The Role Of Depdc5 Deletion-mediated MTORC1 Activation In The Regulation Of Intestinal Epithelial Homeostasis In Mice

BackgroundIntestinal epithelial cells are a type of monolayer special cells,which can be divided into cluster cells,goblet cells and Paneth cells and so on^[1].The main function of cluster cells is to resist the infection of worms and initiate the type 2 immune response,Goblet cells produce mucus,Paneth cells secrete antimicrobial peptides,and Paneth cells in the crypts release various antimicrobial peptides,which have a bactericidal effect^[2,3].Mammalian Target of Rapamycin(m TOR)is a serine/threonine protein kinase and a member of the phosphatidylinositol kinase-related protein kinase family.It mainly regulates cell growth,proliferation,and apoptosis^[4,5].m TOR has two complexes m TORC1 and m TORC2 with different structures and functions.m TORC1 coordinates cell growth and metabolism in response to environmental inputs,including growth factors,amino acids,energy,and stress^[6].DEPDC5 is an inhibitor of the m TORC1 pathway[1].As part of the GATOR1 complex,DEPDC5 is an important negative regulator of m TORC1^[7,8],which is crucial for the m TORC1 mediated signaling pathway.ObjectivesThis study explored the regulation effect of continuous activation of the m TORC1signaling pathway mediated by Depdc5 gene on mouse intestinal epithelium.The mouse model of knocking out the Depdc5 gene(Depdc5^f/f:Villin-cre)in intestinal epithelial cells was established by using the Cre-loxp system of gene knockout to study the knockout of Depdc5 gene in intestinal epithelial cells The effect on the differentiation of intestinal epithelial cells after removal.The completion of this project will provide new theoretical support for the physiology of m TORC1 signaling pathway and the maintenance of intestinal epithelial homeostasis in the case of worm infection.Methods1.To study the role of Depdc5 knockout-mediated overactivation of m TORC1 signaling pathway in the regulation of mouse intestinal epithelial homeostasisTwenty healthy 8-week-old female mice were divided into 4 groups,5 mice/group,namely Ctrl group,IKO group,Rapa-Ctrl group,Rapa-IKO group.Take about 2 cm each of the small intestine and colon tissue for the protein and RNA of the intestinal epithelial cells,and detect the phosphorylation of the downstream target S6 protein and the expression of intestinal epithelial cytokines Depdc5,Dclk1,Trpm5,and Muc2.Another 5cm small intestine tissue was cut longitudinally and rolled into a swiss roll model,and fixed in 4%paraformaldehyde buffer for 48 h for phenotypic analysis.2.Study on the effect and mechanism of Depdc5 knockout of intestinal epithelial cells on intestinal clearance of helminth Hpoly infectionTwenty healthy 8-week-old female mice were divided into 4 groups,5 mice/group,namely Ctrl group,IKO group,H.poly-Ctrl group,H.poly-IKO group,the mice were sacrificed for phenotyping to detect the m TORC1 signal mediated by Depdc5 knockout after worm infection After the pathway is over-activated,cluster cells proliferate and initiate a type 2 immune response,whether it has a certain regulatory effect on the clearance of intestinal worms.Result1.The role of Depdc5 knockout-mediated activation of m TORC1 signaling pathway in the regulation of mouse intestinal epithelial homeostasis(1)The expression of P-S6 protein in the small intestine and colon tissue of the IKO group was higher than that of the Ctrl group(P<0.05);(2)Histomorphology showed that the crypt depth in the IKO group was significantly higher than that in the Ctrl group(P<0.05);(3)The m RNA levels of the marker genes Dclk1,Trpm5,and Muc2 of different types of epithelial cells in the IKO group were lower than those in the Ctrl group(P<0.05);(4)The results of immunohistochemistry and Alcian blue staining showed that the number of positive cells in cluster cells,paneth cells,and goblet cells in the IKO group was significantly lower than that in the Ctrl group,and the number of proliferating cells was significantly higher than that in the Ctrl group(P<0.05).Rapamycin After treatment,the number of cluster cells,paneth cells,and goblet cells increased and the number of proliferating cells decreased,and there was no statistical difference between IKO and Ctrl(P>0.05).2.Study on the effect and mechanism of Depdc5 knockout of intestinal epithelial cells on intestinal clearance of helminth Hpoly infection(1)After worm infection,the number of eggs in the feces and the number of adults in the intestine of the mice in the IKO group were significantly lower than those in the Ctrl group(P<0.05);(2)The expression levels of epithelial cytokines Dclk1,Trpm5,and Muc2 of mice in the Ctrl group and IKO group after worm infection were significantly increased compared with those before infection,but there was no statistical difference between the two groups(P>0.05);(3)The numbers of cluster cells and goblet cells in the Ctrl and IKO groups after worm infection increased significantly compared with those before infection,but there was no statistical difference between the two groups(P>0.05).Conclusion1.DEPDC5-m TORC1 signal is involved in the regulation of the differentiation and proliferation of IECs,and maintains the steady state of IECs.2.After worm infection,the m TORC1 signaling pathway mediated by DEPDC5 may not play a major role in the remodeling of intestinal epithelial cells.