Intestinal Epithelial Fto Regulates FoxO1 And Gut Microbiota-mediated Mice The Role And Mechanism Of UC

Objective : Inflammatory Bowel Diseases(IBD)is a bowel disease characterized by damaged intestinal tissue and chronic inflammation of the intestine,including ulcerative colitis(UC)and Crohn’s disease(CD).With the change of people’s lifestyle and eating habits,the incidence of UC has gradually increased worldwide,which has become a major public health problem.Many studies believe that the occurrence of UC is caused by the interaction between host susceptibility genes and external environmental factors(such as gut microbiota,dietary habits,etc.),but the pathogenesis of UC is not very clear.Fto(fat mass and obesity associated)is the first candidate gene related with obesity,and plays thermogenesis in metabolically related tissues and organs such as the brain and fat.The m6 A regulates immune cell differentiation,such as CD4 + T cell knockout gene METTL3 associated with m RNA m6 A modification,Th1 and Th17 cells decreased,Th2 cells increased,but Treg cells changed unchanged,suggesting that CD4+ T cell differentiation is blocked,and intestinal local immunity is closely related to enteritis,has an important role in enteritis,m6 A modification can affect gut microbiota by regulating Ig A secretion by immune cells.In recent years,the researchers have also found the characteristics of gut microbiota in UC patients,and somewhat alleviate the symptoms of UC patients and reduce mucosal inflammation by regulating the structure of gut microbiota.These suggest that the host gene Fto may participate in the occurrence and development of UC by regulating the gut microbiota.To study whether the intestinal epithelial m6 A demethylase Fto through the interaction with external environmental factors lead to the occurrence of UC,we first constructed the intestinal epithelial Fto specific knockout mice,explore the intestinal epithelial Fto gene and the gut microbiota in glucan sodium sulfate(DSS)induced UC and preliminary mechanism,for the influencing factors and UC treatment to provide certain experimental data and theoretical basis.Methods :1.Construct intestinal epithelial Fto knockout mice(Vinlin Knockout,VKO,Fto-VKO,hereinafter referred to as KO mice)and wild-type mice(wild type,WT)using Cre / Loxp system,and the knockout effect was verified at gene,RNA,protein,immunohistochemical levels.2.The changes of body weight,body fat,colon and length and structure to assess the effects of intestinal epithelial Fto knockout in mice are observed,and the effects of intestinal inflammatory factors(IL-1β,IL-6,TNF-α)and intestinal mucosal barrier,ZO-1,ZO-2,claudin-1,claudin-3,E-cadherin)were assessed by western blot and q PCR.3.In order to observe the composition and metabolites of the gut microbiota,16 S r RNA high-throughput sequencing and LC-MS untargeted metabolomics were used to detect the species,abundance and content of the metabolite SCFAs in the two groups of mice.4.To explore the effect of host genes and the gut microbiota on ulcerative colitis in mice.UC induce with antibiotics,cage separation,cage separation and fecal microbiota transplantation,and record mouse weight change,colon length,disease activity index,histological change score and cytokines related to inflammation IL-1β,IL-6,TNF-αin colon tissue.5.To explore the mechanistic effect of m6 A demethylase FTO on UC occurrence,the relative RNA expression of Fox O1,downstream molecule of Fto in both mouse colon tissues was measured by q PCR,and the protein expression level of Fox O1 in both groups was determined by western blot.Results:1.The mice with tail DNA expressing both Loxp and cre by agarose gel electrophoresis were KO mice;Fto expression levels decreased in the small intestine and colon by western blot and q PCR,without significant changes in the brain,and Fto expression decreased in the colon of KO mice by immunohistochemistry(P<0.05).2.Western blot results showed that the intestinal mucosal barrier of KO mice was significantly enhanced compared with WT mice,immunohistochemistry showed a significant increase of intercrypt goblet cells in the colon of KO mice(P <0.05),HE staining showed no significant change in intestinal epithelial tissue structure in the two groups,and q PCR detected inflammatory factors IL-1β,IL-6,and TNF-αexpression in the two groups(P> 0.05).3.Mouse fecal 16 S r RNA sequencing analysis revealed significant differences in the Fto,the diversity of KO mice and the composition of the flora.The relative abundance of Muribaculaceae,Lawsonibacter and Dysosmobacter increased significantly in the intestine of KO mice,and that of Bacteroides,Eisenbergiella and Erysipelotrichaceae decreased significantly.However,SCFAs found that SCFAs in KO mice increased significantly,especially acetic acid,isobutyric acid and uretopropionic acid(P <0.05).4.After antibiotic clearance of both groups,Then DSS found that KO mice weight ratio decreased significantly(P <0.05)and colon length is significantly shortened from day 4,Colonic tissue structure is seriously damaged;The DAI,histological change score,IL-1β,and IL-6 are all significantly increased(P <0.05);There is no significant difference in the weight ratio loss of KO mice in the cage + DSS group,and the destruction of colon tissue structure is mild,Inflammatory factor IL-1βincreased significantly(P <0.05);The KO mice in the cage + DSS group decrease weight ratio and severe destruction of colon tissue structure;The DAI,histological change score,IL-1β,and IL-6 are all significantly increased(P <0.05);After transplantation of fecal transplantation,DSS found that the weight ratio of KO mice decreased,the colon length is significantly longer than WT mice,the colon tissue structure is also significantly recovered,and the inflammatory factors also decreased to varying degrees.5.RNA and protein levels of Fox O1 in mouse colon tissue found that Fox O1 decreased in intestinal epithelial Fto-specific knockout mice at the RNA level,but is not significantly different(P>0.05),while western blot analysis showed that Fox O1 protein expression is significantly increased in the colon tissue of KO mice(P<0.05).Conclusions:1.This study is the first successful construct of intestinal epithelial Fto-specific knockout mice.2.Fto-specific knockout of the intestinal epithelium do not affect the general situation in mice nor cause spontaneous enteritis in mice.3.Fto-specific knockdown of intestinal epithelium alters the structure and metabolites of gut microbiota in mice.4.The intestinal epithelial Fto regulated Fox O1 and the intestinal microbiota mediated the development of DSS-induced UC in mice.