吡咯里西啶生物碱肝脏毒性机理研究

With the globalization of traditional Chinese medicine (TCM), safety of TCM use and evaluation of toxic TCM as well as toxic components of TCM are in an urgent need. Pyrrolizidine alkaloids (PAs) occur in a wide range of plants, and many TCM herbs contain varieties of PAs. PAs are known to be hepatotoxins, and PA-poisoning cases and even mortality cases are often seen in clinic. PAs are considered to be non-toxic themselves, and they are metabolized by cytochromes P450 to electrophilic species that are responsible for the toxicity. However, the detailed mechanisms of PA-induced hepatotoxicity remain unknown. We hypothesize that PAs are biotransformed to reactive metabolites which adduct proteins and that the dysfunction of critical proteins, resulting from the protein modification, triggers the process of cell death. To probe the hypothesis, we will perform the following studies, including 1) preparation of highly selective and sensitive antibodies that recognize proteins modified by reactive metabolites of PAs; 2) identification of critical adducted proteins associated with PA-induced liver toxicity; 3) verification of the role of P450 3A enzymes in PA-induced hepatotoxicity; 4) examination of the participation of metabolic oxidation in hepatotoxicity of PAs; and 5) determination of structure-activity (toxicity) relationships of PAs. This application combines multiple disciplines that allows us to elucidate the molecular mechanisms of PA toxicity and to provide scientific foundation for clinic treatment of PA poisoning and for the prediction of drug safety as well.

随着中药的全球化，保证中药使用的安全性和评价有毒中药和中药毒性成分已刻不容缓。吡咯里西啶生物碱（PA）存在于许多常用中药里，PA具肝脏毒性，因服用含PA中药引起的急性肝损伤甚至中毒死亡时常发生。一般认为PA本身无毒，进入体内后被P450酶代谢成的活性代谢产物才是真正的致毒物质，但具体致毒机理尚未清楚。我们提出假说：PA在肝脏被代谢成具化学活性的代谢产物，进而与蛋白反应，使蛋白质功能丧失，最终细胞死亡。为验证该假说，本项目将1）研制具高选择性高灵敏度的抗体检测与PA代谢产物结合的蛋白质；2）鉴定与PA肝毒性相关的结合有PA活性代谢产物的蛋白；3）研究P450 3A酶族在PA致肝毒性作用；4）研究代谢氧化在PA致肝毒性中的作用；5）解析PA化学结构－肝脏毒性的相关性。本项目将通过多学科交叉研究，阐明PA肝毒性的分子机理，为临床PA中毒诊断和治疗方案提供理论依据，也为药物安全性预测提供理论基础。

吡咯里西啶化合物（pyrrolizidine alkaloid，PA）是一类生物碱，我国多种常用中药含有PA。PA引起的急性肝炎临床时有报道，已有多起中毒死亡案例。本课题的主要内容是研究PA的反应性代谢产物与生物分子的相互作用，剖析PA肝毒性的作用机制，其目的是通过这些机制研究预防和干预临床上由于PA引起的肝脏损伤。.我们已成功地制备和纯化了具高选择性抗体，用于印迹检测和免疫组织化学检测。通过倒千里光碱和野百合碱的肝毒性差异和不同年龄对PA毒性敏感性的差异深层次地阐明PA致毒机制。在体内外发现PA经代谢活化后与肝脏蛋白质的赖氨酸残基形成共价结合，还发现PA的反应性代谢产物可导致DNA-蛋白质形成交联，DNA的碱基包括脱氧鸟苷和脱氧腺苷，蛋白质的氨基酸残基包括半胱氨酸和赖氨酸。发现PA被代谢活化后与RNA形成共价结合，参与共价结合的RNA碱基包括腺苷和鸟苷。还发现给用PAs可导致RNA-蛋白质形成交联。我们还发现PAs导致发育毒性，PA可在胎肝中代谢活化导致肝毒性。这些研究进展使得我们对PAs致毒机制有了更深的了解。