Anemoside B4 Ameliorates Inflammatory Bowel Disease Through Suppressing The S100A9/NF-?B Signaling Pathway

Objective:Inflammatory bowel disease(IBD)including ulcerative colitis and Crohn's disease,which is characterized by intestinal inflammation and tissue damage.Because of the lack of a thorough cure in the clinic,IBD is called "green cancer",and about 20%of IBD patients will progress to colorectal cancer.Despite the increased morbidity of IBD in China,available treatments remain unsatisfactory,so it's urge to find more effective therapeutic strategies.Pulsatilla chinensis(Bunge)Regel,a Chinese herb with the functions of clearing heat and detoxifying,has been widely used for treating inflamed intestinal disease,including IBD for thousand years in China,but it is unclear which compound of it is responsible for treatment of IBD.In this study,in vivo and in vitro IBD models were established to evaluate the therapeutic effects of anemoside B4 on inflammatory bowel disease,and elucidate the underlying mechanism of its activity,which may provide an active candidate molecule for the development of new drugs for the treatment of IBD.Methods:To evaluate the therapeutic effects of anemoside B4 on treating IBD,three in vivo IBD models were established by acetic acid,TNBS and DSS,body weight,DAI score,coefficient of colon weight,colonic structure and inflammatory cytokines were used as evaluated indicators.To explore its underlying mechanism,label-free quantitative proteomic analysis was used to identify the differentially regulated proteins by anemoside B4 upon the colon tissues of rats with TNBS-induced colitis.Western blotting was used to research the effect of anemoside B4 on the downstream molecular of S100A9;we use qPCR experiment to detect whether anemoside B4 affects the mRNA expression of S100A9 and chemokine,and immunofluorescence was used to investigate the effects of anemoside B4 on colonic mucosal cell proliferation,apoptosis and neutrophil aggregation.Results:In TNBS-induced colitis experiment,anemoside B4 could significantly inhibit the weight loss,the DAI score,intestinal mass coefficient,release of cytokines of TNF-?,IL-6,IL-1?,CXCL-1,CXCL-2 in colon tissue of colitis rats,and ameliorated the pathological structure,cell proliferation,cell apoptosis,neutrophil aggregation and MPO activity in colons with colitis.In acetic acid or DSS-induced colitis model,anemoside B4 also showed similar therapeutic effects,which was comparable to the positive control drug,mesalazine.Label-free quantitative proteomic analyses of six colon samples discovered that 56 proteins were significantly altered by anemoside B4,which were mainly clustered in TCA cycle and respiratory electron transport chain.Among them,S100A9 is the most significantly downregulated protein and associated with NF-?B and MAPK signaling pathways in the pathogenesis of colitis.Further experiments revealed that anemoside B4 suppressed S100A9 and the expression of its downstream genes including TLR4,NF-?B,p-JNK,IL-6 and NOD2 in colon samples.Moreover,we found that anemoside B4 significantly inhibited activation of MAPK/NF-?B pathway after stimulated with LPS or S100A9.Conclusions:Our data indicate that the therapeutic effects of anemoside B4 on IBD may be related to its inhibitory effect on S100A9/NF-?B signaling pathway Therefore,these results could provide experimental basis for B4 treatment of IBD.