基于“血清药物化学-药代动力学-代谢组学”的体外培育牛黄活性成分辨识与机制研究

Revealing the material foundation and mechanism of Chinese medicine is a confusing and difficult task in research and development. Calculus Bovis Sativus (CBS) is one kind of rare Traditional Chinese Medicine (TCM) recorded in state Pharmacopoeia, although people generally believe its pharmacological activities correlate with its major ingredients, the evidence is still untenable and insufficient. Previously we confirmed the potent capacity of CBS to treat intrahepatic cholestasis (IC), based on this work, the integrated three-dimensional “serum pharmacochemistry - pharmacokinetics - metabolomics" system is established hereon. By comparing multi-dimensional fingerprints with spectrum collected from analyzing the transitional compositions into the blood, the absorbed bioactive components could be preliminary identified in mice serum after oral administration of CBS. Furthermore, the pharmacokinetic profiles of absorbed bioactive components are evaluated to understand their internal dynamic process and their potential interactions. Finally, by analyzing the effects of CBS or its single component on the metabolites profiling in serum, urine and liver tissues, metabolomics are employed to discriminate efficacy-correlated biomarkers and construct the molecular network linking efficacy components with disease model (IC). Bringing above results together, we could really understand the material basis which component truly works in CBS and verify whether CBS plays its mechanism by restoring the perturbed metabolic pathways in disease model in a manner of multi-components, multi-channels, multi-targets.

阐明中药药效物质基础和作用机制始终是其研究开发过程中的困惑和难点。体外培育牛黄为国家药典收载的名贵中药，尽管当前普遍认为其药效与其主要成分胆汁酸有关，但仍缺乏强有力证据支持。课题组前期研究发现CBS可有效治疗胆汁淤积症（IC），本项目将在此基础之上，基于“血清药物化学-药代动力学-代谢组学”三维整合体系研究思路，通过分析CBS化学组多维特征指纹图谱和体内血中移行成分，初步鉴定体内直接作用物质，并进一步考察入血胆汁酸药代动力学及其相互作用；通过分析CBS整体和单个化学成分分别干预动物模型前后肝组织、血清及尿液内源性物质变化规律，筛选与药效相关的生物标记物，构建CBS干预后效应物质-病症（IC症）桥连代谢网络，综合所有研究结果，有望阐明CBS的药效物质基础及治疗IC的多组分、多途径、多靶点的整体作用机制。

体外培育牛黄（CBS）为我国药典收载的名贵中药，牛黄中胆酸类是发挥其广泛药理活性的主要成分，但事实证明并非胆汁酸成分含量越高的牛黄品种药效越好，关于其药效物质基础一直缺乏强有力证据支持。本研究首先建立了CBS质量控制方法，可成功应用于鉴别CBS和天然牛黄（CB）；接着采用血清药物化学理论，在参考CBS质量控制方法的基础上，解析模型动物血清样本中胆汁酸谱图信息，鉴别出有牛磺酸（Tau）和5种胆汁酸（CA、DCA、CDCA、TCA、TDCA）移行入血。进一步探索这6中入血胆汁酸在体内的药动学和组织分布特征，结果显示：CBS组和天然CB组血液中均检测到CA、DCA、TCA、TDCA、CDCA 以及Tau， CBS组还检测到GCA。两组之间CA、DCA、TCA、CDCA 、Tau以及GCA的AUC（0-t）具有显著性差异（P < 0.05），而TDCA的AUC（0-t）无显著性差异 （P > 0.05）；天然CB组CA、TCA、TDCA以及Tau的Tmax较CBS组早。CBS组和天然CB组织中均检测到CA、DCA、TCA、TDCA、CDCA、GCA以及Tau。CBS组总体在心和肺分布较好，其中CA在心、脾、肺和肾中浓度在20 min-60 min达到最高，DCA则在5 min时在心、脾、肺和肾中便测得有一定浓度，TCA和TDCA在脾和脑组织中也有一定分布，GCA和Tau在肺组织中分布最高，CDCA在各个时间点各组织分布情况较稳定。天然CB组较CBS组：CA在心、肺和肾中分布较差，GCA在各组织中未测得，Tau分布也较差，而CDCA、TCA和TDCA组织分布情况在天然CB组与CBS组趋势相同。最后在胆汁淤积模型下监测CB对胆汁酸代谢轮廓的干预。初步结果显示：模型组大鼠胆汁酸谱发生显著改变。CBS可在一定程度上使胆汁酸代谢轮廓向正常水平转变；另外，CBS干预的模型组血浆中所有入血胆汁酸浓度都不高；说明CBS并不仅单是通过本身胆汁酸补充大鼠体内含量上的差异。从目前观察到的结果分析，CBS可显著升高亲水性初级或次级胆汁酸浓度水平，下调疏水性次级胆汁酸（尤其是结合型），基于此我们推测CBS可能通过某些特殊调控机制，使体内胆汁酸组成结构发生“精细化”改变，从而逆转毒性胆汁酸对肝细胞和胆管结构的损伤。