The Effect And Mechanism Of Metformin Ameliorating Chronic Colitis-Related Intestinal Fibrosis

Background and Aims:Crohn's disease(CD)is a chronic inflammatory disease that can affect any parts of the gastrointestinal tract,it is one of the main clinical forms of Inflammatory bowel disease(IBD).Since its precise aetiology remains unclear,it is hard to fully control the development of CD in clinic,causing CD patients usually progress to various severe complications such as intestinal fibrosis and seriously threatening human health.Enterectomy,Strictureplasty and Endoscopic Balloon Dilation are the currently available therapies for CD-associated strictures and obstructions,while with 70-90%recurrence within one year unfortunately.Thus,alternative strategies targeting intestinal fibrosis have obtained substantial interests.Metformin has been proved to have satisfactory anti-inflammatory effects in ulcerative colitis(UC)patients.Whether metformin can ameliorate chronic colitis-related intestinal fibrosis and the possible mechanisms remain unclear.Methods:Colitis-related intestinal fibrosis was established in mice by repetitive administration of TNBS or DSS.Meanwhile,the mice were given metformin(200mg/kg/d)by gavage.HE staining,hydroxyproline content determination,Sirius red staining,Western blot,immunohistochemical and immunofluorescence experiments were performed to investigate the preventive effect of metformin on intestinal fibrosis induced by TNBS or DSS in mice.Mice with established intestinal fibrosis were given metformin(200mg/kg/d)to explore whether metformin could reverse established intestinal fibrosis.At the cellular level,to explore effects of metformin on activation,collagen production,proliferation and migration of fibroblasts,metformin(0.1 or 0.5mM)was added to TGF-?1-treated human colonic fibroblast cell line(CCD-18Co)and primary intestinal fibroblasts.And Western blot,immunofluorescence,EdU incorporation and Transwell assay were conducted.Coimmunoprecipitation,siRNA,isolated extraction of cytoplasmic and nuclear protein and Immunofluorescence staining were used to explore underlying mechanisms.Results:Preventive administration of metformin to chronic TNBS or DSS colitis mice indicated that metformin significantly attenuated intestinal fibrosis by reducing mucosal inflammation and submucosal collagen deposition,suppressing the activation of fibroblast,and decreasing expression of fibrosis markers and key proteins of the TGF-?1/Smad3 pathway.Metformin treatment reversed established TNBS-and DSS-induced intestinal fibrosis in mice,presenting with reduced mucosal inflammation,submucosal collagen fiber deposition and fibroblast activation,and decreased expression of fibrosis markers and key proteins of the TGF-?1/Smad3 pathway.In vitro studies with human colon fibroblast cell(CCD18Co)and primary intestinal fibroblast treated with TGF-?1 confirmed the antifibrotic function of metformin for fibroblast activation,proliferation,migration and collagen production.Mechanistically,metformin particularly inhibited phosphorylation and nuclear translocation of Smad3 by blocking the interaction of Smad3 with T?RI.And we also found that the anti-fibrotic functions of metformin might be independent of AMPK activation.Conclusion:Metformin protected against intestinal fibrosis induced by TNBS or DSS in vivo and inhibited activation and collagen synthesis in colon fibroblasts.The underlying mechanisms may be associated with the inhibition of TGF-?1/Smad3 pathway.These findings provide the evidence of the application of metformin as a promising new therapeutic medicine for intestinal fibrosis.