白头翁皂苷B4治疗结肠炎作用的新发现及其调节中性粒细胞募集的分子机制研究

炎症性肠病（IBD）在我国的发病率呈逐年上升趋势。治疗上，包括氨基水杨酸、激素、免疫调节剂和生物制剂等。耐药性、反复复发以及并发症是IBD治疗中的主要问题。白头翁是治疗结肠炎的常用中药。我们发现白头翁皂苷B4（AB4，药材中的指标性成分）可抑制S100A9蛋白的表达，抑制中性粒细胞募集，对结肠炎的治疗作用略优于美沙拉嗪。据此，我们推测S100A9/MAPK/NF-κB是AB4治疗结肠炎的关键信号通路。为证实该假说，我们采用了生物素-AB4探针结合蛋白质组学技术钩钓并鉴定出8个AB4的直接结合蛋白，其中蛋白plakophilin-1的改变最为显著，且能多次重复。本课题拟进一步采用多种分子生物学技术筛选并确证AB4的靶蛋白，深入探讨靶蛋白对S100A9所在的信号通路和中性粒细胞募集的影响，阐明其治疗结肠炎的分子机制。本项目的顺利实施将为结肠炎治疗新药的研发提供作用机制独特的候选药物分子。

The incidence of inflammatory bowel diseases (IBD) is increasing and has become a common digestive disease in recent years in China. The treatment of IBD includes aminosalicylic acid, hormones, immunomodulators and biological agents. Drug resistance, repeated relapses, and complications are major issues in the treatment of IBD. Pulsatilla chinensis (Bunge) Regel is commonly used for the treatment of colitis in Traditional Chinese medicine. In our preliminary study, we found that anemoside B4, the most abundant in this herb, exhibited significant anti-inflammatory activity and inhibited neutrophils recruitment, had a good therapeutic effect on trinitrobenzene sulfonic acid-induced colitis in rats. Its effect was slightly better than that of mesalazine. Our preliminary results showed that anemoside B4 inhibited the expression of S100A9 protein and neutrophils recruitment. Therefore, we speculate that anemoside B4 may regulate neutrophil recruitment by inhibiting the activation of the S100A9/MAPK/NF-κB pathway during the treatment of colitis. In order to prove this hypothesis, this project will explore the roles of anemoside B4 in the activation of S100A9/MAPK/NF-κB signaling pathway and the influence of neutrophil recruitment. To confirm this hypothesis, we used biotin-AB4 probe combined with proteomics to hook and identify several direct-acting proteins of AB4. The data showed that there were 8 proteins which belonged to direct-acting proteins of AB4. Among them, the expression of plakophilin 1 protein significantly decreased, compared with free AB4 group from 3 repeated experiments. In this project, we plan to further use a variety of molecular biology techniques to screen and confirm the target protein of AB4, and further study the effects of target protein on the S100A9/MAPK/NF-κB signaling pathway and neutrophil recruitment. Then, the molecular mechanism of AB4 for treatment of colitis will be elucidated. In all, the successful implementation of this project may provide a candidate drug molecule for the research and development of new drugs treating colitis with a new target.