The Effects And Mechanisms Of Autophagy On Intestinal Epithelial Barrier Function In Inflammatory Bowel Disease

ObjectiveTo detect the change of tight junction protein claudin-2,autophagy related protein,the status of autophagy and the effects of autophagy on intestinal epithelial barrier function in well conformed intestinal barrier injury cell model of inflammatory bowel disease(IBD)as well as the mechanisms.MethodsAfter the cell model of intestinal epithelial barrier constructed by Caco-2 or IEC-6 cells well conformed,10 ng/mL or 20ng/mL tumor necrosis factor alpha(TNF-α)were treated for 48 h to simulate IBD condition in vitro.Western blot analysis was used to examine the protein level of claudin-2,LC3B-II and P62.Cells were transiently transfected with Ad-mCherry-GFP-LC3 B adenovirus to construct a monitor system of LC3 B fluorescence,which then used for evaluating autophagy under laser scanning confocal microscope.Lysotracker Red DND-99 and Lysosensor Yellow/Blue DND-160 were used for qualitative and quantitative analysis of lysosomal pH,respectively.The expression of claudin-2 was detected by immunofluorescence staining qualitatively.Intestinal barrier function was assessed by permeability of fluorescein isothiocyanatedextran(FITC-Dextran)and transepithelial electrical resistance(TER).Results 1.Research on claudin-2 protein and the status of autophagy in IBD cell model.The claudin-2 protein levels were increased in IBD cell model constructed by TNF-α;meanwhile,both the expression of LC3B-II and P62 increased in this model.3-methyladenine(3-MA)decreased the levels of LC3B-II while bafilomycin A1 enhanced it;TNF-α was still enhanced the amount of LC3B-II though pretreated with 3-MA,but could not further promote the expression of LC3B-II even when pretreated with bafilomycin A1,which indicated that the autophagic degradation process was inhibited in IBD cell model;both TNF-α and bafilomycin A1 largely enhanced the yellow dots while it did not increase the number of red dots in cells transfected with admCherry-GFP-LC3 B adenovirus,which further determined the fact that the autophagic degradation process was inhibited in IBD cell model.2.The mechanism study of claudin-2 change and function test in IBD cell modelBoth the qualitative and quantitative analysis of lysosomal pH indicated that the acid environment of lysosome was disrupted in IBD cell model;the mTOR inhibitor PP242,which has been shown to promote lysosomal biogenesis,rescued this change of lysosomal pH.In addition,PP242 also partly alleviated the disruption of autophagy flux and the elevation of claudin-2 level.Further function test demonstrated that the permeability of FITC-Dextran increased and TER decreased in IBD cell model,and PP242 partly rescued the change of intestinal barrier function.ConclusionOur study reveals that TNF-α-induced claudin-2 increase and impairment of intestinal epithelial barrier function are partly attributed to the inhibition of autophagic degradation in vitro,and the inhibition may related to the impairment of lysosomal acid environment,while the mTOR inhibitor PP242 partly alleviated these changes.Therefore,mTOR pathway and autophagy may be a new target for IBD therapy.