its well-recognized gastrointestinal side effect profile.…”

Pirfenidone Modulates Macrophage Polarization and Ameliorates Radiation-induced Lung Fibrosis by Inhibiting the TGF-β1/Smad3 Pathway

“…The anti brotic effect of PFD was rst established in a bleomycin-induced IPF animal model in 1995 [10] . Later, it was shown that PFD could inhibit brosis by downregulating the expression of brogenic growth factors and inhibit the production and release of in ammatory cytokines [11][12][13] . PFD can also reduce the occurrence of lipid peroxidation and oxidative stress injury [14][15] .…”