Effect And Mechanism Of TRPV4 Regulating Colonic Epithelial Barrier Function

Background Ulcerative colitis(UC)is a gastrointestinal disorder characterized by an inflammatory response and mucosal damage.Uncontrolled inflammation disrupts the epithelial line and results in mucosal ulceration in the bowel wall.Previous investigations have demonstrated that a reduction of tight junction(TJ)strands is possibly the cause of barrier dysfunction in UC.The disrupted morphology of TJ is often the result of changes in TJ protein expression.However,these studies were unable to determine whether the downregulation of Claudins drives or is a consequence of colitis.Researchers have demonstrated that the phosphorylation of TJ proteins causes allosterism,endocytosis and changes in the polarity of the epithelium,thus affecting the barrier function.Claudins are also subject to regulation by post-translational modifications,including phosphorylation.Modest phosphorylation of certain groups in Claudins is necessary for the maintenance of their function.However,abnormal phosphorylation of Claudins will change the manner in which they combine with each other and thus affect the aggregation and structural stability of TJs,which leads to changes in trans-epithelial resistance and epithelial permeability and ultimately affects the epithelial barrier function.Lipopolysaccharide(LPS)is an activator for immune cells,such as macrophages.LPS can induce acute lung injury,followed by the downregulation of Claudin-4.In cholangiocyte monolayers,LPS induces the redistribution of Claudin-1 and Claudin-4.To date,few studies have demonstrated the effect of LPS on Claudin phosphorylation in intestinal epithelial cells.In the present study,we first assessed the phosphorylation of colonic Claudins in DSS induced colitis in rats.Morphological examination,intestinal permeability studies and serum C-reactive protein analyses were also performed.Second,we assessed the effect of LPS on the phosphorylation of Claudins in T84 colonic cells.Objective 1.To investigate the phosphorylated status of Claudins in experimental colitis rats.2.Study the effect of LPS on phosphorylated Claudins in T84 cells.Method 1.Experimental colitis Female SD rats were randomly divided into four groups: one control and three experimental colitis groups which has six rats in each group.The control rats received drinking water for seven days and the experimental colitis rats were given 4 % DSS in drinking water for four,six and eight days.Six rats of each group were studied at each time point.Samples of the blood and colon from each group were collected,and intestinal segments from the ileocecal valve to the anus(5-6 cm in length)were collected for subsequent assays.2.ELISA Use ELISA method measure C-reactive protein(CRP)in rat serum.3.immunostaining Use immunostaining to detect the localization and expression of phosphorylation Claudins in experimental colitis rats.4.Western blotting Western Blotting was used to detect phosphorylation level of Claudins in experimental colitis rats.In vitro,use western blotting to detect the expression of Claudins and phosphorylated Claudins in T84 cells.Result 1.The colonic damage degree of the rat administered DSS was ignificantly higher than that of the control group.With DSS stimulation time increasing,the degree of colonic injury increased and colonic mucosal permeability increased gradually.2.As inflammatory cytokines,the levels of serum CRP were increased at days 4 then decreased at days 6 and 8 after DSS administration stimulation.3.Phosphorylated Claudin-3 and phosphorylated Claudin-6 immunostaining predominantly distributed in the colonic epithelium at lateral crypts and the luminal colonic epithelium.Phosphorylated Claudin-5 was distributed in the colonic epithelium at the tip and later of crypts and the luminal colonic epithelium.Phosphorylated Claudin-7 was distributed in the colonic epithelium at the tip of crypts and in the luminal colonic epithelium.4.Compared with the control rats,phosphorylated Claudin-4,Claudin-7 in colon were increased at days 6 and 8 in the rats administered DSS.Colonic phosphorylated Claudin-6 was increased at days 4 and 8.Phosphorylated Claudin-5 was slightly decreased at day 4 in the rats administered DSS but markedly increased at day 8.5.Compared with the control group,the level of phosphorylated Claudin-3 was increased at 48 h but decreased at 72 h.The level of phosphorylated Claudin-6 remained was increased and phosphorylated Claudin-5 was decreased at 72 h.These were in accordance with the results form in vivo study.Conclusion 1.The colonic mucosal permeability gradually increased in course of DSS induced colitis rats,and the phosphorylation status of Claudins was generally increased,phosphorylation Claudin-5 was decreased in severe colitis.2.The phosphorylated status of Claudins was increased on colonic epithelial cells administered LPS,but decreased with over stimulation by inflammatory cytokines.Background UC,ulcerative colitis,a chronic non-specific colonic inflammation,is characterized by repeated abdominal pain,diarrhea,mucus blood and pus,Clinically.Pathological damage is mainly located in the mucosa and submucosa,with unknown pathogenesis.Colonic epithelial barrier is the primary physiological defense of the intestine,dysfunction leading to the occurrence aggravation of intestine inflammation.TJs,Tight junctions,create a physiological intercellular barrier to regulate the paracellular permeability of various solutes and restrict the uncontrolled entry of luminal antigens.Claudins interact in a tissue-specificity manner to play an important role in the maintenance of the epithelial barrier function.Modest phosphorylation of certain groups in Claudins is necessary for the maintenance of the barrier function.However,abnormal phosphorylation of Claudins will change the manner in which they combine with each other and thus affects the aggregation and structural stability of TJs,which leads to changes in trans-epithelial resistance and epithelial permeability and ultimately affects the epithelial barrier function.TRPV4 is one of the most important members of the transient receptor potential vanilloid(TRPV),which is a non-selective calcium channel.Regarded as a cell receptor,TRPV4 expressed widely in the gastrointestinal tract and be activated easily by factors to regulate the permeability of epithelial cells.Therefore,this study attempts to observe the expression of TRPV4 in DSS colitis and explore the effect of TRPV4 channel on colonic epithelial barrier function and the phosphorylated status and expression of Claudin-7.Objective 1.To observe the expression of TRPV4 in experimental mice,and to investigate the effects of TRPV4 on colonic epithelial barrier function.2.To observe the association between TRPV4 and claudins,and the phosphorylated status and location of Claudin-7 by regulating TRPV4 channel,thus to investigate primarily the effect and mechanism of TRPV4 regulating colonic barrier function.Method 1.Preparation of colitis mice Female C57 BL / 6 mice were randomly divided into four groups: one control and three experimental colitis groups which has six rats in each group.The control group received drinking water for seven days and the experimental colitis mice were given 4 % DSS in drinking water for seven days.The experimental colitis mice were randomly gavage administrated DMSO,TRPV4 agonist and TRPV4 antagonist from 3rd day to 7th day.Samples of the blood and colon from each group were collected,and intestinal segments from the ileocecal valve to the anus(5-6 cm in length)were collected for subsequent assays.2.immunostaining Use immunostaining to detect the localization and expression of TRPV4,Claudin-7 and phosphorylated Claudin-7 in experimental mice colon.3.Western blotting Western blotting was used to detect phosphorylation level of Claudin-7 in TRPV4 channel agonist/antagonist administrated NCM460 cells and the expression of TRPV4 in experimental mice.Co-immunoprecipitation to examine the association between TRPV4 and claudins.5.Wild-type Claudin-7 stable transfection NCM460 cells were stable transfected with wild-type Claudin-7 plasmid.The location of Claudin-7 on the cells membrane was observed after TRPV4 agonist treatment and antagonist Pretreatment by confocal microscopy.6.Intracellular calcium concentration measurement NCM460 cells were stimulated by TRPV4 channel agonist/antagonist.Use Calcium imaging fluorescence microscope system to detect changes of intracellular Ca2+ concentration.7.The permeability of the colonic The levels of FD4 in serum were measured,which reflected the damage of colonic epithelial barrier function.8.Cell wound scratch assay The wound scratch assay of NCM460 cells monolayer after TRPV4 agonist/antagonist administrated was used,which reflected the function of the colonic epithelial monolayer repair.4.Co-immunoprecipitationResult 1.Compared with the control,the level of TRPV4 expression was downregulated in colitis model group and TRPV4 antagonist intervention group.compared with colitis mice,the level of TRPV4 expression in colitis mice administrated TRPV4 agonist significantly increased.2.The interaction between TRPV4 and Claudin-4,Claudin-5,Claudin-7 and Claudin-8 in colonic epithelial cells was detected.3.TRPV4 agonist increases the phosphorylation status of Claudin-7,and TRPV4 antagonist decreases the phosphorylation status of Claudin-7.4.TRPV4 agonist induces Claudin-7 to accumulate on the cell membrane,and TRPV4 antagonist pretreatment reduces the aggregation of Claudin-7 on the cell membrane.5.TRPV4 agonist increased the influx of calcium in NCM460 cells.the effect of agonist was significantly inhibited with TRPV4 antagonist pretreatment.6.Compared with the control,the colonic permeability in colitis model group and TRPV4 antagonist intervention group were significantly higher;compared with colitis mice,the mucosal permeability in colitis mice administrated TRPV4 agonist significantly decreased.TRPV4 agonist slow the progression of bloody stool.7.The result of the cell wound scratch assay TRPV4 antagonist decreased the function of the colonic epithelial monolayer repair.Conclusion 1.TRPV4 agonist would reduce the epithelial barrier dysfunction in DSS colitis.2.TRPV4-Claudin-7 change the phosphorylation status and the aggregation of Claudin-7 on the cell membrane,to regulate the permeability of colonic epithelial cells,thus affects the epithelial barrier function.