基于NLRP3炎症小体的淫羊藿致免疫特异质肝损伤的多成分协同效应及其分子机制研究

Epimedii Folium（EF）is a traditional Chinese medicine that is non-toxic and restorative. However, liver injury caused by EF-related preparations has been reported frequently in recent years. In the past, studies on Hepatotoxicity of EF rigidly adhered to traditional toxicological models, and little attention has been paid to idiosyncratic TCM-induced liver injury, which may be mediated by organism factors. Preliminary studies of the research group confirmed the properties of immunological idiosyncratic liver injury in terms of EF and its preparation Zhuanggu Guanjie pills, and screened out four components of EF that can increase the activity of NLRP3 inflammasome induced by ATP, etc. To systematically elucidate the mechanism of EF-induced liver injury, the project intends to draw on the ample clinical resources in 302 hospital combined with the NLRP3 inflammasome activation model and mouse evaluation model of the idiosyncratic drug-induced liver injury to study the role of target components and their combinations in regulating NLRP3 inflammasome and its relationship with liver injury, clarify susceptible components of EF-induced liver injury and its synergistic effect, analyze the effect of susceptible components of liver injury on the expression, assembling and activation of the constituent proteins of NLRP3 inflammasome, and systematically explain its mechanisms and targets for inducing liver injury. On this basis, combined with possible susceptible population of EF-induced liver injury screened and verified through clinical samples, the prevention and control countermeasures for the clinical risk of liver damage in EF-related preparations were preliminarily proposed and formulated, which provides scientific basis for ensuring clinically safe and rational drug use.

淫羊藿为传统无毒补益类中药，但近年来相关制剂致肝损伤现象屡见报道。既往多拘泥于传统毒理学模式研究其毒性，对可能由机体因素介导的中药特异质肝损伤鲜有关注。课题组前期研究证实淫羊藿及其制剂壮骨关节丸的免疫特异质肝损伤属性，并且筛查发现淫羊藿中四种成分均可增加ATP等诱导的NLRP3炎症小体活性。为系统阐明淫羊藿致肝损伤的机制，本项目拟利用302医院丰富的临床病例资源并结合NLRP3炎症小体活化模型和药物特异质肝损伤小鼠评价模型，研究目标成分及其组合调控NLRP3炎症小体的作用及其与肝损伤的关系，明确淫羊藿肝损伤易感成分及其协同效应，分析肝损伤易感成分对NLRP3炎症小体组成蛋白表达及组装活化的影响，系统阐释其诱发肝损伤的机制和靶标。在此基础上，结合临床样本筛查验证淫羊藿肝损伤可能的易感人群，初步提出并制定淫羊藿相关制剂肝损伤临床风险防控对策，为保障临床安全合理用药提供科学依据。

近年来，中草药肝损伤事件频发，尤其是何首乌、补骨脂和淫羊藿等传统无毒中药致肝损伤问题引起了国内外广泛关注，严重制约了中医药事业健康发展和国际化。前期课题组研究证实淫羊藿所致肝损伤具有免疫特异质肝损伤属性且其发生与NLRP3炎症小体活化相关，为此，本项提出从NLRP3炎症小体角度开展淫羊藿致免疫特异质肝损伤的成因机制研究。本项目首先结合临床病例研究揭示了淫羊藿相关制剂致肝损伤的免疫特异质属性，并且利用免疫特异质肝损伤评价模型研究发现了淫羊藿和补骨脂为一对新的中药配伍禁忌组合并揭示了其产生的病证基础；利用构建的NLRP3 炎症小体活化模型筛查发现淫羊藿成分淫羊藿次苷II、淫羊藿次苷I和朝藿定B均可特异性增强NLRP3炎症小体活性诱发肝损伤并分别揭示其活化炎症小体机制，同时研究发现淫羊藿次苷II具有一定直接毒性，从而证实淫羊藿所致肝损伤是机体免疫应激状态下，淫羊藿中直接毒性成分与免疫活性成分协同所导致的，由此提出了淫羊藿致免疫特异质肝损伤“三因致毒”机制假说；进一步，针对淫羊藿配伍补骨脂致肝损伤机制研究发现，补骨脂多个成分可通过活化不同炎症小体类型诱发肝损伤，由此揭示了淫羊藿和补骨脂多成分协同诱发免疫特异质肝损伤的效应机制，为两者配伍用药风险防控对策制定提供了证据。针对淫羊藿致肝损伤成因机制，本项目提出了基于成分效应靶标互作的中西药配伍/联用控毒策略和方法，发现了系列抑制炎症小体活性的中药及其活性成分，同时发现了多个靶向活化NLRP3炎症小体诱导肝损伤的化药，由此提出了淫羊藿配伍/联用控毒策略和方法，本项目为淫羊藿临床安全合理用药提出了科学依据，研究将促进淫羊藿临床精准用药，降低临床用药风险。