Effects Of Constitutive TL1A Expression On Intestinal Epithelial Barrierin DSS-induced Colitis And Effection Of ASIV

Inflammatory bowel disease(IBD)describes conditions with idiopathic,chronic,relapsing and remitting inflammation of the gastrointestinal tract.Crohn’s disease(CD)and ulcerative colitis(UC)are the two most common types of IBD,the incidence of IBD continues to rise steadily.The pathogenesis of IBD remains incompletely understood.Genetic,infectious and other environmental factors may play a role in the dysregulation of intestinal immunity,leading to gastrointestinal injury.This barrier separates harmful luminal substances such as microorganisms,toxins and antigens from the body and thus plays a critical role inmucosal homeostasis.Intestinal epithelial barrier damage is an important pathological change of IBD,the proinflammatory cytokines released can not only activate the invasion of immune cells to inhibit microorganism,but also act on intestinal mucosal barrier,which affect the expression,distribution and composition of tight junction,and affect the function of intestinal barrier.Integrity of the intestinal epithelial barrier depends on a complex network of intercellular tight junctions(TJs)and cytoskeletal structures,and disruption of this network results in impaired barrier function.In inflammatory bowel disease,TNF-αis associated with intestinal TJs damage and cytoskeletal rearrangements,which lead to increased intestinal permeability and promote the release of other inflammatory factors,such as IL-18 and IL-17.Both the myosin light chain kinase(MLCK)/p-MLC pathway and nuclear factor(NF)-κB activation have key roles in TNF-α-mediated damage to the intestinal barrier.Activation of the MLCK/p-MLC pathway leads to downregulation of the expression of TJ proteins,alterations in cell adhesion and migration,and cytoskeletal rearrangements.It was found that the expression of TLR in the intestinal epithelial cells of CD and UC increased,and the exposure to flagellin could increase the expression of TNF-αto further damage the intestinal epithelial barrier function.The tumor necrosis factor-like ligand 1 aberrance(TL1A)is a protein product of TNF super family member 15(TNFSF15),which modulates the immune reponse as costimulatory factor of T lymphocyte activation.The latest researches have proved that variants in the TNFSF15 gene are associated with IBD,and TL1A is elevated in the intestinal mucosa of IBD patients.Many studies have shown that TL1A is the main regulator of mucosal immunity,which connects innate immunity and adaptive immunity,promotes the production of inflammatory factors.DC TL1A transgenic mice ileum developed striking goblet cell hyperplasia that was associated with the small intestine interleukin(IL)-13 levels elevated.The cytokines IL-15 or the DR3ligand tumor necrosis factor(TNF)-like cytokine 1A(TL1a)induced mucosal tissues memory IL-18Rα~+DR3~+CD4~+T cells to produce interferon-γ,TNF-α,IL-6,IL-5,IL-13,GM-CSF and IL-22 in the presence of IL-12/IL-18.Otherwise,it’s not clear that TL1A how to influnce the intestinal epithelial barrier.Wild-type(WT)and Myeloid Tl1a-Tg(Tg)C57BL/6 mice were administrated with DSS for 4 weeks to establish the chronic experimental colitis model.Caco-2 cell lines was to establish intestinal epithelial barrier model in vitro.Part one Effects of constitutive TL1A expression on intestinal epithelial barrier in DSS-induced colitisObjective:To investigate the damage of intestinal mucosal barrier by TL1A.Methods:Evaluate the changes of inflammation and the permeability of intestinal mucosal barrier.Intestinal mucosal damage was observed with transmission electron microscopy.Results:Significant increased DAI and reduced BW were observed in DSS/Tg group mice as compared to DSS/WT group mice.The changes of mucous membrane permeability changes in chronic colonic experimental colitis was detected.Through transmission electron microscopy(TEM)we found that DSS/Tg tight junctions and colon microvilliin mucosal epithelia disruption were more obviously.Conclusions:TL1A impaired intestinal epithelial barrier in chronic experimental colitis.Part two The damage mechanisms of constitutive TL1A expression on intestinal epithelial barrier in DSS-induced colitis in vivo and in vitro.Objective:To investigate the damage mechanisms of intestinal mucosal barrier by TL1A in vivo and in vitro.Methods:Evaluate the expression of TL1A,TJ,p-MLC,MyD88,TRAF6 through RT Q-PCR and western blot,TNF-αwere measured by RT Q-PCR.Evaluate the permeability of Caco-2 cells monolayers barrier.The damage was observed with transmission electron microscopy.Evaluate the expression of TJ,p-MLC,MyD88,TRAF6 through RT Q-PCR and western blot.Results:Compared with DSS/WT mice;DSS/Tg group mice intestinal tissue TNF-αmRNA increased significantly;Occludin,JAMA,ZO-1 and TL1A expression significantly reduced,p-MLC,MyD88,TRAF6 increased significantly.Caco2+LV-TNFSF15+TNF-α,Caco2+LV-TNFSF15+LPS sepe-rated compared with Caco2+TNF-α,Caco2+LPS,Caco-2 cells monolayers barrier permeability increased significantly,TJ expression significantly reduced,p-MLC,MyD88,TRAF6 increased significantly.Conclusions:TL1A regulated TJ expression may via MLCK/p-MLC pathway and LPS mediated MyD88/TRAF6 pathway.Part three Effects of ASIV on intestinal epithelial barrier in constitutive TL1A expression DSS-induced colitis.Objective:To investigate the protection of ASIV on intestinal mucosal barrier in constitutive TL1A expression DSS-induced colitis.Methods:Evaluate the changes of inflammation and the permeability of intestinal mucosal barrier.Intestinal mucosal damage was observed with transmission electron microscopy.TJ,p-MLC,MyD88 and TRAF6expressions were dected in vivo and in vitro.Results:Compared with control group,intestinal epithelial barrier permeability increased significantly,TJ expression significantly increased,p-MLC,MyD88,TRAF6 decreased significantly in vivo and in vitro.Conclusions:ASIV repaired intestinal epithelial barrier via intestinal epithelial barrier MLCK/p-MLC and MyD88/TRAF6 pathway in TL1A-associated experimental colitis.