TAK-242 Ameliorate Dss-induced UC And Effect On TLR4/JAK2/STAT3 Signaling Pathway And Gut Microbiota

Background:Ulcerative colitis(UC,Ulcerative Colitis)is characterized by chronic recurrent colonic mucosal inflammation.Intestinal flora has become a new therapeutic target of UC.Resatorvid(TAK-242)is a drug that specifically inhibits the small molecule TLR4(Toll-like receptors 4)and has anti-inflammatory effects.In UC,the imbalance of inflammatory response,microbial interaction and immune mechanism are rarely discussed.Therefore,the role of resatorvid(TAK-242)in ulcerative colitis is of valuable.Objective:To investigate the effects of TAK-242 on the gut microbiota and TLR4/ JAK2/STAT3 signaling pathway in DSS-induced mice colitis.Method:Fifty C57BL/6J mice were randomly divided into five groups(A= Normal group,B = DSS-induced colitis model group,C = DSS + TAK-242 3mg/kg,D = DSS +TAK-242 10mg/kg,E= 5-ASA group).All groups except group A were given 3% DSS and different doses of TAK-242 were intraperitoneally injected in TAK-242 treatment groups every other day for a total of 7 days.Group E was given 5-ASA(500mg/kg)once a day for 7 days.On the 8th day,eyeball blood was collected,mice were sacrificed,and colon and fresh colic feces were collected for analysis.Serum IL-17 and IL-10 levels were determined by ELISA.Intestinal histopathological changes were detected by HE staining,m RNA expressions of TLR4,JAK2 and STAT3 were detected by real-time PCR,protein expressions of TLR4,JAK2,STAT3,p-JAK2 and p-STAT3 were detected by Western blot,and intestinal flora was determined by16 Sr DNA analysis.Results:1.The surface of intestinal segment in the normal group was smooth with occasional edema.The overall response degree of colon inflammation in DSS group was more obvious than that in normal group.There were edema,redness and ulcers on the surface of intestinal segment.In the TAK-242 group,the colonic mucosa in the low-dose group showed slight hyperemia,edema and less ulcer,slightly less than that in the DSS model group.At high doses of TAK-242,compared with the model group,there was slight hyperemia and edema in the colonic mucosa,and the ulcer was reduced.In the 5-ASA group,compared with the model group,colon mucosal congestion and ulcer were significantly reduced.HE staining results showed that the structures of glands and crysts in the normal group were completely arranged and orderly,with a few inflammatory cells scattered in the tissues.In the DSS model group,the intestinal structure was severely damaged,the glands and crypts were severely reduced,and the arrangement was disordered.There were a large number of inflammatory cells infiltration,indicating that the ulcerative colitis model was successfully constructed.In the low-dose TAK-242 group,compared with the model group,the glands and crypts were clearer and more orderly,with inflammatory cell infiltration.In the high-dose TAK-242 group,compared with the model group,the glands were clear and orderly,and the crypts were reduced compared with the low-dose group,with inflammatory cell infiltration.Compared with the model group,the glands and crysts in the 5-ASA group were more clearly visible and arranged in a more tidy manner,with a few inflammatory cells infiltration.2.ELISA results showed that in the model group,the serum IL-17 level was higher compared with normal group,compared with model group,TAK-242 low-high-dose and the 5-ASA group was significantly lower(p < 0.001).Compared with normal group,serum IL-10 level in the model group was lower;compared with model group IL-10 level in the low-dose TAK-242 group was higher,but not significantly;IL-10 level in the high-dose TAK-242 group and 5-ASA group was significantly higher(p < 0.01).3.At the phylum level,Bacteroidetes,Firmicutes,Actinobacteria,,Epsilonbacteraeota and Proteobacteria were main in the five groups.The relative abundance of Bacteroidetes was similar between normal and DSS induced colitis mice gut,which was obviously decreased after treatment of TAK-242 and 5-ASA.The ratio of Bacteroidetes to Firmicutes got imbalanced in the fecal microbiota of DSS+3mg/kg TAK-242 group and DSS+10mg/kg TAK-242 group.Compared with normal group,Proteobacteria showed marked high relative abundance in DSS-induced mice but significantly decreased in the low and high dose of TAK-242 group.Cyanobacteria,Epsilonbacteraeota and Proteobacteria were significantly increased in DSS induced colitis mice,which were decreased after treatment of TAK-242 and 5-ASA.TAK-242 and 5-ASA significantly enhanced the relative abundance of Verrucomicrobia,while not detectable in normal and DSS colitis mice gut.Some specific bacterial biomarkers were identified at the genera level,Enterobacter,Paraprevotella,Oscillibacter and Ruminiclostridium showed significantly high relative abundance in DSS induced colitis mice,which was significantly decreased by TAK-242 treatment.Moreover,TAK-242 significantly promoted the growth of Akkermansia.Taking together,these results showed that TAK-242 could regulate the microbial composition in the intestine and enhanced the proliferation of certain bacteria such as Akkermansia.Conclusion:TAK-242 ameliorates DSS-induced colitis via TLR4/ JAK2/STAT3 signaling pathway.