基于关联分析及系统生物学方法探索何首乌中不同提取分离组分致肝损伤的生物学特性

Numerous studies on Polygonum Multiflorum which could cause liver injury raised widely concern, but the knowledge about the transaction of components, the relevance and mechanism of the active ingredients resulting in liver injury and the diverse biological properties of hepatotoxicity were not distinct. Data mining methods such as association analysis, and the LC/MS were used to further analyze the liver injury, injury characteristics and the compatibility for the extract sites of Polygonum Multiflorum, determining the biological properties, the compatibility between the ingredients and the components of the ingredients which result in diverse liver injury. And the study validated the above results by monoclonal antibodies specific knockout, high content screening, etc. A number of methods such as gene chips technology, proteomics and metabonomics were applied to investigate the mechanism of different types of liver injury caused by diverse extract sites, and then the results were verified by zebrafish as the model organism. The studies are able to reveal what ingredients cause the liver injury, and the compatibility and transaction of the ingredients, the indicated ingredients and components. This research is expected to provide an academic evidence for the reasonable application of Polygonum Multiflorum, supply the amended content under the Polygonum Multiflorum caption of in Pharmacopoeia of The People’s Republic of China with usage and dosage, indicatons, etc.

关于何首乌致肝损伤的报道引起广泛关注，但关于何首乌致肝损伤的指征成分确证及与肝损伤不同生物学特性的关联性及机制、药物组份之间相互影响等并不清晰。本研究在已有研究的基础上采用关联分析等数据挖掘手段、液质联用技术进一步对何首乌中提取分离部位进行肝损伤-损伤生物学指标-成分及配伍进行分析，明确导致不同肝损伤的生物学特性成分及组份之间配伍影响；同时采用单克隆抗体特异性敲除技术、高内涵筛选等对结果进行验证；采用基因芯片技术、蛋白质及代谢组学等研究致肝损伤的作用机制，同时采用模式生物斑马鱼对相关研究结果进行验证。通过研究可以揭示何首乌中致肝损伤的物质基础与作用机制、主要成分、指征成分及组份配伍影响，为何首乌准确、合理应用，为修订或增加药典何首乌项下及含何首乌中成药用法用量、使用注意等提供理论基础和实验依据，也为类似研究提供参考借鉴。

本项目针对何首乌致肝损伤的问题，开展了何首乌致肝损伤成分、毒性作用机制及代谢通路研究。采用体外细胞实验、UPLC-QTOF/MS、相关性分析等技术手段对何首乌中肝毒性成分进行推测并验证，结果表明何首乌的肝毒性与其含有的蒽醌类化合物关联性强，尤其是大黄素及其衍生物；采用单克隆抗体特异性敲除技术解析大黄素等蒽醌类成分与何首乌肝细胞毒性的相关性，结果表明大黄素等蒽醌类成分的缺少会降低何首乌的肝细胞毒性，反向证明了何首乌中大黄素等蒽醌类成分是其导致肝损伤的潜在风险成分。.采用高内涵筛选、iTRAQ定量蛋白质组学、Non-targeted代谢组学等方法，从细胞、斑马鱼、整体动物三个层面对何首乌提取物及其主要单体成分的肝损伤机制及代谢通路进行深入探究，结果发现致肝损伤机制主要与线粒体功能的氧化磷酸化、TCA循环信号通路传导异常相关；其他花生四烯酸代谢通路，与胆汁酸及类固醇类化合物生成、代谢相关的类固醇合成途径、类固醇激素生物合成途径等都与何首乌致肝损伤相关。.项目研究成果发表了SCI论文7篇，核心期刊1篇，为何首乌、含大黄素中药的安全用药、新药研发提供了合理化建议。