Effect And Mechanism Of TLR4 Antagonist TAK-242 Intervention On Experimental Colitis In Mice

Background and objective:Inflammatory bowel diseases?IBD?are characterized by chronic inflammation of the intestinal tract.Crohn'sdisease?CD?and ulcerative colitis?UC?are the most widely known types of IBD and have been the focus of attention due to their increasing incidence.Both Crohn's disease and ulcerative colitis are associated with multiple pathogenic factors including environmental changes,an array of susceptibility gene variants,a qualitatively and quantitatively abnormal gut microbiota and a broadly dysregulated immune response.Abnormal activation of immune in intestinal mucosal plays an important role of IBD.Nowadays,many researches on TLR4 signal transduction pathways are carried out,which offers a new opportunity to the treatment of IBD.TAK-242 is a kind of TLR4 antagonist and its effect on IBD has not been reported,so this study aims to investigate the effect of TAK-242 on DSS or TNBS-induced experimental colitis in mice and elucidate its possible mechanisms.Methods:?1?Groups:Eighty BALB/c mice were randomly divided into five groups?n=16 in each?as follows:?1?Control?2?DSS:DSS model+ddH₂O i.p.?3?DSS+TAK-242:DSS model+TAK-242 i.p.?4?TNBS:TNBS model+ddH₂O i.p.?5?TNBS+TAK-242:TNBS model+TAK-242 i.p.?2?Induction of Colitis:?1?DSS-induced colitis:BALB/c mice were given 5%dextran sodium sulfate?DSS?in drinking water for 7 days to induce colitis.?2?TNBS-induced colitis:BALB/c mice were fast but drink freely 24 hoursfirstly,ethyl ether anesthesia.While in a supine position,3.5F intravenous catheter was inserted 5.5cm from the anal verge,and TNBS 2.0mg/50%ethanol 100?l was delivered slowly.The mice were kept in a head-down position for 1 min.?3?Administration of TAK-242 intervention:On day 5,DSS+TAK-242 group and TNBS+TAK-242 group were givenTAK-242 solution?concentration:3 mg/kg?,i.p.injection,once daily for 3 days.The DSS group and TNBS group was given sterile water,i.p.injection,once daily for 3days.?4?Observation and detection of each index:?1?Observe the weight and disease activity index?DAI?of each group everyday;?2?Observe the macroscopic shape of colon in mice,and the change of pathology through HE staining;?3?The levels of IL-6,IL-1?,IFN-?,TNF-?in serum were measured by multiplex beads enzyme immunoassay.?4?The TLR4,MyD88,NF-?B p65,HMGB1,Foxp3 protein expression of colon tissues were detected by immunohistochemistry.?5?The TLR4,MyD88,NF-?B p65 and HMGB1 protein expression of colontissues were detected by western blot.Results:?.Effect of TAK-242 Intervention on Experimental Colitis Symptoms and Pathological Changes in Mice:1.Mice in the DSS and TNBS model groups developed typical symptoms of colitis such as diarrhea,bloody stools,and weight loss.On the 7^th day,with or without TAK-242 intervention,DAI in DSS and TNBS model groups were higher than the Control group?P<0.01?.With TAK-242 intervention,DAI was decreased in DSS+TAK-242 group compared with DSS group?P<0.01?.DAI was decreased in TNBS+TAK-242 group compared with TNBS group,but there was no significant difference?P>0.05?.2.The pathohistological injury score of colon characterized by the degrees of colonic inflammation,pathological depth and crypt destruction,in DSS and TNBS model group and their corresponding TAK-242 intervention group were higher than that in Control group?P<0.01?;In TAK-242 intervention group were lower than the corresponding model group?P<0.01?.?.Effects of TAK-242 Intervention on Serum Cytokines in Experimental Colitis in Mice:The levels of serum IFN-?,IL-1?and TNF-?in the DSS model group and the DSS+TAK-242 intervention group were significantly higher than that Control group?P<0.001,P<0.01?;The levels of IL-1?and TNF-?in the TAK-242 intervention group were lower than DSS model group?P<0.01?;The level of IL-6 in the DSS model group were higher than the Control group and the TAK-242 intervention group?P<0.001?,while there was no difference between the Control group and the TAK-242 intervention group?P>0.05?.The levels of IL-6,IFN-?,IL-1?and TNF-?levels in the TNBS model group were higher than Control group?P<0.01?.In the TNBS+TAK-242 group,IL-6 and IFN-?levels were lower than TNBS model group?P<0.001?;IL-1?and TNF-?levels were lower than TNBS model group but had no statistical significance?P>0.05?;As for IL-6,there was no significant difference between TNBS+TAK-242 group and Control group?P>0.05?.IFN-?,IL-1?,TNF-?levels were significantly higher than the Control group?P<0.001,P<0.001,P<0.01?.?.The effect of TAK-242 on TLR4/NF-?B signaling pathway in experimental colitis in mice:1.TLR4 expression in colonic mucosa:Immunohistochemical staining:the DSS and TNBS model groups were significantly higher than the Control group?P<0.001?.TAK-242 intervention group was significantly lower than the corresponding model group?P<0.001?,but still higher than the Control group?P<0.01?.Western blot:The expression of TLR4 in the DSS model group was significantly higher than that in the Control group?P<0.01?;the TAK-242 intervention group was significantly lower than the model group?P<0.01?,and there was no significant difference compared with the Control group?P>0.05?.The expression of TLR4 in the TNBS model group and the TNBS+TAK-242 intervention group was significantly higher than that in the Control group?P<0.01?.After TAK-242 intervention,compared with TNBS group,TLR4 expression was down-regulated,but had no statistical significance?P>0.05?.2.MyD88 expression in colonic mucosa:Immunohistochemical staining:the DSS and TNBS model groups were significantly higher than the Control group?P<0.001?.TAK-242 intervention group was significantly lower than the corresponding model group?P<0.001?,but still higher than the Control group?P<0.01?.Western blot:the expression of MyD88 in the DSS model group was significantly higher than that in the Control group?P<0.01?;the TAK-242intervention group was significantly lower than the model group?P<0.01?,and there was no significant difference compared with the Control group?P>0.05?.The expression of MyD88 in the TNBS model group and the TNBS+TAK-242intervention group was significantly higher than that in the Control group?P<0.01?.After TAK-242 intervention,compared with TNBS group,MyD88 expression was significantly down-regulated?P<0.01?.3.NF-?B p65 expression in colonic mucosa:Immunohistochemical staining:The DSS and TNBS model groups were significantly higher than the Control group?P<0.001?.TAK-242 intervention group was significantly lower than the corresponding model group?P<0.001?,but still higher than the Control group?P<0.01?.Western blot:The expression of NF-?Bp65 in the DSS model group was significantly higher than that in the Control group?P<0.01?;the TAK-242intervention group was significantly lower than the model group?P<0.01?,and there was no significant difference compared with the Control group?P>0.05?.The expression of NF-?Bp65 in the TNBS model group and the TNBS+TAK-242intervention group was significantly higher than that in the Control group?P<0.01?.After TAK-242 intervention,the expression of NF-?B p65 was significantly down-regulated compared with TNBS group?P<0.01?.4.HMGB1 expression in colonic mucosa:Immunohistochemical staining:the DSS and TNBS model groups were significantly higher than the Control group?P<0.001?.TAK-242 intervention group was significantly lower than the corresponding model group?P<0.001?,but still higher than the Control group?P<0.01?.Western blot:the expression of HMGB1 in the DSS model group was significantly higher than that in the Control group?P<0.01?;the TAK-242intervention group was significantly lower than the model group?P<0.01?,and there was no significant difference compared with the Control group?P>0.05?.The expression of HMGB1 in TNBS model group and TNBS+TAK-242 intervention group was significantly higher than that in Control group?P<0.01?.After TAK-242intervention,HMGB1 expression was significantly down-regulated compared with TNBS group?P<0.01?.5.Foxp3 expression in colonic mucosa:Immunohistochemical staining:the DSS and TNBS model groups were significantly lower than the Control group?P<0.001?.TAK-242 intervention group was significantly higher than the corresponding model group?P<0.001?,but still lower than the Control group?P<0.001?.Conclusion:1.The TLR4 antagonist TAK-242 has a therapeutic effect on experimental colitis in mice.2.The anti-inflammatory mechanism of TAK-242 on experimental colitis in mice may be inhibiting the TLR4/NF-?B signaling pathway and blocking the feedback loop of HMGB1/TLR4.