基于循环exosomes的川楝子肝毒性生物标志物发现及毒性机制研究

Drug-induced liver injury has been the most frequently adverse reaction of traditional Chinese medicine. Nevertheless, it is difficult to accurately identify toxic components and mechanisms of traditional Chinese medicine-induced hepatotoxicity as there is a lack of effective screening and evaluation methods, which is the key bottleneck of modernization and internationalization of traditional Chinese medicine. As exosomes are expected to bring a major breakthrough in the field of drug-induced hepatotoxicity, this project first proposes that miRNAs distribution patterns inside or outside of circulating exosomes may reflect the mechanisms of traditional Chinese medicine-induced hepatotoxicity and identify new biomarkers. As traditional Chinese medicine is a multi-component complex system, this project will investigate the dose- and time-dependent phenotypes and mechanisms of the components or single component of fructus toosendan-induced hepatotoxicity. The expression levels of miRNAs inside or liver-specific miRNAs outside of circulating exosomes of each treatment will be detected. The key molecular events and the mechanisms of fructus toosendan-induced hepatotoxicity will be determined. In order to identify cross-species biomarkers, exosomes-based potential biomarkers will be verified on cellular level. This project will establish a new method to evaluate traditional Chinese medicine-induced hepatotoxicity based on circulating exosomes.

药物性肝损伤是中药的主要不良反应，但当前由于缺乏有效的中药肝毒性评价方法，难以对中药肝毒性的物质基础及毒性机制进行准确识别与研究，阻碍了中药现代化与国际化的发展。本项目基于exosomes有望为药物肝毒性评价领域带来重大突破的前景，提出一种利用循环exosom-es内部和外部miRNAs的分子模式来识别中药肝毒性机制及发现毒性生物标志物的全新方法，从中药“多组分复杂体系”的特点出发，着重研究川楝子组分/单一成分在作用时间及剂量依赖下的小鼠肝毒性表型及机制的异同，系统分析血清exosomes内部miRNAs分子的表达谱及exos-omes外部肝特异性miRNAs的表达水平，筛选川楝子肝毒性发生、发展过程中关键的分子事件，揭示毒性机制，对exosomes内部特征性表达的miRNAs进行细胞水平跨种属的验证研究，寻找发现潜在的肝毒性生物标志物，建立基于循环exosomes的中药肝毒性评价新方法。

药物性肝损伤是中药引起的主要不良反应之一，但当前由于缺乏有效的中药肝毒性评价方法，难以对中药肝毒性的物质基础及毒性机制进行准确识别与研究，阻碍了中药现代化与国际化的发展。鉴于外泌体中的miRNA被认为是目前最有希望认识和区分药物肝毒性机制的生物标志物，本研究基于循环外泌体miRNA着重研究了川楝子组分或成分在作用时间及剂量依赖下的小鼠肝毒性表型及机制的异同。结果发现：川楝子水提醇沉液导致的肝细胞损伤可能是由胞内活性氧升高，进而通过p53依赖的线粒体损伤和S期周期阻滞致细胞凋亡造成，同时Nrf2介导的抗氧化反应也被激活，而血清外泌体中miR-370-3p可能通过升高p21和Cyclin E的表达而加重川楝子水提醇沉液的毒性作用；川楝子乙酸乙酯萃取物导致的小鼠肝损伤可能与p53、PI3K/Akt、PTEN等信号通路及线粒体功能异常引起肝细胞凋亡相关，而下调血清外泌体中miR-222的水平可能会促进PTEN和PPP2R2A的表达，从而调节与凋亡相关的信号通路，引起肝脏损伤；川楝素导致的小鼠肝损伤机制涉及肝细胞S期阻滞进而导致肝细胞凋亡，而miR-106b-5p在川楝素引起的肝损伤适应性反应中发挥重要作用，提高该miRNA的表达能显著减轻川楝素导致的小鼠肝损伤，结合临床数据分析表明该miRNA可能是跨种属的治疗肝损伤的新靶点。综上所述，循环外泌体miRNA能指示川楝子组分和成分肝毒性机制的异同，其中细胞凋亡是这些组分或成分共同涉及的通路，且川楝素是川楝子中致肝毒性的主要成分之一。本项目相关研究结果有助于拓展中药安全性研究方法，也有望进一步推动和加快中医药领域中exosomes生物功能的研究。