The Role And Mechanism Of Orai1 Channel In The Development Of Intestinal Epithelial Barrier Dysfunction In Experimental Colitis Model

Objective:Ulcerative colitis(UC)is an inflammatory bowel disease that is prone to relapse,and its incidence rate increases every year.The purpose of this study was to investigate the role of Orai1 channel in the impairment of intestinal epithelial barrier function in experimental colitis.Methods:We used the TNF-α-induced NCM460 cell inflammation model and detected the expression of relevant proteins such as Orai1,Orai2,Orai3,PKD2,Claudin-1,Claudin-4,Claudin-7 by western blot.The interaction between Orai1 and Claudin-1,Claudin-4,Claudin-7 was detected by immunoprecipitation.Immunofluorescence was used to colocate Orai1 with Claudin-1,Claudin-4,and Claudin-7.The cell scratch test was used to test the migration ability of NCM460 cells.The gap permeability of epithelial cells was detected by fluorescein permeation assay.Change of intracellular calcium ion concentration detected by calcium ion image experiment.The experimental colitis model was established using DSS,and the expressions of Orai1,Claudin-1,Claudin-4,Claudin-7 and other related proteins were detected by western blot.Tunel staining was used to detect the apoptosis of colonic epithelial cells.The histological localization and expression of Orai1,Claudin-1,Claudin-4,Claudin-7 and other related proteins were detected by immunohistochemistry.HE staining was used to detect the degree of histopathological damage of his colon.Results:In the NCM460 cell inflammation model induced by TNF-α,the expression levels of Orai1,Orai2 and PKD2 proteins were increased with the increase of calcium ion concentration,while the expression levels of Orai3,Claudin-1,Claudin-4 and Claudin-7proteins were decreased.There was interaction and co-localization between Orai1 protein and Claudin-1,Claudin-4,and Claudin-7 proteins.Orai1 inhibitors can weaken cell migration ability,and reduce cell gap permeability.In the DSS-induced experimental colitis mouse model,compared with the control group,the model group mice lost significantly weight,bloody feces appeared,and fluorescein permeability was significantly increased,indicating that its mucosal barrier might be damaged.HE staining showed that its mucosa was indeed damaged,and the apoptosis of colonic epithelial cells was increased,as well as the expression of Claudin-1,Claudin-4 and Claudin-7 proteins.The phosphorylation levels of Claudin-1,Claudin-4 and Claudin-7 were increased,while in the Orai1 inhibitor treatment group,the mice lost some weight,delayed bleeding,and significantly decreased fluorescein permeability.The HE staining showed that the mucosal damage was improved,indicating that the Orai1 inhibitor up-regulated the expression of Claudin-1,Claudin-4,Claudin-7 and other proteins.And the phosphorylation levels of Claudin-1,Claudin-4 and Claudin-7 are reduced,the apoptosis of colonic epithelial cells is reduced,and the pathological damage of colonic mucosa is alleviated.Conclusion:1.Claudin-1,Claudin-4,Claudin-7 and Orai1 can form a complex in NCM460 cells;Orai1 inhibitors reduce the phosphorylation of Claudin-1,Claudin-4 and Claudin-7,and up-regulate the expression of Claudin-1,Claudin-4 and Claudin-7 proteins,thus enhancing the colonic epithelial barrier function.2.In the DSS-induced colitis mouse model,the permeability of colonic mucosa was increased in the model group,and the phosphorylation levels of Claudin-1,Claudin-4 and Claudin-7 were increased.The expression levels of Claudin-1,Claudin-4 and Claudin-7were decreased.The Orai1 inhibitor reduced the permeability of colonic mucosa,upregulated the expression levels of Claudin-1,Claudin-4,Claudin-7 and other proteins,and weakened their phosphorylation levels.