Mechanisms Of WNT2b Promotes Intestinal Mucosal Damage In IBD

BackgroundInflammatory bowel disease(IBD)is a chronic non-specific intestinal inflammatory disease,mainly including ulcerative colitis(UC),Crohn’s disease(CD),and unclassified inflammatory bowel disease(IBDU).In recent years,the number of patients with childhood IBD in China has gradually increased,and becomes one of the clinical common digestive tract diseases.Its etiology may be related to the genetic susceptibility population,environment,infection,immune function,mental psychology,dietary allergy and so on.The study of the IBD’s pathogenesis has always been a hotspot and difficulty in digestive system diseases research.The current pathogenesis study mainly revolves around the immune cells.Macrophages are the key gatekeeper of intestinal immune homeostasis.The body is stimulated by external antigen,which activates macrophages to initiate abnormal immune response to secrete inflammatory factors and promote the occurrence of intestinal inflammation.However,the infiltration of IBD inflammation,the loss of mucosal barrier function of intestinal epithelium is one of the important features of pathological changes.The intestinal epithelial mucosal barrier is mainly formed by the intestinal epithelial cells and the connections between the cells,which are composed of desmosomes,tight junction proteins,and adherens junctions.Tight junction proteins are mainly composed of ZO,occludin,and claudin proteins,which directly limit paracellular permeability.The most important component of adherens junctions is the E-cadherin protein,and several studies have shown that E-cadherin protein expression is downregulated in the inflammatory epithelia of IBD patients.The intestinal epithelial mucosal barrier function is currently assessed by detection of tight junction proteins,adhesion protein levels,and transmembrane resistance values.ObjectiveIn these experiments,we explored the mechanism why highly expressed WNT 2B fibroblasts activate macrophages to produce inflammatory factors,disrupt the intestinal mucosal barrier function,lead to intestinal mucosal damage,promote IBD progression,and sought new targets for the treatment of IBD.Methods1.Single-cell sequencing and pathway analysis of IBD.2.Samples were collected from colitis and normal sites of IBD patients,inflammation was assessed by HE staining and histopathological score,and colocalization of macrophage markers CD11b and CXCL12 was performed by tissue immunofluorescence.3.The control group was DMEM culture medium,while the experimental group added SKL2001 agonist of the WNT/β-catenin pathway,the CXCL12 expression was determined by qPCR,ELISA.4.The expression levels of β-catenin,GSK 3 β,and P-GSK 3 β were determined using western blot.5.A model of the action of fibroblasts on Caco-2 cells was constructed by in vitro cell co-culture and conditional culture.The experimental groups included 20%fibroblast conditioned medium or co-culture with WNT 2B highly expressing fibroblasts,and the control groups were common conditioned medium or with wild-type fibroblasts,to assess the change of Caco-2 cell barrier permeability by determining the transmembrane resistance and fluorescent yellow permeability.6.After co-culture with high WNT 2B expression intestinal fibroblasts,we use immunofluorescence to measure the β-catenin entry of Caco-2 cells.7.To examine the expression of tight junction proteins such as ZO-1 and E-Cadherin by Western blot assay.8.C57 mice were treated with DSS(dextran sulfate)to establish IBD-like enteritis.The experimental group used Salinomycin(5mg/kg,intraperitoneal injection)while the control group with corresponding solvent saline to evaluate the therapeutic effect of the inhibitor on enteritis.Results1.Macrophages were enriched in colitis tissues from IBD patients.2.The macrophage markers CD11b and CXCL12 are colocalized,and the macrophages can secrete CXCL12.3.SKL2001 is an agonist of the WNT/β-catenin pathway,promoting the infiltration of macrophages and CXCL12 secretion in colitis,thus aggravating intestinal inflammation.4.When WNT 2B was overexpressed,GSK 3 β phosphorylation was enhanced,indicating the activation of the WNT/β-catenin pathway.5.Compared with control group,overexpression of WNT2B gene could cause a significant increase in secretion of WNT 2b protein by fibroblasts and promote β-catenin entry into the nuclear in Caco-2 cells(P-value 0.01),and decrease in expression of tight junction protein.6.This effect was reversed by knockdown of FZD4 expression in Caco-2 cells,with reduced β-catenin nuclear entry and upregulation of tight junction proteins.7.The addition of 20%fibroblast conditioned medium or co-culture group with WNT 2B highly expressing fibroblasts significantly reduced the transmembrane resistance and fluorescent yellow permeability values,indicating the increased barrier permeability of Caco-2 cells.8.The level expression of tight junction proteins,including ZO-1 and E-Cadherin,was enhanced in the overexpressed WNT 2b group compared with the control group.9.IBD enteritis model was constructed,and both HE and inflammation scores indicated significant inflammatory infiltration in the intestinal tract of mice.10.Salinomycin can significantly reduce the intestinal inflammation in DSS mice and increase the expression of tight junction proteins in the intestinal mucosa.conclusionHigh expression of WNT2B activates macrophages to promote CXCL12 expression and secretion and destroys intestinal mucosal barrier function by WNT/β-catenin signaling pathway,which may lead to the development of IBD inflammation and have important practical significance to find new targets for the treatment of IBD.