TLR4 Gene Deletion Promotes DSS-induced Intestinal Epithelial Injury And Changes Of Intestinal Flora

Objective: Informatory bowel disease(IBD),a specific inflammatory disease of the gastrointestinal tract whose cause is unknown and whose pathogenesis is unclear,refers to Crohn’s disease(CD)and ulcerative colitis(UC).According to literature reports,disturbances in the intestinal flora may be an important cause of the occurrence of IBD.IBD places a huge burden on human health,and our main focus at this stage is to provide potential therapeutic strategies for the clinic.The objective of this study was to explore the use of TLR4 as a new therapeutic target to provide a relevant basis for the treatment of inflammatory bowel disease.method:1.Experiment grouping:Six 6-week-old C57BL/6 wild type(WT)mice and six Toll-like receptor 4-Knock out(TLR4-KO)mice were selected for phenotypic experiments.Another twelve WT mice and twelve TLR4-KO mice were selected for survival experiments.2.Construction of DSS-induced IBD mouse model:Establishment of a DSS-induced mouse model of inflammatory bowel disease: WT mice and TLR4-KO mice fasted and drank for 1 day,and dextran sulfate sodium(DSS,40,000 k Da,ICN biomass,USA)was added to the mice’s drinking water to make the concentration 1.5%(1.5% DSS),and after 7 days of continuous molding,the mice were independently reared and drank normally.3.Observe and detect various indicators of mice:1)Record the amount of DSS water and food intake of the two groups of mice in the phenotypic experiment(unspecified are observation indicators in the phenotypic study experiment);2)changes in the Disease Active Index(DAI)of two groups of mice;3)Peripheral blood cell analysis of two groups of mice;4)Colonic bleeding in two groups of mice;5)changes in the histopathology of the colon in two groups of mice;6)Record the number of deaths of two groups of mice in the survival experiment daily to calculate the survival rate;7)16S r RNA sequencing of two sets of mouse fecal samples.Result:(1)The amount of DSS and the amount of food eaten by the two groups of mice:After 1.5% DSS orally for 5 consecutive days,the feeding and drinking capacity of TLR4-KO mice was significantly worse than that of WT mice(P<0.05).(2)Changes in DAI of two groups of mice: THE DAI integral of TLR4-KO mice was significantly greater than that of WT mice(P<0.05).(3)Peripheral blood cell analysis of two groups of mice in phenotypic experiments:After 5 days of oral administration of 1.5% DSS,the peripheral red blood cell count of TLR4-KO mice suggested more severe anemia(P<0.05).(4)Changes in colonic bleeding in two groups of mice: oral administration of 1.5%DSS for 7 days,the degree of colonic bleeding in TLR4-KO mice was more severe than that of WT mice(P<0.05).(5)Colonic histopathological changes in the two groups of mice: the degree of colonic inflammation,crypt destruction and lesion depth of TLR4-KO mice were significantly higher than those of WT mice(P<0.05).(6)The survival rate of two groups of mice in the survival experiment: the survival rate of TLR4-KO mice was significantly reduced compared with WT mice(P<0.05).(7)16S r RNA sequencing results of two sets of mouse fecal samples:(1)Changes in the number of intestinal flora in miceThe intestinal flora of mice was significantly different,and the number of intestinal flora in TLR4-KO mice was smaller than that of WT mice.(2)Changes in the diversity of intestinal microflora in miceThe diversity of microflora within the intestinal community of TLR4-KO mice was not significantly different from that of WT mice,and the diversity of microflora between different communities of TLR4-KO mice was significantly different from that of WT mice.(3)Differences in intestinal microbial species in TLR4-KO mice and WT miceWT mice were significantly more abundant at the genus level than TLR4-KO mice,while deferroid bacteria,nematodes,and Proteus were significantly more abundant than WT mice in TLR4-KO mice.Conclusion:1.TLR4 gene deletion significantly promotes DSS-induced intestinal epithelial injury.2.TLR4 gene deletion lead to intestinal flora disorder in mice and may further aggravate DSS-induced intestinal epithelial damage.