SLC26A3(DRA)maintains The Intestinal Epithelial Barrier In Ulcerative Colitis

BACKGROUND&AIMS:Ulcerative colitis is one type of inflammatory bowel disease(IBD)and a non-specific intestinal inflammatory disease with increasing incidence in recent years.Indeed,a growing body of evidence indicates that UC is associated with or may be secondary to defects in the gut barrier.The intestinal epithelial barrier is composed of a layer of columnar cells that separate internal tissues from the luminal content.The paracellular pathway between cells are sealed by tight junctions(TJs)and adherens junctions,which modulate the integrity and permeability of epithelial monolayers.TJ disruption results in increased mucosal permeability,which allows bacteria and toxins to pass from the lumen into the submucosa,triggering inflammation.TJ disruption is considered as a vital event in the pathogenesis of intestinal inflammation,highlighting the importance of exploring TJ protein dynamics.DRA(downregulated-in-adenoma)is a Cl^-/HCO₃^-exchanger encoded by the gene SLC26A3 that contributes to intestinal fluid absorption and enterocyte acid/base balance.Attention has largely focused on its ion exchange properties.DRA anion exchange activity and expression are both significantly decreased in UC and related animal models.Recent work suggested that the loss of DRA is associated with decreased mucosal HCO₃^-secretion,potentially due to inflammation-induced changes in the crypt and villous architecture or to the effect of proinflammatory cytokines on epithelial ion transporters.We previously identified that DRA deficiency is associated with the absence of a firmly adherent mucus layer and with HCO₃^-/mucus barrier impairment in mice.This change in mucus layer renders SLC26A3^-/-mice susceptible to dextran sulfate sodium(DSS)-induced colitis.DRA was also found to be involved in tumor suppression by maintaining gastric mucosal integrity.DRA regulation of the intestinal epithelial barrier remains to be elucidated.Previous work in TNF-?-overexpressing mice demonstrated that TJ-associated proteins are altered,and DRA expression in the colon was effected.These findings led us to hypothesize that TNF-?induced TJ disruption occurs in part through effects on DRA expression,ultimately exacerbating colon inflammation.To test this hypothesis,we examined DRA localization within TJs in a Caco-2BBe cell monolayer.In addition,mice with DSS-induced colitis were infected with adenovirus encoding DRA and treated with a TNF-?monoclonal antibody(m Ab).Epithelial permeability,TJ protein expression,colitis severity,and histological inflammation were then studied in these mice.Based on these studies,we discuss the mechanism by which TNF-?regulates DRA to further influence the intestinal epithelial barrier.METHODS:(1)The expression of DRA in the colon tissues of UC patients and mice was detected by Western blot,RT-PCR and immunofluorescence.(2)The mouse colitis model was established.Immunohistochemistry and immunofluorescence were used for structural,morphological and biochemical analyses of DRA in DSS-induced colitis mice model.(3)The localization of DRA and tight junction protein in Caco-2BBe was detected by immunofluorescence.(4)The relationship and expression of DRA and tight junction protein in Caco-2BBe cells were detected by co-immunoprecipitation and western blot.(5)Establish mouse model animal:purified adenovirus of DRA was injected by coloclysis in DSS-induced colitis model.Then,TEER was used to detect epithelial barrier function.The localization of DRA and tight junction protein was detected by immunofluorescence.Histological changes were detected by HE staining.Intestinal permeability was measured by Evans blue method.Inflammatory factors were detected by cytometric bead array.Transmission electron microscopy was used to observe the differences of colonic epithelial barrier structures.(6)Serial deletion,site-directed mutagenesis and luciferase report assays were used to determine the NF-?B binding sites in DRA promoter region.The co-location relationship between DRA and TJs was analyzed by co-immunoprecipitation and immunofluorescence.The changes of DRA and TJs were investigated by constructing a mouse model of colitis treated with TNF-antibody.RESULTS:Part ?:The expression of DRA was detected in the colon lesion tissues of UC patients and health tissues by Western blot,and then a DSS-induced acute enteritis model was established to further explore the expression and localization of DRA in the animal model.The results were as follows:(1)The expression of DRA in the colon tissues of UC patients was significantly down-regulated.(2)In DSS-induced colitis mice,the serum TNF-?level increased compared with control mice,and the expression of DRA m RNA and protein significantly decreased.(3)RT-PCR,Western blot,immunofluorescence and immunohistochemical experiment results showed that the expression of DRA in the normal control group was significantly lower than that in the DSS group.These results showed that the expression of DRA in colitis was significantly down-regulated.Part ?:(1)In the polarized Caco-2BBe monolayer,DRA is located at the tight junction.(2)Double immunoflurence staining for DRA and ZO-1 and endogenous co-immunoprecipitation assays demonstrate that they were co-localized in Caco-2BBe cells.DRA knockdown can affect the normal localization of TJ proteins and down-regulate of tight junction proteins ZO-1,occludin,claudin 1 and claudin 5,and up-regulation of claudin 2.In addition,DRA knockdown caused a decrease in TEER value,indicating that DRA downregulation impair TJ.(3)In overexpressed cells,ZO-1and occludin were increased,while claudin 2 was decreased,claudin1 and claudin 5levels were not significantly changed,and TEER values were increased,indicating that overexpression of DRA could strengthen TJ.(4)Caco-2BBe cells were incubated with TNF-?(100 ng/ml)for 24h to simulate the pathophysiological process of epithelial barrier destruction during the pathogenesis of UC.Immunofluorescence results showed that overexpression of DRA can strengthen intestinal epithelial TJ and resist the failure of tight junction proteins ZO-1,occludin and F-actin.After treatment with TNF-?for 24h,the expression of ZO-1,occludin,claudin 1 and claudin 5 in the DRA overexpression group increased,compared with the control group.While the expression levels of claudin2 decreased.TEER experiments further confirmed that overexpression of DRA enhanced intestinal epithelial function and partially reversed the destruction of barrier function induced by TNF-?.Part ?:(1)By constructing the colitis mice model,purifying adenovirus by enema injection of DRA,and conducting DAI score,it was confirmed that Ad-DRA ameliorated colitis induced by DSS in mice.(2)Western results showed that the loss of TJ protein decreased.HE staining of Ad/DRA group showed a significant improvement in epithelial inflammatory cell infiltration and a significant decrease in histopathological double-blind score.(3)Ultrastructure of epithelial cells by transmission electron microscopy showed that the tight intercellular structure of Ad/DRA group was closed.(4)Ad/DRA adenovirus intervention increased the expression of DRA in colonic epithelial cells and stabilized the expression of TJ structure and tight junction protein.(5)The treatment with Ad/DRA significantly inhibited the levels of pro-inflammatory cytokines TNF-?,IL-6 and KC,while the expression of anti-inflammatory factor IL-10 increased.Part ?:(1)TNF-?induces a decrease in DRA expression.DRA expression significantly increased after adding the NF-?B specific inhibitors BAY-11-7082.(2)Gene mutations and luciferase report confirmed the NF-?B transcriptional binding site on DRA promoter region.DRA was down-regulated by TNF-?/NF-?B pathway activation.(3)Western blot and immunofluorescence results showed that neutralization of TNF-?in dextran sulfate sodium(DSS)-induced colitis mice demonstrated improved the outcomes,and increased the expression of DRA in colonic epithelial cells and stabilized the expression of TJ structure and tight junction protein.These data suggest that DRA may be one of targets of TNF-?.CONCLUSIONS:The role of DRA in the tight junction of intestinal epithelial barrier can be summarized as follows:(1)DRA is poorly expressed in the colonic epithelial tissues of patients with inflammatory bowel disease and in the DSS-induced mouse enteritis model.(2)DRA localizes to TJs in polarized Caco-2BBe cell monolayers and is necessary for maintaining epithelial barrier integrity.DRA overexpression strengthens the epithelial barrier and mitigates TNF-?-induced damage.Delivery of Ad-DRA ameliorates DSS-Induced experimental colitis in mice and reduces TNF-?levels.(3)Anti-TNF-?treatment prevents the loss of DRA in DSS-Induced colitis.NF-?B mediates the TNF-?-induced decrease in DRA.In conclusion,our in vitro and in vivo data show that DRA plays a central role in protecting the epithelial barrier in intestinal inflammation.The present study shows that in addition to participating in fluid-electrolyte absorption and acid/base balance in the gut,as well as mucus layer barrier formation,DRA is involved in the epithelial barrier and in preventing the progression of inflammation.Our study characterized a new mechanistic pathway in UC,in which DRA interacts with the TNF-?/NF-?B pathway by targeting TJ proteins.The multiple roles of DRA may inform novel therapeutic strategies for the management of UC in the future.