The Anti-fibrotic Effect And Mechanism Of Adiponectin On Intestinal Fibrosis In Crohn’s Disease

BackgroundCrohn’s disease(CD)is an inflammatory disease of the gastrointestinal tract with unknown etiology,which often recurs and is difficult to cure.Intestinal stenosis and obstruction caused by intestinal fibrosis is a common complication that causes patients with CD to require surgical treatment.Surgery can only relieve the symptoms of intestinal stenosis,but cannot prevent the recurrence and progression of fibrosis.Mesenteric thickening and creeping fat are one of the characteristic manifestations of CD.The mesenteric thickening and intestinal fibrosis often show a one-to-one relationship.Adipocytes in the mesentery are closely related to intestinal fibrosis,muscle layer thickening and intestinal stenosis,but the specific role and mechanism are still unclear.Adipocytes in the mesentery have endocrine and immune regulation functions,and a large amount of adipokines secreted by them are important factor in regulating intestinal function.Among the adipokines,adiponectin is an important molecule with multiple functions,which has been confirmed to be involved in the occurrence and development of intestinal inflammation,but its effect on CD-related intestinal fibrosis has not yet been reported.Objective1.Analyze the relationship between the expression of adiponectin in different intestinal tissues or corresponding mesentery and intestinal fibrosis in patients with CD;2.Verify the therapeutic effect of exogenous adiponectin on CD-related intestinal fibrosis;3.To explore the potential mechanism of adiponectin influencing CD-related intestinal fibrosis.MethodsSurgical specimens from patients with CD intestinal stenosis were prospectively collected.Intestinal wall and its corresponding mesenteric tissue from the stenosis site and the relatively normal intestinal wall and its corresponding mesenteric tissue from the same patient were respectively collected.The intestinal specimen was embedded in paraffin and made into sections.The intestinal structure difference and fibrin deposition of the stenotic and non-stenotic intestines were observed by hematoxylin-eosin(H&E)and Masson trichrome staining.The expression of adiponectin and collagen I in the stenosis and non-stenosis intestine,hypertrophy and normal mesenteric was observed by immunohistochemistry(IHC).Real-time polymerase chain reaction(PCR)and western blotting(WB)were used to verify the expression of adiponectin and collagen I in the stenotic and non-stenotic intestines and the corresponding mesentery.A total of 36 female 8-week-old Balb/c mice without specific pathogens were randomly divided into control group,modeling group and treatment group.The mice in the model and treatment groups were administered increasing concentrations of2,4,6-trinitrobenzene sulfonic acid(TNBS)via enema throughout the 7-week study period.Mice in the treatment group were intraperitoneally administered recombinant full-length adiponectin at the third day after the last TNBS enema.One week after the intraperitoneal injection,the mouse enteroscopy was performed to observe the inflammation and stenosis of the intestines,and scored under the endoscopy.Then take the colon tissue,record the length of the colon and the weight of the last 7 cm.H&E and Masson trichrome staining method was used to observe the intestinal muscular layer thickness and fibrin deposition of mice in each group,and the fibrosis score was performed.IHC,PCR and WB were used to compare the expression of adiponectin and collagen I in the intestines of mice in each group,and detect the activity of myeloperoxidase and the expression of inflammatory factors.The fibroblasts of the patient’s intestinal specimens were extracted for primary culture.Flow cytometry was used to detect cell surface markers,and immunofluorescence was used to detect the expression of vimentin and smooth muscle actin in cells.Using transforming growth factor(TGF)-β1 to stimulate primary fibroblasts to construct a fibrotic cell model.PCR was used to detect the expression of vimentin and smooth muscle actin to observe the effect of adiponectin co-culture on fibrotic cell models.WB was used to detect the changes in signal pathways in primary fibroblasts affected by adiponectin intervention.Use chemical pathway inhibitors to reversely verify the signaling pathways affected by adiponectin.Results1.Compared with the normal mesentery,the adipocytes were smaller,more numerous and denser in the hypertrophic mesentery corresponding to the narrow intestine.2.The basic structure of the narrow intestine was destroyed,losed the normal hierarchical structure,and presented a scarred state with obvious deposition of fibrotic protein and collagen.3.The expression of adiponectin in the hypertrophic mesenteric was significantly higher than that in the normal mesenteric.The expression of adiponectin in the stenosis intestine was higher than that in the normal intestine,but this increase was not statistically different.4.Exogenous infusion of adiponectin can significantly reduce the intestinal inflammation and fibrosis changes in fibrosis model mice induced by TNBS.5.Adiponectin co-culture can inhibit the transformation of fibroblasts to myofibroblasts induced by TGF-β1.6.Adiponectin can inhibit the deposition of collagen by inhibiting the phosphorylation of Smad2 protein induced by TGF-β1,and this effect can be blocked by the pathway inhibitor of adenosine monophosphate-activated protein kinase(AMPK).ConclusionsAdiponectin can protect against intestinal fibrosis by enhancing AMPK phosphorylation and inhibiting the activity of the TGF-β1/Smad signaling pathway,thereby achieving the effect of preventing and treating fibrosis.Adiponectin is expected to become a new type of drug for treating CD-related intestinal fibrosis.