大黄蒽醌-半胱氨酸加合诱导的中药肝损伤机制研究

Anthraquinone-induced hepatotoxicity is the risk factor that affecting the security use of anthraquinone-containing herbs, such as rhubarb and radix polygoni multiflori. The mechanism of anthraquinone hepatotoxicity is still unclear and whether anthraquinones are the toxic factors or not is controversial. Therefore, researches on the hepatotoxicity of anthraquinone and the hepatotoxicity mechanism need to be further studied. We first discovered the anthraquinones-cysteine adduct in liver, which showed dose-and time-dependent along with emodin hepatotoxicity. Based on these findings, we proposed an original hypothesis that anthraquinone hepatotoxicity is probably induced by cysteine –related metabolic pathway disorder resulting from cysteine depletion. In this research, SD rats and human liver cells will be administered different doses of anthraquinone, anthraquinone mixture or anthraquinone-containing herb extracts. Anthraquinone –cysteine adduct, standard liver toxicology index, metabolic changes and proteome changes will be detected at different time points. Specially, cysteine-related metabolic pathway, such as glutathione synthesis, taurine metabolism and cysteine-containing protein metabolism, will be mainly detected. Based on these findings, we will systematically elaborated the mechanism of anthraquinone hepatotoxicity and discover specific anthraquinones hepatotoxicity biomarkers. This finding will provide scientific basis for toxicity evaluation and rational administration of anthraquinones containing drugs.

何首乌、大黄中大黄蒽醌的肝毒性问题是影响相关产品安全使用的风险因子，其致毒机制不清楚，是否致肝毒也存在争议，需要加强对其有可能产生的肝毒性及致毒机制研究。本课题首次发现大黄素可与肝脏内源性半胱氨酸形成加合物，而这种加合物呈现剂量和时间依赖性，与大黄素引起的肝细胞毒呈正相关。藉此提出大黄蒽醌肝损伤可能是由其引起半胱氨酸相关物质过度消耗及代谢异常，进而诱发肝毒性的原创性假说。本项目将应用体外人源性肝细胞模型和动物模型，给予不同剂量蒽醌单体、蒽醌混合物及含大黄蒽醌中药提取物，对不同时间点的生物样品进行蒽醌-半胱氨酸加合物检测、常规毒理指标检测、代谢组学和蛋白组学检测，筛选肝毒性差异标志物和差异代谢通路，阐述大黄蒽醌肝毒性机制。重点检测谷胱甘肽代谢通路、牛磺酸代谢通路异常及含半胱氨酸蛋白质的变性引起的功能异常及相关肝毒标志物，为含大黄蒽醌中药的毒性评价及安全、合理使用提供科学依据。

大黄蒽醌是何首乌、大黄致肝毒的风险因子，前期发现大黄蒽醌可与肝细胞内源性半胱氨酸形成加合物，导致谷胱甘肽代谢紊乱，引发肝细胞毒性，但其机制尚不清楚。基此，本项目利用蛋白组学和代谢组学技术对大黄蒽醌进行肝毒性机制研究并发现：1、大黄素与半胱氨酸结合形成加合物，通过抑制γ-GCS活性干扰GSH代谢进而引起肝损伤；2、大黄素可与核糖体蛋白直接结合，干扰蛋白质的正常合成和正确折叠，抑制富含半胱氨酸的组织蛋白酶的蛋白表达，导致内质网应激，启动固醇级联反应，干扰脂质代谢和花生四烯酸代谢，造成脂质在肝细胞内的过量堆积，诱发细胞炎症反应；3、大黄素可与ATP合酶直接结合，抑制TCA、脂肪酸β氧化和氧化磷酸化，抑制质子传输和ATP代谢，导致线粒体功能紊乱和ATP合成下降，进而开启了线粒体凋亡通路，导致肝细胞死亡；另外，线粒体功能紊乱也会促使ROS升高，导致氧化应激损伤；4、除大黄素外，代谢组学研究发现大黄酚可特异性调节胆汁酸合成和初级胆汁酸生物合成，大黄酸干扰亚麻酸和亚油酸代谢、柠檬烯和蒎烯降解和糖鞘脂代谢，大黄素甲醚对泛素和其他萜烯类-醌生物合成、络氨酸、苯丙氨酸、精氨酸、脯氨酸色氨酸、核黄素代谢具有调节作用。本项目共发表文章12篇，4篇为SCI杂志收录论文，7篇为中文核心期刊论文，1篇为会议论文，培养博士后1名，硕士3名。