基于代谢物组定量表征的胆类中药改善肝内胆汁淤积的药效物质及作用机制研究

The therapeutic benefits of bile-class traditional Chinese medicines (TCMs) towards intrahepatic cholestasis (IC) have been well-defined for centuries; however, the effective materials as well as the underlying mechanisms haven’t been revealed because of the unavailability of practical work-flow. In current study, we aim to develop a new technique roadmap to address these issues. The key procedures of the systematic experimental design include: 1) proposing a new strategy to characterize the quantitative metabolome of various bile and biological samples, in particular those from IC rats; 2) defining the migration of the quantitative metabolome of IC rats following bile administration in combination with traditional pharmacological indicators as “efficacy”, and constructing “spectrum-efficacy relationships” with the quantitative metabolome of various bile samples to screen out those potential effective components which will undergo pharmacological confirmation with several in vitro and in vivo models; and 3) revealing the underlying mechanisms being responsible for correcting the IC metabolome with direct and indirect manners following the oral administration of effective bile samples via the dynamic pattern profiling of IC metabolome, the quantitative determination of key enzymes, transporters and nuclear receptors, and the construction of pharmacological network. In brief, the strategy for quantitative metabolome profiling as the primary tool, together with the construction of “spectrum-efficacy relationships” and dynamic pattern profiling, comprise this new technique roadmap for clarifying the effective materials and the underlying mechanisms of TCMs. The findings obtained are expected to provide promising scientific clues for the therapeutic principles of bile-type TCMs namely "heat-clearing and detoxifying, and dispersing stagnated liver qi" , indicating great academic values as well as bright application prospects.

胆类中药具有明确改善肝内胆汁淤积（IC）的功效。本项目针对其药效物质和作用机制不明的科学问题，以IC模型大鼠为病理体系，在LC-MS平台上构建代谢物组定量表征策略，表征各胆类中药的化合物组和生物样品代谢物组；以起效时大鼠代谢物组的迁移结合传统药效指标作为“效”，与各胆类中药定量化合物组构建“组效关系”，筛选潜在药效成分，并通过体、内外模型确认活性；有效中药给药后，通过综合分析大鼠代谢物组的动态变化轨迹以及关键酶、转运体和核受体的转录和表达，并结合网络药理学等分析，揭示胆类中药以直接和间接两种方式纠正IC大鼠代谢物组的作用机制。本研究构建了以代谢物组定量表征策略为基础，结合“组效关系”及动态轨迹分析的中药药效物质和作用机制研究的方法体系，诠释胆类中药“清热解毒、疏肝利胆”的科学内涵，促进中医药理论发展和临床合理用药，具有重要学术价值和应用前景。

胆类中药具有明确改善肝内胆汁淤积（IC）的功效。本项目针对其药效物质和作用机制不明的科学问题，以疗效确切的名贵中药熊胆粉为代表，开展了系列研究，主要取得以下重要进展：1）通过系统的化学成分定性分析，结合在线能量分辨质谱技术，建立了复杂体系化学成分组定量表征策略；2）利用定量表征策略，对熊胆粉、牛黄、蛇胆、猪胆、羊胆、鹅胆等十余种胆类中药进行了深入的化学成分组比较，阐明各胆类中药的特征性成分；3）成功构建了ANIT诱导的IC大鼠模型，以熊去氧胆酸为阳性药，验证了熊胆粉的药效；4）利用定量表征策略，深入地研究了熊胆粉、熊去氧胆酸和牛磺熊去氧胆酸给药后大鼠血浆中胆汁酸组的定量动态变化规律，发现三者均可以造成整个胆汁酸组的迁移；5）非靶标代谢组学及非靶标蛋白组学研究结果均表明，熊胆粉主要通过调节胆汁酸代谢网络（由胆汁酸组及胆汁酸合成、转运等相关蛋白组成）；6）广泛靶标代谢组学结合广泛靶标蛋白组学研究揭示种结合型胆汁酸含量显著升高，其中牛磺酸结合型胆汁酸和磺酸化胆汁酸可作为胆汁淤积的潜在生物标志物，UDCA和熊胆粉通过下调病理状态下表达升高的CYP4A2、CYP2C11和BAAT等蛋白的表达水平发挥药效。因此，熊胆粉改善IC的药效物质为熊去氧胆酸、牛磺熊去氧胆酸等胆汁酸类成分，作用机制为通过吸收直接调节胆汁酸组，以及通过调节相关蛋白的表达水平间接修复病理状态下受扰动的胆汁酸组。依托本项目，共发表期刊论文15篇，其中SCI 10篇，第一作者或通讯作者11篇。在分析化学领域顶级期刊Anal Chem和Anal Chim Acta，以及色谱领域顶级期刊J Chromatogr A发表研究性文章多篇。申请发明专利2项。培养科研骨干4名，博士3名，硕士8名。较好地完成了项目预期目标，所获得的成果具有较高的学术价值和应用前景。