基于核受体FXR调控的胆宁片中利胆活性组分的辨识和作用机制研究

Cholestasis-related disease is with high incidence in China. The etiology and pathogenesis of Cholestasis-related disease is complex. Nowadays there is no ideal drug except ursodeoxycholic acid. Danning tablets as the protected traditional chinese medical product, is one of the main chinese herbal prescriptions used for hepatoprotection and cholaneresis clinically. Its main effect is to "dispersing stagnated liver qi for promoting bile flow, clear away heat and purge the bowels". Danning tablets is composed of seven medicinal materials and complicated chemical compositions. The less knowledge on the bioactive constituents of Danning tablets had severely affected its quality control. Our pervious research indicated that Danning tablets exerted hepatoprotective and cholagogic effect on ANIT-induced cholestasis with liver injury in rats, and suggested that this protective effect was likely due to up-regulation of nuclear receptors farnesoid X receptor (FXR) mRNA level to stimulated the mRNA expression of bile acid transporters in ANIT-induced Cholestasis model. Therefore, in this project, active components in Danning tablets will be obtained by luciferase reporter gene assay and their effects on FXR target genes will be explored. Furthermore, cholagogic effects of active components based on FXR-regulated pathway will be validated using cholestasis and FXR gene knock-out animal model. Identification of choleretic effect compositions in Danning tablets will help to establish active components-based quality standard, to lay the foundation for Pharmacopoeia standards of Danning tablet improvement. The elaboration of cholagogic effect mechanism will provide new ideas for traditional cholagogic herbs with modern theory.

胆汁淤积型疾病在我国发病率较高，病因病机较复杂，目前临床上除熊去氧胆酸等少数药物外，尚无其他理想药物。胆宁片作为国家级中药保护品种，是临床用于保肝利胆的主要复方之一，主要功效为疏肝利胆、清热通下，但其组方药味较多，化学成分复杂，药效物质基础并不完全明晰。前期工作发现胆宁片对α-荼基异硫氰酸盐（ANIT）诱导的胆汁淤积模型显示出良好的保护作用，其作用机制主要是通过上调胆汁酸合成、代谢和转运相关的核受体-法尼醇受体(FXR)的表达。鉴于此，本项目采用荧光素酶报告基因实验快速筛选胆宁片中具有调控FXR表达作用的活性组分并研究其对FXR靶基因的作用。进一步用胆汁淤积以及FXR基因敲除的动物模型验证活性组分基于FXR调控的利胆作用。胆宁片利胆有效组分的辨识有助于建立以有效成分为主的质量标准，为胆宁片药典标准的提升和完善奠定基础。而其利胆作用机制的阐述为利胆中药用现代理论阐释提供新思路。

胆宁片作为国家级中药保护品种，是临床上用于保肝利胆的主要复方之一，主要功效为疏肝利胆、清热通下，但其组方药味较多，化学成分复杂，其保肝利胆物质基础及作用机理国内外尚未有研究报道。法尼醇受体(FXR)作为胆汁酸核受体，对体内的胆汁酸稳态及炎症反应具有重要调节作用。因而，本课题以FXR为切入点，利用荧光素酶报告基因测试方法快速筛选到胆宁片中的指标性成分姜黄素及白藜芦醇对核受体FXR具有激动作用，体外细胞实验证实姜黄素及白藜芦醇是FXR的配体。体内研究证实姜黄素与白藜芦醇对ANIT所致胆汁淤积小鼠具有一定的保护作用。其作用机制是基于调控FXR而对肝肠循环中的胆汁酸稳态和肝脏与小肠中的炎症反应的进行调节。而在基因敲除小鼠中，姜黄素与白藜芦醇对于ANIT导致的胆汁淤积型肝损伤小鼠的保护作用消失。说明FXR是胆宁片活性成分姜黄素与白藜芦醇保肝利胆作用的重要靶点。本课题的实施有助于建立以有效成分为主的质量控制指标，科学、客观地进行胆宁片的质量评价; 提高产品的加工与生产质量，促进该产品的临床应用；胆宁片有效成分及作用机制的阐明有助于促进胆宁片国际化应用。