SLC9A8 Regulates Differentiation Of Intestinal Epithelial Cells To Maintain Gut Mucosal Barrier And Its Molecular Mechanism

Backgroud and objective:The gut mucosal barrier is not only the place where organism exchanges substances with the outside environment,but also prevent pathogenic microorganisms into the body effectively.Dysfunction of the gut mucosal barrier has been reported to be closely related to occurrence and development of various diseases such as inflammatory bowel disease.Several factors have been proved involved in mucosal barrier protection,among which precisely regulated proliferation and differentiation of intestinal epithelial cells plays a vital role in maintaining the integrity of the mucosal barrier.The function of gut mucosal barrier would be weakened when intestinal stem cell injury,decreased number of goblet cells,insufficient mucus protein synthesis and secretion,and decreased number and dysfunction of paneth cells take place.Notch signaling pathway has been reported to be responsible for differentiation regulation of intestinal stem cells.When the notch signaling pathway is activated,the number of secretory epithelial cells in the intestine decreases,while the number of absorptive epithelial cells increases;conversely,when the notch signaling pathway is inhibited,the intestinal stem cells tend to differentiate into secretory epithelial cells.Solute Carrier Family 9 Member A8（SLC9A8）,a member of the sodium/hydrogen exchanger family,participates in the absorption of water and sodium and maintaining the intracellular pH.It has been confirmed that SLC9A8 is associated with the intestinal biological barrier,mucus barrier and mechanical barrier.However,the mechanism by which SLC9A8 affects the gut mucosal barrier function is not clear.In the present research,we take WT mice,SLC9A8^-/-mice and enteroids as research objects to explore the influence of SLC9A8 on proliferation and differentiation of intestinal epithelial cells and notch signaling pathway,and investigate the underlying mechanism how SLC9A8regulates the notch signaling pathway.We hope that our research findings could provide new theoretical basis for gut mucosal barrier regulation.Materials and methods:1.SLC9A8^+/-mice was generated by knockout of exon 2 of SLC9A8 with CRISPR/Cas9 system,and SLC9A8^+/-mice were breeded to obtain SLC9A8^-/-mice.According to the genotype,the mice were divided into WT group and SLC9A8^-/-group,with 6 mice in each group.We compared the differences of the intestinal gross morphology,tissue structure,cell proliferation and apoptosis,stem cell and epithelial cell composition and mucosal barrier function between WT mice and SLC9A8^-/-mice,with the methods of histopathological techniques（including transmission electron microscopy,HE staining,PAS staining,IHC staining,IF staining and Tunel staining）,molecular biology techniques（including Western Blot and qRT-PCR）along with transcriptomics sequencing of colon tissue and intestinal permeability testing.2.Intestinal stem cells from WT mice and SLC9A8^-/-mice were isolated for enteroids culture.We compared the differences of histological structure,cell proliferation,stem cell and epithelial cell composition,and mucus secretion between enteroids from WT mice and SLC9A8^-/-mice,with the methods of histopathological techniques（including HE staining,PAS staining,IHC staining and IF staining）and qRT-PCR.3.Mice were allocated into 4 groups,i.e.,WT group,SLC9A8^-/-group,WT+DSS group and SLC9A8^-/-+DSS group,with 6 mice in each group.Mouse colitis model was established by drinking 2%DSS water.We compared the differences of histopathological damage,levels of inflammatory factors,cell apoptosis,intestinal mucus barrier and bacterial adhesion among 4 groups,with the methods of histopathological techniques（including HE staining,PAS staining,IHC staining,IF staining,Tunel staining and fluorescence in situ hybridization staining）and molecular biology techniques（including ELISA and qRT-PCR）.4.Mice were allocated into 4 groups,i.e.,WT group,SLC9A8^-/-group,WT+LY group and SLC9A8^-/-+LY group,with 6 mice in each group.Mice were gaveged with LY411575 to inhibit notch signaling pathway.We compared the differences of tissue structure,cell proliferation,stem cell and epithelial cell composition and mucosal barrier function among 4 groups,with the methods of histopathological techniques（including HE staining,PAS staining,IHC staining,IF staining）and molecular biology techniques（including Western Blot and qRT-PCR）.Results:1.The results of agarose gel electrophoresis and SLC9A8 qRT-PCR indicated that SLC9A8^-/-mice were successfully constructed.Compared with WT mice,the length of the intestine of SLC9A8^-/-mice were shortened,while the depth of the crypt and the number of proliferating cells in the crypt increased.Sequencing data of colonic transcriptomics showed that SLC9A8 was related to epithelial cell development and differentiation.Loss of SLC9A8 led to increased numbers of intestinal stem cells and absorptive epithelial cells,but decreased numbers of goblet cells and Paneth cells.Loss of SLC9A8 led to less mucin secretion in the intestine,thinner mucus layer,and increased permeability of the intestine,accompanied by changed expression of tight junction proteins including Claudin2,Occludin and Zo-1.2.Compared with enteroids from WT mice,enteroids form SLC9A8^-/-mice demonstrated increased numbers of proliferating cells and stem cells,decreased numbers of goblet cells and Paneth cells,less mucus protein and glycosylated mucus protein secretion.Moreover,results by qRT-PCR demonstratd that the mRNA levels of Alpi,Sis and Slc2a5 in enteroids from SLC9A8^-/-mice were significantly increased when compared with enteroids from WT mouse.3.After drinking 2%DSS water,the mice presented with higher DAI score,intestinal histological score,inflammatory cytokine levels and apoptosis rate.Compared with WT+DSS group,the DAI score,intestinal histological score,inflammatory cytokine levels and apoptosis rate in the SLC9A8^-/-+DSS group increased significantly.The intestinal mucus barrier was damaged more severely,with more bacterial adhesion.4.Loss of SLC9A8 resulted to increasd protein levels of NICD and Hes1 in the colon tissue and enteroids.Afterγ-secretase inhibitor LY411575 administration,the numbers of proliferating cells and stem cells in the intestine were unaffected,however,the numbers of goblet cells and Paneth cells increased significantly with more mucus protein and glycosylated mucus protein in the intestine,while the number of absorptive epithelial cells decreased.We found that endocytosis in enteroids was enhanced after the loss of SLC9A8Conclusion:1.SLC9A8 was related to proliferation of intestinal stem cells and involved in differentiation regulation of the absorptive epithelial cells and secretory epithelial cells.Loss of SLC9A8 would lead to dysfunction of intestinal mucus barrier and mechanical barrier.2.Loss of SLC9A8 aggravated the intestinal mucus barrier damage and induced epithelial cell apoptosis in mice with DSS-induced colitis,and subsequently increased the severity of intestinal injury.3.SLC9A8 regulated differentiation of intestinal epithelial cells through Notch signaling pathway.4.Loss of SLC9A8 enhanced endocytosis in enteroids from SLC9A8^-/-mice, which was possibly related to regulation of Notch signaling pathway.