The Roles And Mechanisms Of Deacetylase Sirtuin 6 In Intestinal Epithelial Cells

Background The gut is the body's important digestive organ as well as the body's largest detoxification organ.In intestinal diseases,ulcerative colitis is a chronic non-specific inflammatory disease of the colon and rectum with unknown etiology.The lesions are mostly confined to the mucosa and submucosa of the colon,and the location of the disease is more common in sigmoid colon and rectum and can extend to the descending colon or even the whole colon.The intestinal epithelial cells(IECs)are regenerated continuously from stem cells reside in crypt.The mammalian IECs are composed of 7 different types of highly specialized cells,which cooperate to absorb nutrients,regulate intestinal peristrophy,and maintain the mucosal barrier.Epithelial villi contain only non-proliferating,differentiated cells,including intestinal epithelial cells,goblet cells,intestinal endocrine cells,and tuft cells.IECs can sense and response to stimuli,i.e.helminth,and then proceed epithelial remodling to clear helminth infection.Tuft cells are a class of brush shaped epithelial cells in the intestinal epithelium,accounting for about 0.5% of the total epithelial cells in the small intestine.Tuft cells can promote the proliferation of intestinal epithelial cells and enhance the integrity of intestinal epithelial cells in the intestine.During worm infection,tuft cells induce type 2 immune response to induce intestinal epithelial remodeling.As a member of the Sirtuins family of mammals,Sirtuin 6(SIRT6)is involved in regulating many important physiological functions,including metabolism,inflammation,and aging.The role of SIRT6 in IEC has not been extensively studied,and the mechanism is still unclear.Objectives This study intends to establish a mouse model of Sirt6 gene knockout in intestinal epithelial cells(Sirt6f/f: Villin-cre)by using a widely used gene knockout system called Cre-loxp to study the role of SIRT6 in intestinal epithelial cells.Meanwhile,the systemic overexpression of SIRT6 in transgenic mice(Tg SIRT6)was used as the research object to investigate whether SIRT6 directly affects the intestinal epithelial cells after overexpression.The completion of this project will provide a new theoretical basis for the prevention and treatment of inflammatory bowel diseases and parasitic infections.Methods1.Study on the role of SIRT6 in ulcerative colitis(1)Wild type(WT)mice were fed with normal distilled water and distilled water containing 2.5%DSS to establish a model of ulcerative colitis,and the expression of SIRT6 in normal colon tissues and ulcerative colitis colon tissues was detected by Western Blot.(2)Use of Sirt6 floxed(Sirt6flox/flox)mating and Villin-cre mice,mice get Sirt6f/f:Villin-cre(Sirt6-IKO)in mice.Namely,mice with specific knockout of intestinal epithelial cells by Sirt6.The knockout efficiency of SIRT6 was detected by Western Blot and q RT-PCR,and Sirt6 knockout mice,namely Sirt6-IKO mice,were selected for the study.(3)The female mice of WT(Ctrl)and Sirt6-IKO were intermitentially fed with distilled water containing 2.5%DSS for a long time,and sacrificed after 3 months to collect blood and obtain colon tissues for phenotypic typing.Total RNA from Colon was extracted from two groups of mice,and the expression levels of proinflammatory factors IL6 and TNFa in Colon tissues of two groups of mice were detected.2.Study on the role and mechanism of SIRT6 in intestinal tuft cell differentiation(1)The study was conducted in WT(Ctrl)mice and Sirt6-IKO mice to detect whether Sirt6 affects the development and differentiation of tuft cells.(2)According to H.polygyrus parasite growth curve of lavage WT mice get parasitic infection 0 day,7 days,15 days for 11 days,four groups of different number of days the parasite infection in mice,detect worms after intestinal infection,tuft type2 cells the cell immune response to intestinal worms away play an important role,and the intestinal epithelial cells of STAT6 phosphorylation level and intestinal epithelial cells within the overall SIRT6 levels were positively correlated.(3)10 male and 10 WT(Ctrl)mice only 8 weeks Sirt6-IKO male mice,equal weight to each,mouse using 200 H.polygyrus L3 larvae of infected mice in lavage way,15 days after the sacrifice mice phenotype analysis,detect worms infected SIRT6 whether to tuft cell hyperplasia and start type 2 immune factors on the removal of the intestinal worms have played a role in regulating.(4)The systemic overexpression of SIRT6 in Tg SIRT6 mice and WT(Ctrl)mice was used to detect that the overexpression of SIRT6 in intestinal epithelial cells could not affect the differentiation of intestinal tuft cells.(5)Organoid from cultured WT(Ctrl)mice and Sirt6-IKO mice were used to detect whether intestinal epithelial SIRT6 directly regulated the differentiation of tuft cells and reduced the phosphorylation level of STAT6.IL-13 up-regulated SIRT6 levels in cells,thereby increasing the phosphorylation level of STAT6,were detected by stimulating Organoid IL-13(10ng/ml,2h).(6)To detect whether SIRT6 regulates the transcriptional activity of STAT6 and changes the acetylation level of STAT6 by Luciferase assay and CO-IP method.Result1.Study on the expression and effect of SIRT6 in ulcerative colitis(1)Compared with mice fed with normal distilled water,the expression of SIRT6 at m RNA and protein levels in colon tissues of 2.5%DSS mice decreased(P< 0.05).(2)2.5%DSS long-term feeding of WT(Ctrl)and Sirt6-IKO mice(10/group)showed more severe ulcerative colitis phenotype in terms of body weight and DAI score in Sirt6-IKO mice(P< 0.05).(3)2.5%DSS long-term feeding of WT(Ctrl)and Sirt6-IKO mice(10/group),immunohistochemical results and HE staining results showed that the infiltration degree of macrophages and the number of positive cells in the colon tissues of Sirt6-IKO mice were higher(P< 0.05).(4)2.5%DSS long-term feeding of WT(Ctrl)and Sirt6-IKO mice(10/group),colon q RT-PCR results showed that the pro-inflammatory factors in the colon tissues of Sirt6-IKO mice were higher than that of WT mice(P< 0.05).2.Role of SIRT6 in intestinal tuft cell differentiation and its mechanism(1)The absence of SIRT6 in intestinal epithelial cells resulted in a significant decrease in the number of intestinal tuft cells(P< 0.05).(2)The expression of SIRT6 and phosphorylation of STAT6 increased with the increase of tuft cells on different days of parasite infection(P< 0.05).(3)When the expression levels of DCLK1 in jejunum of WT(Ctrl)mice were significantly increased after parasite infection,there was no significant change in the expression levels of DCLK1 in jejunum of Sirt6-IKO mice(P< 0.05).(4)There was no significant change in the number of intestinal tuft cells after SIRT6overexpression(P > 0.1).(5)Intestinal epithelial cell SIRT6 directly regulates tuft cell differentiation and affects STAT6 phosphorylation.(6)SIRT6 and STAT6 interact and regulate the activity of STAT6,but SIRT6 does not change the acetylation level of STAT6.Conclusion1.Deficiency of SIRT6 in intestinal epithelial cells increases susceptibility to ulcerative colitis.2.The absence of SIRT6 in intestinal epithelial cells significantly reduces the number of tuft cells,thereby impelling the intestinal epithelial remodeling mediated by type 2immune responses after worm infection.