Study On The Role And Mechanism Of LAMP2A In Inflammatory Bowel Disease

Background Inflammatory bowel disease(IBD)is a chronic nonspecific intestinal inflammatory diseases,including ulcerative colitis(UC)and Crohn's disease(C D).At present,the pathogenesis of IBD is still not clear.However,it is generally accepted that IBD is a multifactor disease,which is triggered by dysbacteriosis or other disease-causing agents in the environment,effects on people who are susceptible,leads the intestinal natural immune and specific immune response,thereby causing intestinal mucosa barrier damage.Recent research shows that the incidence and prevalence of IBD in C hina had an increasing trend gradually.Immune disorder and dysbacteriosis in intestine are parts of pathogenesis of IBD.More and more studies show the reciprocal nature of the regulation of the immune system and microbial community structure.In addition,autophagy plays an important role in the innate immunity and specific immunity,which contains three types: macroautophagy,microautophagy,and chaperone-mediated autophagy(CMA).CMA only exists in mammalian cells.The substrate proteins of CMA must expose KFERQ sequence to be recognized by heat shock protein 70.Then,the complex binds to lysosomal membrane and the substrate translocate in lysosome through the lysosome-associated membrane protein type 2a(LAMP2A)and finally been hydrolyzed.LAMP2 A acts rate-limiting molecule of CMA,and the abnormal expression or inactivation of LAMP2 A will definitely affect disease.Therefore,we aim to explore the role of LAMP2 A in inflammatory bowel disease.Methods: 1.Using Cre-Lox P system to generate specific intestinal epithelial cell Lamp2 a knock-in mice(LAMP2A-IECKI mice).LAMP2A-IECKI mice genetype were confirmed by standard PCR.2.Using 4% dextran sulfate sodium(DSS)-induced colitis mouse model to simulate human IBD.Mice were monitored daily to assess change in body weight,stool consistency,fecal occult blood and activity.3.The mice were sacrificed on day 10.The entire colon was removed from the cecum and measured the colon length.Using he matoxylin-eosin staining(HE),transmission electron microscope(TEM)and immunohistochemistry to detect the regeneration,damage degree and inflammatory infiltration of colonic mucosa.4.Intestinal microbiome of LAMP2A-IECKI and WT mice were analyzed by using fecal 16 S r DNA sequencing.Results: 1.Specific intestinal epithelial cell Lamp-2a knock-in mice was set up successfully.2.LAMP2A-IECKI+DSS group showed significant weight loss,severe fecal occlusion,and the length of the colon were shortened.Both HE and TEM showed overexpression of LAMP2 A aggravates DSS-induced colitis in mouse.3.The inflammatory infiltration in LAMP2A-IECKI+DSS mice was more obvious,among which macrophages,CD4+T cells,and C D8+T cells increased significantly.And the release of IL-1 leads to inflammatory cascade reaction.4.16 S r DNA results show the abundance of IBD related pathogenic bacteria in the intestinal tract of the miceConclusion: Overexpression of LAMPA2 A in intestinal epithelial cells enhanced CMA function.On the one hand,it increased colonization of pathogenic bacteria.O n the other hand,excessive autophagy activation,release a large number of inflammatory mediators to recruit more inflammatory cell,thus aggravating symptom colitis in mice.