基于人类白细胞抗原HLA-B*35:01的何首乌肝毒性物质基础研究

The reports of Polygonum multiflorum (PM)-induced liver injury have been increasing in China in recent years. There still have been insufficient studies about PM-induced liver injury so far, due to the unclear material basis, in vivo process, targets and mechanisms. According to clinical reports, PM-induced liver injury is probably a typical Idiosyncratic liver injur. Human leucocyte antigen, which distinguishes between xenogenic and autogeneic substances, is highly polymorphic. HLA has been proved to be related to a few drug adverse rections in clinic. In our previous study, HLA-B*35:01 was found to be associated with PM-induced liver injury. Herein, the research starts from pharmacogenetics and pharmacogenomics. Firstly, liquid chromatography tandem mass spectrometry analysis methods of Polygonum multiflorum components and hepatotoxicity models will be established in vitro and in vivo. The next step is to qualitatively and quantitatively investigate the main material basis of PM-induced hepatotoxicity from the perspective of pharmacokinetics and pharmacodynamics. Finally, the relationship between HLA susceptibility gene and the specific components of Polygonum multiflorum which induce the liver injury will be elucidate. The study will provide novel and common methods for studies of material basis and mechanisms of drug adverse reaction induced by Chinese herbal medicines.

近年来国内有关何首乌肝毒性的报道日益增多。目前针对何首乌致肝损害的研究仍比较匮乏，其肝毒性物质基础，体内过程，作用机制等不明确。临床调查提示，何首乌可能为典型的特异质肝损害。人类白细胞抗原是人类最丰富的多态性基因，是免疫系统区分本体和异体物质的基础。临床研究表明，药物不良反应与HLA介导的免疫反应、基因的易感性有关。本项目前期研究发现人类白细胞抗原等位基因位点HLA-B*35:01与何首乌肝毒性相关。本研究以遗传药理学与药物基因组学为切入点，首先建立基于液质联用技术的何首乌体内外物质成分分析方法及基于HLA-B*35:01的何首乌致肝损害的药效评价模型；然后从药代、药效两方面对定性、定量地探究基于HLA-B*35:01的何首乌肝毒性主要物质基础；最终阐明引起肝毒性的物质成分与肝毒性HLA易感基因的关系，为中药不良反应发生机制和物质基础研究提供新的的研究思路和方法。

何首乌是引起药物性肝损伤的最具代表性中药之一，明确其肝损伤的生物标志物及物质基础对何首乌肝损伤的防治有重要意义。本项目运用药物基因组学方法，通过病例对照研究和队列研究发现并证实了HLA-B*35:01是何首乌肝损伤的特异性生物标志物，此外还发现HLA-DRB1*15:01和 HLA-DRB5*01:01与HLA-B*35:01携带者何首乌肝损伤的严重程度相关。我们采用肝S9体外代谢孵育体系结合液质联用技术建立了何首乌主要成分及其代谢产物的定性定量方法，构建了特异性表达不同HLA-I类分子的C1R细胞株用于基于HLA-B*35:01的何首乌肝损伤物质基础的体外筛选，我们利用免疫亲和层析技术，分离纯化不同C1R细胞的HLA-B*35:01分子和HLA-B*35:03分子，超滤离心使何首乌原型成分和/或代谢成分与HLA分子解离。分离的成分经HPLC-MS/MS测定，对比与HLA-B*35:01分子和HLA-B*35:03分子结合的成分差异，筛选并鉴定出与HLA-B*35:01分子和HLA-B*35:03分子结合的差异性成分，主要是何首乌中的蒽醌类衍生物及其II相代谢产物如大黄素、大黄素葡萄糖苷、大黄素葡萄糖醛酸苷等。我们利用表达HLA-B*35:01的人源化转基因小鼠对HLA-B*35:01特异性的何首乌肝毒性成分进行确证，发现HLA-B*35:01转基因小鼠对大黄素引起的肝损伤的敏感性以及CD8细胞增殖水平明显高于野生型小鼠，表明大黄素可能通过HLA-B*35:01特异性途径激活CD8 T细胞引起细胞毒作用产生肝损伤。