基于CYP450酶表达调控及代谢组学的五味子醋制保肝作用机制研究

The central role played by Chinese herbal pieces in the clinic of Chinese medicine is increasingly apparent, but the theory of traditional Chinese medicine (TCM) processing which guides the reasonable application of Chinese herbal pieces still lacks modern scientific arguments, and it has become a bottleneck problem that hinders the modernization of TCM which must be solved urgently. Schisandra chinensis processed with vinegar has already achieved significant clinical curative efficacy in the treatment of liver disease, but the research has not seen on the mechanism of its hepatoprotective effect. The key mechanisms of hepatoprotective effect of medicines were related to metabolic enzymes, metabolic pathways and signaling pathways. According to the above issues and the existing research foundations, we introduced LC/TOFMS, RTFQ-PCR and microdialysis frontier technologies to the processing theory of Chinese herbal medicine field innovatively for the first time. Taking P450 enzymes, Nrf2/ARE pathway and dynamic changes of exogenous and endogenous metabolic components as the starting point, we used biological information database to analyze the change rule of metabolic components by means of the overall identification and target analysis, and evaluate the correlation of metabolic enzyme, metabolic component and the body. We will study and clarify the scientific connotation of the hypothesis that Schisandra chinensis processed with vinegar expressed a hepatoprotective effect by regulating CYP450 expression and Nrf2/ARE pathway, producing changes in time and space of exogenous and endogenous metabolic components as well as changes in specific metabolic pathways in organism from the the overall, tissular and cellular levels. The research interprets traditional processing theories with modern scientific language and it will not only provide a model for the modern research of processing theory of Chinese herbal medicine, but also offer a scientific basis for the development of the hepatoprotective new drugs of Schisandra chinensis processed with vinegar.

中药饮片在中医临床发挥的核心作用正日益显现，但指导其合理应用的中药炮制理论尚缺乏现代科学论据，已成为制约中药现代化的瓶颈。五味子醋制治疗肝病取得显著临床疗效，但其保肝作用机制未见研究。药物保肝关键机制与代谢酶、代谢途径及信号通路等有关。基于上述问题及已有研究基础，项目创新性地将LC/TOFMS、RTFQ-PCR、微透析等前沿技术首次引入炮制理论研究领域。以P450酶、Nrf2/ARE通路和内外源性代谢组分动态变化为切入点，通过整体辨识和靶标分析，利用生物信息数据库解析代谢组分的变化规律，评价代谢酶、代谢组分与机体之间的关联性。从整体、组织、细胞水平，研究阐明五味子醋制是通过调控P450酶表达与Nrf2/ARE通路、机体内外源性代谢组分时空变化与特异代谢通路改变而发挥保肝作用假说的科学内涵。以现代科学语言诠释传统炮制理论，为中药炮制的现代研究提供示范，也为醋五味子的保肝新药开发提供科学依据。

本项目采用UPLC-Q-TOF/MS及多元统计分析的方法研究了五味子醋制前后差异化学标志物，通过对质谱数据进行PCA和OPLS-DA，结果表明五味子醋制后其化学成分显著变化。通过对s-plot图分析，筛选出五味子醋制前后8个差异化学标记物，鉴定并确认了其中的5个化学成分，分别是5-羟甲基糠醛、五味子甲素及其同分异构体、五味子乙素和五味子酯丁。实验采用免疫组化等方法分别对肝损伤大鼠脂质代谢、炎症因子及氧化应激等指标进行评价，结果表明五味子醋制后提高了抗脂质水平紊乱，抑制了炎症因子的反应导致的损伤，增强了ADH活力，同时增加了SOD、GSH-Px、CAT活性和GSH含量等而提高抗氧化能力，降低了ROS等自由基在肝组织中的蓄积，减少MDA等有害物质的产生，从而达到抗肝损伤的作用。采用cocktail、western blot及RT-PCR技术对生、醋五味子抗肝损伤作用机制进行研究，结果表明：五味子醋制增强其抗肝损伤效应与CYP450酶及Nrf2/ARE通路的表达调控密切相关。通过建立的UPLC-QTOF-MS方法，对五味子醋制前后外源性代谢物及内源性代谢组学进行了深入研究，结果表明：在大鼠血浆、胆汁、尿液及粪便中鉴定了五味子6种木脂素原型成分及它们的20种代谢产物；五味子经醋制后改变了总木脂素的组成配比，导致部分木脂素成分的吸收、分布及代谢差异。大鼠血浆及胆汁代谢组学研究结果显示：醋制前后五味子醇提取物对大鼠血浆、胆汁代谢物谱的具有显著影响，鉴定得PE、PC、LysoPE、LysoPC、PG(18:0/18:1)、12-ketodeoxycholic acid、TG(64:2)、13-HETE 等41个与酒精性肝损伤相关的潜在生物标记物，代谢通路分析表明五味子醋制增强抗肝损伤效应的机制可能与一些相关的酶、代谢及信号通路密切相关（如CYP450酶，PI3K-AKT、mTOR、NF-κB等信号通路）。.综上所述，本课题以P450酶、Nrf2/ARE通路和内、外源性代谢组分动态变化为切入点，通过整体辨识和靶标分析，利用生物信息数据库解析代谢组分的变化规律，评价代谢酶、代谢组分与机体之间关联性。从整体、组织、分子水平，研究阐明五味子醋制是通过调控P450酶表达与Nrf2/ARE通路、机体内外源性代谢组分时空变化与特异代谢通路改变而发挥保肝作用假说的科学内涵。