Formononetin Alleviates Dextran Sulfate Sodium Induced Acute Colitis In Mice Via NLRP3 Inflammasome Pathway

Inflammatory bowel disease(IBD)is a kind of recurrent and chronic noninfectious inflammatory disease,mainly consisting of ulcerative colitis and Crohn's disease.Epidemiological data showed that IBD has become a global disease,although the incidence of IBD in high-risk areas tends to be stable,the incidence in low-risk areas such as southern Europe,Asia and most developing countries is increasing.The specific mechanism of IBD is still unclear,but there is no doubt that intestinal mucosal immune dysfunction plays an important role in the pathogenesis of IBD.Hence,seeking for novel immunomodulatory drugs has been the focus of the clinical prevention and treatment of IBD.Formononetin(For)is a kind of isoflavone compound and widely exists in natural plants(mainly exists in red clover).The existing research evidences indicate that For owns the effects of regulating lipid metabolism,anti-inflammatory,anti-oxidative,anti-tumor and promoting apoptosis.While there has no report on whether For plays a protective role in acute colitis in mice.In this study,we aim to explore the protective effects of formononetin on DSS-induced acute colitis in mice and the potential mechanism.Part 1.Formononetin protects dextran sulfate sodium induced acute colitis in miceObjective.In this study,we aim to explore the protective effects of For on dextran sulfate sodium(DSS)induced acute colitis in mice.Methods(1)The mice acute colitis was induced by 2.5%DSS then different doses of For(25g/kg,50g/kg,100g/kg)were administrated to the colitis mice.Body weight,the disease activity index(DAI)score,pathological injury,the expressions of colonic tight junction protein ZO-1,claudin-1,occludin in translation levels were examined and compared in mice of different groups.(2)TNF-a was used to establish the cell injury model and HCT116 cells were incubated with different concentrations of For(25M?50M)or 0.1%DMSO(vehicle).Then the expressions of inflammatory cytokines IL-1? and IL-6 mRNA level and the protein expressions of colonic tight junction protein ZO-1,claudin-1,occludin were examined and compared between different groups.Results(1)As expected,DSS feeding could cause colonic injury and weight loss,bloody stool and watery diarrhea could be observed in mice.While,For could alleviate the mice colitis in a dose-dependent manner,mainly manifesting as relieved clinical symptoms of colitis,mitigated colonic epithelial cell injury,up-regulations of colonic tight junction protein ZO-1,claudin-1,occluding in translation levels.(2)TNF-a was used to establish the in vitro colonic cell injury model,and For could prevent the colonic cells from injury dose-dependently,specifically manifesting as the increased expressions of colonic tight junction proteins ZO-1,claudin-1,occludin together with the decreased expressions of inflammatory cytokines IL-1? and IL-6 mRNA.Conclusion.For could protect against DSS-induce mice acute colitis in mice.Part2.The mechanism of For protects dextran sulfate sodium induced acute colitis in miceObjective In this study,we aim to explore the potential mechanism of For on dextran sulfate sodium(DSS)induced acute colitis in mice.Methods(1)The mice acute colitis was induced by 2.5%DSS then different doses of For(25g/kg,50g/kg,100g/kg)were administrated to the colitis mice.the expressions of NLRP3 pathway proteins(NLRP3,ASC,IL-1?,Caspase-1)in colonic epithelial cells were examined and compared in mice of different groups.(2)TNF-a was used to establish the cell injury model and HCT116 cells were incubated with different concentrations of formononetin(25M.50M)or 0.1%DMSO(vehicle).Then the expressions of NLRP3 pathway proteins(NLRP3,ASC,IL-1?,Caspase-1)were examined and compared between different groups.(3)In the mechanism research,NLRP3 inhibitor MCC950 was adopted to elucidate the mechanism underlying the protective effects of For on mice acute colitis.The DSS-induced colitis mice were disposed to three different groups:control group,MCC950(50mg/kg)group,and MCC950+For group(100mg/kg For+50mg/kg MCC950)respectively,and body weight,the DAI score and pathological injury in mice were observed.Results(1)For down-regulation of NLRP3 pathway proteins(NLRP3,ASC,IL-1?)in a dose-dependent manner in mice.(2)For decreased expressions of NLRP3 pathway proteins NLRP3,ASC,IL-1?.(3)NLRP3 specific inhibitor MCC950 could alleviate the DSS-induced mice acute colitis.however,in the basis of administrating MCC950,we failed to observe the protective effects of 100mg/kg For on mice acute colitis,mainly manifesting as no significant changes in the body weight,clinical symptoms and colonic injury of mice.ConclusionFor protect against DSS-induce mice acute colitis via negative regulation of NLRP3 pathway.