从核受体—代谢调控网络新视角探索雷公藤复方配伍“异类相制”减毒的分子机制

The project takes Qingluo Tongbi formula (Tripterygium wilfordii compound, TW compound), which is developed from the experience of professor Zhou Zhongying as the object, and performs the research on the clinical prominent problem--hepatotoxicity, and integrates several methods and techniques, such as pharmacology and toxicology, metabonomics, molecular biology and other disciplines. Our previous finished project found that the detoxicity effect of compound compatibility is possibly relevant to the metabolism process of Tripterygium wilfordii(TW) and regulation of endogenous glucolipide metabolism. Thus, we put forward an innovative idea----"nuclear receptor-metabolism regulatory network", and will observe the influence of TW compound compatibility on exogenous metabolic enzymes, by comparing the difference of the main metabolites and toxicity of TW before and after compatibility with other drugs, both in vivo and in vitro; moreover, analyze the change of endogenous metabolism before and after the compound compatibility by metabonomics; Furthermore, study the interaction and regulation network of endogenous/exogenous metabolic enzymes with relevant nuclear receptors by gene knockout and silencing technology. The project combines the overall characterization of metabolic fingerprint and the discovery and verification of specific key metabolic pathways, in order to reveal the molecular mechanism of detoxicity by “Diversity Restriction” of compound compatibility from the "nuclear receptor-metabolism regulatory network" point of view, and provide clinical guidance of safe and reasonable application of TW and other toxic herbs.

本项目以国医大师周仲瑛经验方—清络通痹方（雷公藤复方）为载体，选择雷公藤临床突出的肝毒性为切入点，集成药理毒理学、代谢组学、分子生物学等多学科的方法和技术开展研究。已结题项目的研究发现，复方配伍减毒效应的实现可能与影响雷公藤药物代谢过程和调节内源性糖、脂的代谢相关。在此基础上进一步提出“核受体－代谢调控网络”的研究思路，通过雷公藤与不同功效药物的配伍，结合体内外实验，比较复方配伍前后雷公藤主要代谢产物的变化及毒性差异，观察雷公藤复方配伍对外源性代谢酶的影响；采用代谢组学方法分析复方配伍前后内源性物质代谢的变化；并通过基因敲除和基因沉默等技术沟通内/外源代谢酶与相关核受体交互调控的网络关系。本项目将代谢指纹图谱的整体表征与特定关键代谢通路的发现、验证相结合，从核受体-代谢调控网络角度揭示复方配伍“异类相制”减毒的分子机制，为指导雷公藤类有毒中药的临床安全合理应用提供示范。

雷公藤是治疗风湿免疫病的常用药物，但其毒副作用明显。本项目根据复方配伍应用雷公藤的临床实践所提出的“异类相制”理论，在前期研究的基础上，以清络通痹方为载体，选择临床常见的肝毒性为切入点，从核受体-代谢调控网络角度揭示复方配伍减毒的分子机制。.研究结果显示：雷公藤或雷公藤甲素可引起大鼠肝功能指标异常，提高大鼠血清糖脂水平，损伤肝细胞亚细胞结构（线粒体、内质网），降低肝脏中抗氧化酶活性，诱发过氧化损伤，导致肝细胞出现坏死、水肿、脂肪变性和炎细胞浸润等为主的病变；而清络通痹方或其组分配伍均能有效改善上述异常改变，尤以清络通痹全方效果最优。通过研究清络通痹方对雷公藤代谢产物的影响，分析血浆中生物碱的暴露水平，发现了雷公藤生物碱成分55个，表明大部分的生物碱成分能够入血，发挥效应，其中22个成分暴露时间超过12h,可能是雷公藤引起肝毒性的物质基础，而雷公藤组和清络通痹方组大鼠粪便和尿液中的生物碱成分并无显著差异，提示复方配伍未改变雷公藤生物碱在体内的暴露。从代谢轮廓的角度，分析雷公藤配伍前后HepaRG细胞及大鼠代谢谱的变化，进一步筛选雷公藤肝毒性潜在生物标志物，发现清络通痹方组各生物样本观测值距离空白对照组最近，说明清络通痹方能够纠正雷公藤引起的肝损伤，并鉴定出雷公藤在血清中及肝组织匀浆中的特异性代谢生物标志物，多为脂肪、蛋白质和糖三大物质代谢的中间产物。减毒机制的研究结果提示：清络通痹方配伍能调节药物代谢酶及多种亚酶活性、表达和定位；也可调节代谢酶基因表达及肝脏核受体转录水平，通过调控核受体-代谢酶减轻雷公藤肝毒性，发挥“异类相制”配伍减毒作用。.项目研究从核受体-代谢调控网络角度阐明了中药复方配伍减轻雷公藤肝毒性的机制，较深入地阐释“异类相制”理论的科学内涵，对雷公藤的临床应用及有毒中药的配伍减毒研究具有指导作用和重要的理论意义。