The Role Of Fibroblasts Er Stress In The Development Of Ibd-related Intestinal Fibrosis And Cell-type-specific Reshaping

Inflammatory bowel disease(IBD)is an inflammatory lesion of the intestine,which primarily involves in ileum,ileocecal region,colon and the rectum.The patients have no specific clinical manifestations,but usually diarrhea,abdominal pain,bloody stools,and some patients have symptoms of intestinal obstruction.IBD includes ulcerative colitis(UC)and Crohn's disease(CD).For UC,the intestinal walls are chronically continuous inflamed and the inflammation is confined to the mucosal layer and sub-mucosal layer.Lesions usually start on the proctosigmoid region,and spreads to the entire colon for severe cases.For CD,inflammation can affect the whole digestive tract,with all layers of the intestinal wall are involved.Ileocecum and terminal ileum are the mostly involved.Intestinal fibrosis is one of the most severe complications for IBD,and its related bowel stricture is the main indication for operation.It is characterized by the excessive synthesis and deposition of the extracellular matrix(ECM)in the intestinal wall and once started,it is not reversible.Preventing the fibrosis process could improve the prognosis and decrease the operation rate of IBD.As the main effector cells of the intestinal fibrosis,fibroblasts often involved in the healing of inflammation.But the over-growth of fibroblasts in injury site and the imbalance between synthesis and degradation of ECM are the pathologic basis of abnormal intestinal fibrosis formation.In recent years,accumulating evidence proved that endoplasmic reticulum stress(ERS)played an important role in cell proliferation,differentiation and apoptosis.Continuous ERS seemed to be associated with fibrosis of multiple tissues,such as liver,lung,kidney and blood vessels.But the role of myofibroblast ERS in the formation of intestinal fibrosis was largely unknown.In this study,we established an IBD mouse model by 2,4,6-Trinitrobenzenesulfonic acid(TNBS),and also an in vitro model of ERS in fibroblasts by tunicamycin(TM).They are summarized in details as follows:Part 1 The expression of ERS-related protein in myofibroblasts of TNBS-induced fibrosis mice modelObjective:To explore the role of the sustained ERS in intestinal fibrosis of IBD-related TNBS mice model.Methods:C57BL/6 mice were randomly assigned into three groups:the control group(C group),the TNBS group(T group)and the TNBS+TUDCA group(TT group),with 18 mices in each group.The C group received the saline as control;the T group received TNBS enema once a week;and the TT group received TNBS enema and oral TUDCA.Results:TNBS caused intestinal fibrosis in mouse models,the ERS proteins are significantly increased,and TUCDA can ameliorate intestinal fibrosis.Conclusions:TUDCA can decrese intestinal fibrosis caused by TNBS.Sustained ERS is one of the mechanisms of intestinal fibrosis caused by TNBS.Part 2 Phenotypic changes of myofibroblast safter TM stimulation and the regulation effect of TUDCAObjective:This in vitro experiment was to explore the role of the sustained ERS in the phenotypic changes of the fibroblast after TM stimulation.Methods:Six-week-old mices(C57BL/6)was used for the separation and extraction of myofibroblasts.Using the adherenence culture technique,the colonic myofibroblasts were cultured in vitro.Then,ERS was induced by tunicamycin(TM).The cells were divided into three groups:the control group(C group),the TM group,the TUDCA group and the TM+TUDCA group,and the optimal dose of TM were determined by CCK-8 test.Results:In the in vitro ERS model of myofibroblast,the expressions of a-SMA protein in TM group were significantly increased,and reducing ERS can significantly decrease the expressions of a-SMA protein.Conclusions:With the involvement of ERS,differentiation of myofibroblast into fibroblast is an important mechanism of intestinal fibrosis,and TUDCA could ameliorate intestinal fibrosis.