基于PDZK1蛋白互作网络及胆汁酸代谢轮廓研究体外培育牛黄的利胆机制

Intrahepatic cholestasis (IC), which is mainly caused by impaired bile acid formation and secretion, falls into the category of “jaundice” in traditional Chinese medicine and still can not be effectively treated hitherto. Calculus Bovis Sativus (CBS) has definite effectiveness on IC, whereas its underlying mechanism and material foundation remain unclear. CBS up-regulated PDZK1 expression and enhanced the activity of bile acid transporter protein in our previous study. Adapter protein PDZK1 can interact with various bile acid transporters and its nuclear receptors, thus modulating the metabolite profile of bile acids (MPBA). Hence, the changes of MPBA characteristics caused by PDZK1-mediated protein interaction network constitute a promising mechanism for cholaneresis of CBS. MPBA of rats was also established and CBS disposition in vivo was preliminarily explored. This project intends to reveal the role of PDZK1 protein interaction network in PDZK1 knock-out rats and classic IC animal models by using proteomics tools. Further mechanisms for CBS-regulated PDZK1 protein interaction network will be investigated from the whole to the cellular and molecular levels. We will also analyze the effect of CBS on the changes of MPBA characteristics in IC by using metabolomics technology. The results will elucidate the cholaneresis mechanism and profile the material basis of CBS, which will provide evidences for its clinical treatment.

肝内胆汁淤积（IC）以胆汁酸摄取和分泌障碍为主要病因，在中医学中多属“黄疸”范畴，目前尚缺乏特效药物。体外培育牛黄（CBS）治疗IC效果显著，但作用机制和物质基础尚未阐明。本课题组发现CBS可上调肝脏接头蛋白PDZK1表达，进而提高胆汁酸转运蛋白活性。国内外研究表明PDZK1参与多种胆汁酸转运蛋白及其核受体的相互作用，进而改变机体胆汁酸代谢轮廓（MPBA），由此推测PDZK1蛋白互作网络介导的MPBA改变可能是CBS利胆的重要机制。前期我们还构建了大鼠的MPBA，并研究了CBS的指纹图谱与体内过程。本项目拟利用蛋白组学手段，结合PDZK1敲除大鼠和经典IC动物模型，揭示PDZK1蛋白互作网络在IC中的关键靶点，从整体、细胞和分子等水平探讨CBS调控该互作网络的作用，并应用代谢组学技术研究CBS对IC发病中MPBA特征的影响，最终阐明CBS的利胆机制和物质基础，为促进其临床应用提供依据。

肝内胆汁淤积（IC）以胆汁酸摄取和分泌障碍为主要病因，在中医学中多属“黄疸”范畴，目前尚缺乏特效药物。本课题在已经证实体外培育牛黄（CBS）具有治疗IC的前期基础上，发现PDZK1介导的蛋白互作在CBS利胆作用中扮演了重要角色，并发现CBS通过PDZK1途径增强肝脏MRP2和BCRP的功能来缓解肝内胆汁淤积。进一步地，我们基于生物信息学分析和分子生物学手段筛选了肝脏和肠道相关核受体，发现CBS是通过激活肠肝FXR-PDZK1信号通路改善肝脏胆汁酸平衡而缓解雌激素诱导的胆汁淤积性肝损伤。.课题组建立了LC-MS/MS测定胆汁酸类成分（BAs）的方法，该方法灵敏度高、特异性强，从而确定了CBS的成分分析和质量控制标准，为研究结果的可重复性奠定了基础。我们对CBS和天然CB进行了入血成分的经时特征以及组织分布研究，探索了该药在机体发挥作用的潜在物质基础。同时我们构建了大鼠体内的胆汁酸代谢轮廓（MPBA）测试技术，在IC动物模型中给予CBS后，通过MPBA分析，确定该药对机体胆汁酸成分和比例的调节作用，从代谢组学角度阐述了CBS利胆的作用机制。.此外，我们发现IC大鼠除肝脏炎症外，还存在肠肝氧化应激和凋亡反应，且机制与肠肝AKT与MAPK（ERK1/2和P38）信号通路异常表达有关。CBS通过调控AKT与MAPK通路显著改善肝脏炎症、肠肝氧化应激和凋亡反应，显著减轻EIC诱导的肠肝损伤，同时整体逆转胆汁酸肠肝循环紊乱。总之，本研究从PDZK1介导的转运体调节、MPBA为代表的靶向代谢组学和肠肝轴等角度阐述了CBS治疗IC的机制，为该药进一步应用于临床提供了理论基础。