Preliminary Study On The Role And Mechanism Of Nur77 In Regulating Susceptibility Of Intestinal Pathogens

Nur77 is an orphan nuclear receptor that plays an important role in inflammation and immune regulation.However,the relationship between Nur77 and mucous layer,antimicrobial peptide and intestinal flora in inflammatory diseases caused by intestinal pathogen infection is still unclear.Therefore,this study firstly compared the differences in Citrobacter rodentium susceptibility between wild-type and Nur77^-/-mice.Then use of C57BL/6 mice,explores the Nur77 on intestinal mucous layer thickness,antibacterial peptides expression and the influence of intestinal flora composition,using the mode-k small intestinal epithelial cells in mice and the original generation colon epithelial cells in mice to explore Nur77 in intestinal epithelial cells and the effect of antibacterial peptide gene expression reveals Nur77 and antimicrobial peptides and the potential links between the intestinal flora,in order to clear Nur77 of mucous layer,antibacterial peptides and the readjustment of intestinal flora.The main research contents and results are as follows:The first part:Effects of Nur77 on Citrobacter rodentium susceptibility in miceTwenty male mice of Wild type?WT?and Nur77^-/-C57BL/6 aged 10 weeks were selected and divided into two groups.Slaughter samples were collected on days 8 and 21after administration of Citrobacter rodentium.During the experiment,body weight and death were recorded every day,and the amount of Citrobacter rodentium in feces was measured.After slaughter,the liver,spleen,and kidney were weighed,the length of the colon was recorded,and the number of Citrobacter rodentium in the liver,spleen,and kidney was measured.The results showed that compared with WT mice:There was no significant change in the mortality of Nur77^-/-mice,but the weight loss was rapid and the recovery was slow.On the 7th and 8th days of Citrobacter rodentium infusion,there was a significant difference in the rate of weight change?P<0.05?.On the8th day of Citrobacter rodentium infusion,the colon of Nur77^-/-mice was significantly shortened?P<0.05?,the expression of inflammatory factors IL-13 and IL-22 in the colon was not significantly up-regulated,and it was significantly different from wild-type mice?P<0.05?;On day 8 after administration of Citrobacter rodentium,there was no difference in visceral index?liver,spleen and kidney?,but on day 21,Nur77^-/-kidney index significantly increased in mice?P<0.05?.The colonization amount of Citrobacter rodentium was measured by using the coated dilution counting method.The results showed that there was no significant difference in fecal bacterial load of Nur77^-/-mice,but the bacterial load of liver,spleen and kidney increased significantly?P<0.05?,indicating an increased susceptibility of Nur77^-/-mice to Citrobacter rodentium.The second part:Effects of Nur77 on intestinal mucous layer and antimicrobial peptides in miceThirteen 10-week-old WT and Nur77^-/-C57BL/6 male mice were selected,and tissue samples of jejunum and colon were collected after slaughter to study the effects of Nur77on intestinal mucus layer and antimicrobial peptide of mice.The main results are as follows:1. Effects on mucous layer in the colon of mice:PAS staining results showed that there was no significant difference in the thickness of mucous layer and the number of goblet cells secreting mucous in the colon of Nur77^-/-mice compared with WT.Expression of MUC2 m RNA in colon tissues was significantly down-regulated?P<0.05?,but there was no significant difference in protein expression.2. Effects on the intestinal antimicrobial peptides and the number of Paneth cell in mice:HE staining results showed that the number of Paneth cell in the jejunum of Nur77^-/-mice was significantly reduced compared with WT.QPCR results showed that the m RNA expression of antimicrobial peptides?including Defa1,Defa5,cryptdin1,cryptdin2,cryptdin5,lyz-1 and lyz-2?derived from Paneth cell in jejunum of Nur77^-/-mice was significantly down-regulated?P<0.05?,and m RNA expression of non Paneth cell derived antimicrobial peptides,including Defb2 and PLA2 genes,was significantly up-regulated in jejunum and colon?P<0.05?,while pglyrl-1 and Ang4 were significantly down-regulated?P<0.05?;MRNA expressions of Defb6 and CAMP in jejunum and reg3b and reg3g genes in colon were significantly down-regulated?P<0.05?.Western blot results showed that the expressions of Defb2,reg3g and Ang4 proteins in the colon were consistent with q PCR results.3.Primary colonic epithelial cells of mice were isolated and cultured,and q PCR was used to detect the predicted expressions of Nur77 target genes Ang4,reg3b and reg3g m RNA.The results showed that m RNA expressions of reg3b and reg3g genes in primary colonic epithelial cells of Nur77^-/-mice were significantly down-regulated compared with WT?P<0.05?,which was consistent with q PCR results of living colon tissues.Overexpression of Nur77 of Nur77,The m RNA expression of reg3b and reg3g genes were significantly up-regulated in mode-k cells?P<0.05?.The third part:Effects of Nur77 on intestinal flora in miceThirteen male mice aged 10 weeks WT and Nur77^-/-C57BL/6 were selected,and colonic chyme was collected after slaughter to study the effect of Nur77 on intestinal flora of mice.The main results are as follows:1.Effects on intestinal microbial composition of mice:WT and Nur77^-/-mice showed no difference in fecal-diversity of colon flora,but significant difference in bray-curtis distance reflecting fecal-diversity?P<0.05?.Compared with WT,the abundance of Rikenella,Corynebacterium and Erysipelotrichaceae.Clostridium decreased in the intestinal microbial of Nur77^-/-mice,while that of Shigella,Allobaculum and Clostridiaceae.Clostridium increased.The results showed that the anti-inflammatory flora in the colon of Nur77^-/-mice decreased and the pro-inflammatory flora increased.2. The mice intestinal microbial composition and antibacterial peptide m RNA expression correlation analysis:Clostridiaceae.Clostridium in colonic chyme.Clostridium and jejunum by cell source of antimicrobial peptides?Defa1?Defa4?Defa5?cryptdin-1?cryptdin-2?cryptdin-4 and lyz-2?m RNA expression was significantly negative correlation?P<0.05?,but positively correlated with PLA2 and Defb2 m RNA expression in the colon?P<0.05?;Corynebacterium and Erysipelotrichaceae Clostridium were negatively correlated with the expression of colon Defb2 m RNA?P<0.05?;Shigella was negatively correlated with colonic lyz-2,reg3b and reg3g m RNA expression?P<0.05?,but positively correlated with PLA2 m RNA expression in the colon?P<0.05?,suggesting that Nur77 may regulate intestinal microbial by affecting the expression of antimicrobial peptides.3. Effects on intestinal microbial function in mice:KEGG analysis showed that,compared with WT mice,the biosynthetic function of betbettin and isoflavones in anti-inflammatory and anti-oxidant functions of the intestinal flora of Nur77^-/-mice may be weakened.In summary,the main conclusion of this study is:Nur77 is a key factor to maintain intestinal health.It improves the anti-infection performance of the intestinal against pathogenic bacteria by improving the number of jejunum Paneth cell and the expression of antimicrobial peptide gene in intestinal epithelial cells in mice,and by regulating the abundance of anti-inflammatory and antioxidant-related bacteria in the intestinal.