基于柴胡降脂保肝与肝脂毒性的“毒-效-证-物”关联特性研究和脂质代谢网络构建

Bupleuri radix, the primary effects of which are lowering lipid and hepatoprotective effect, has been recorded “Bupleurum robbing hepatic yin” according to traditional Chinese medicine theory. During the last decade, our laboratory has confirmed the main effects, toxic substance and function characteristic of Bupleuri radix found the preliminary biological transformation process, and clarified the relationship of “does-time–efficacy-toxicity”. In addition, the expression mechanism of gene and hepatotoxicity in the decoction liquid, volatile oil and saikosaponins of Bupleuri radix was investigated. The differential protein expressionin liver induced by saikosaponins, indicated the involvement of CREBH mediated lipid gene metabolism abnormity. Therefore, we proposed a working hypothesis that the underlying mechanism of hepatotoxicity of Bupleuri radix is closely correlated with its pharmacological effects and the physiological condition or pathological syndrome of a model, which can be scientifically cognized and synthetically characterized by integrated analysis of lipideomic, proteome and genomic approaches. The employment of chromatograph lipideomic, genomic approaches and other modern biological technology, as well as “efficacy-toxicity” characteristics of both normal, non-alcoholic fatty liver disease (NAFLD) and liver-depression syndrome models, in combination of biological evaluation targeting integrated analysis of lipideomic, proteome and genomic approaches, such as CREBH singaling pathway, will provide us significant evidence of its eventually establish potential connections and scientific annotations for “shuganjieyu” and “Bupleurum robbing hepatic yin”. In conclusion, all evidence and findings will further provide a practical research-folw for the investigation the relationship between toxicity, efficacy and syndrome theory, combined with reasonable identification and scientific control of Quality marker(Q-marker) in Bupleuri radix, and subsequently the safer use of it.

柴胡为“疏肝解郁”要药，又有“柴胡劫肝阴”之载。前期研究获知其主要效毒物质、生物转化过程、量-时-效/毒关系及柴胡水煎剂、挥发油、总皂苷基因/蛋白水平应答机制。证实柴胡总皂苷兼具降脂保肝和大剂量可诱导肝毒性，其机制与肝脏CREBH介导的脂质代谢基因差异表达有关。据此，提出“柴胡肝毒性与降脂保肝功效、证候和物质基础密切相关，可被脂质/蛋白/基因组学的整合分析科学认知和综合表征。”的工作假说。拟用色谱分离、系统生物学、多组学整合分析手段，通过正常、非酒精性肝损伤、肝郁模型效毒特征，基于生物转化物质基础的毒效关系，CREBH相关效毒靶点多组学整合分析探讨柴胡“疏肝解郁”功效和“柴胡劫肝阴”的科学内涵和“毒-效-证”发生机制与关联特性。结合效毒关联评价的柴胡质量标志物（Q-Marker）的合理辨识，提出中药功效和病证背景下，效、毒Q-Marker精准控制的柴胡临床安全及合理用药策略。

柴胡具解表退热，疏肝解郁，升举清气之功；药理活性明显，临床应用广泛；但也有一定的毒副作用。项目组前期研究表明，柴胡总皂苷具有降脂保肝作用并在大剂量下发生肝毒性，诱导肝脏脂肪代谢和脂肪酸氧化基因差异表达，但其机制与毒效内在联系并不清楚；故本课题提出“柴胡肝毒性与降脂保肝功效、证候、生物转化、物质基础密切相关，可被脂质组学、蛋白/基因组学的整合分析科学认知和综合表征。” 的工作假说。.本课题通过对柴胡基于HPLC-MS指纹图谱技术物质基础表征和效、毒指标的研究和基于灰色关联分析的谱-效-毒分析，对降脂活性及肝毒效毒成分进行预测-验证，确认柴胡水提物效毒成分具有较高的一致性，柴胡皂苷类作用显著。通过对柴胡在人工胃、肠液，肠道菌群，肝微粒体的HPLC-MS，对其体外生物转化情况的研究发现部分柴胡效毒成分在人工肠液及肠道菌群发生转化，在其他部位较为稳定。继而采用系统生物学、多组学整合分析的等技术手段，通过不同动物模型进行对比实验，确认效毒作用特征，证实柴胡发挥降脂作用主要是通过促进肝脏对甘油三酯的代谢以及抑制胆固醇的合成来实现的，发生肝毒作用可能与诱导肝细胞氧化应激损伤与细胞凋亡有关；后通过CREBH相关效毒靶点多组学整合分析探讨了柴胡在“毒性-功效-证候”背景下脂代谢调节网络，发现柴胡皂苷调节相关基因如Lipe和Lipg来调节甘油脂代谢。通过下调Fasn和Acaca的表达深度抑制脂肪酸合成，通过诱导Acox1和Cpt1a的表达促进脂肪酸降解，也进一步发现柴胡皂苷A、D和特异性代谢产物可通过激活转录因子网络调控肝脏脂质代谢。上述研究结果为临床安全合理应用柴胡和基于效毒评价的柴胡质量精准控制奠定基础，也为后期开发基于柴胡传统功效和现代研究的安全、高效、利于吸收的降脂保肝创新药物打下坚实的基础。依托本项目申报的“中药“效-毒整合”评价体系构建与应用”获得2020年度山东省科技进步一等奖。