Abstract
BackgroundIntestinal fibrosis is a serious complication of inflammatory bowel disease, including Crohn’s disease and ulcerative colitis. There is no specific treatment for intestinal fibrosis. Studies have indicated that peroxisome proliferator-activated receptor- γ (PPAR-γ) agonists have anti-fibrogenic properties in organs besides the gut; however, their effects on human intestinal fibrosis are poorly understood. This study investigated the anti-fibrogenic properties and mechanisms of PPAR-γ agonists on human primary intestinal myofibroblasts (HIFs).MethodsHIFs were isolated from normal colonic tissue of patients undergoing resection due to colorectal cancer. HIFs were treated with TGF-β1 and co-incubated with or without one of two synthetic PPAR-γ agonists, troglitazone or rosiglitazone. mRNA and protein expression of procollagen1A1, fibronectin, and α-smooth muscle actin were determined by semiquantitative reverse transcription-polymerase chain reaction and Western blot. LY294002 (Akt inhibitor) was used to examine whether Akt phosphorylation was a downstream mechanism of TGF-β1 induced expression of procollagen1A1, fibronectin, and α-smooth muscle actin in HIFs. The irreversible PPAR-γ antagonist GW9662 was used to investigate whether the effect of PPAR-γ agonists was PPAR-γ dependent.ResultsBoth PPAR-γ agonists reduced the TGF-β1-induced expression of α-smooth muscle actin which was integrated into stress fibers in HIFs, as determined by actin microfilaments fluorescent staining and α-smooth muscle actin-specific immunocytochemistry. PPAR-γ agonists also inhibited TGF-β1-induced mRNA and protein expressions of procollagen1A1, fibronectin, and α-smooth muscle actin. TGF-β1 stimulation increased phosphorylation of downstream signaling molecules Smad2, Akt, and ERK. TGF-β1 induced synthesis of procollagen1A1, fibronectin, and α-smooth muscle actin through a phosphatidylinositol 3-kinase/Akt-dependent mechanism. PPAR-γ agonists down regulated fibrogenesis, as shown by inhibition of Akt and Smad2 phosphorylation. This anti-fibrogenic effect was PPAR-γ independent.ConclusionsTroglitazone and rosiglitazone suppress TGF-β1-induced synthesis of procollagen1A1, fibronectin, and α-smooth muscle actin in HIFs and may be useful in treating intestinal fibrosis.
Highlights
Intestinal fibrosis is a serious complication of inflammatory bowel disease, including Crohn’s disease and ulcerative colitis
Effect of peroxisome proliferator-activated receptor- γ (PPAR-γ) agonists on cell viability in human primary intestinal myofibroblasts To rule out the possibility that the putative effect of PPAR-γ agonists were mediated by cytotoxicity, we performed a cell viability assay after administration of the PPAR-γ agonists (TRG or RSG) in increasing concentrations for 24 h
Effect of PPAR-γ agonists on TGF-β1-induced expression of α-smooth muscle actin (α-SMA) in human primary intestinal myofibroblasts To determine whether PPAR-γ agonists inhibit fibrogenic activation of myofibroblasts, HIFs were treated with an increasing concentration of TRG or RSG and simultaneously stimulated with TGF-β1
Summary
Intestinal fibrosis is a serious complication of inflammatory bowel disease, including Crohn’s disease and ulcerative colitis. Studies have indicated that peroxisome proliferator-activated receptor- γ (PPAR-γ) agonists have anti-fibrogenic properties in organs besides the gut; their effects on human intestinal fibrosis are poorly understood. Intestinal fibrosis is a common complication of inflammatory bowel disease (IBD) occurring in both Crohn’s disease (CD) and ulcerative colitis (UC), but is clinically more apparent in CD [1]. ECM proteins including collagen and fibronectin are synthesized by activated myofibroblasts, which are the key effector cells of intestinal fibrosis [5,6,7]. Incorporation of α-SMA into stress fibers enhances the contractile activity of myofibroblasts [13] leading to the formation of specialized contacts with the ECM [14]
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