大黄素通过靶向结合BCL-10抑制脓毒症小鼠中性粒细胞胞外诱捕网激活的机制研究

脓毒症是ICU内致死的重要疾病，免疫失衡和凝血功能障碍是核心病机。中性粒细胞胞外诱捕网（NETs）是介导免疫-凝血的关键环节。中药大黄是治疗危重症疾病的明星药物，大黄素是其重要物质基础，但目前缺乏大黄素关键作用靶点的研究。前期证实大黄素具有抗炎和抗血栓作用，抑制NETs标志物NE生成，B细胞淋巴瘤因子10(BCL-10)是潜在靶点。本研究旨在阐明大黄素对NETs生成的干预和机制，体内实验通过构建EμSR-BCL10和BCL10-/-小鼠，CLP手术复制脓毒症模型，考察大黄素以BCL-10为靶点对脓毒症中性粒细胞激活、凝血障碍和NETs生成的调控，阐明BCL-10参与NETs形成的机理。体外借助计算机模拟和蛋白位点突变等技术研究大黄素靶向结合BCL-10及调控CBM复合体/NF-κB信号通路的作用，揭示大黄素抑制NETs形成的机制，为大黄素在临床的合理应用和脓毒症防治提供科学依据和潜在靶点。

Sepsis is a fatal disease in ICU. Immune imbalance and coagulation disorder are two core pathogenesis. Neutrophil extracellular trap nets (NETs) play key role in dysfunction of immune and coagulation system. Emodin is the principal constituent of Rhubarb, which has long been recorded as a star drug for the treatment of critical diseases in traditional Chinese medicine. However, there are very limited researches focused on the direct target and regulatory mechanism of emodin. Our previous studies have confirmed that emodin has promising anti-inflammatory and anti-thrombotic properties. Moreover, we found that emodin treatment significantly suppresses the production of neutrophil elastase (NE), a well documented NETs marker. Via detection of HuProt(TM) microarray and pulldown experiment, we identified that B cell lymphoma/leukemia 10 (BCL-10) is a potential target of emodin. The aim of this study is to elucidate the intervention of emodin on NETs production in sepsis and its underlying mechanisms. In vivo experiments, CLP operation is employed to promote sepsis in mice. We constructed EuSR-BCL10 overexpression and BCL10-/- knockout mice to better understand the role of BCL-10 in the generation of NETs and in the regulatory effect of emodin on excessive activation of neutrophils, coagulation dysfunction and over-production of NETs in septic mice. In vitro experiments, neutrophils are harvested from wildtype, EuSR-BCL10 overexpression and BCL10-/- knockout mice. By employing computer simulation and protein site-directed mutagenesis, we tried to further characterize the interaction between emodin and BCL-10. Moreover, we attempt to understand how domdin prevents production of NETs in sepsis via detecting regulatory effect of emodin on CBM complex/NF-kappa B signaling pathway. This study could bring new insight into the molecular mechanism of emodin on inhibiting NETs formation, and thus provide noval target and scientific basis for rational application of emodin when treating septic patients.