基于核受体PXR-转运体OATPs/MRPs-胆汁酸通路的苦参肝毒性机制研究

Sophora flavescens (SF) is widely used in TCM for the treatment of various diseases. Previous investigation indicated that SF was hepatotoxic. However, the mechanisms of SF-induced hepatotoxicity remained unclear. Our group found that SF-induced hepatotoxicity was related to perturbation of the bile acid pool and SF may interact with hepatic bile acid uptake transporters OATPs and efflux transporters MRPs. Nuclear receptor PXR-transporters OATPs/MRPs- bile acids pathways might play an essential role in SF-induced hepatotoxicity. On this basis, in this project, Real-time PCR and Western Blot will be used to determine the expression levels of PXR, OATP1B1, OATP1B3, MRP2 and MRP3 genes and proteins in rat liver after SF administration in vivo. LC-MS/MS will be used to quantify 21 kinds of bile acids in rat serum, liver and bile. In vitro, the influence of SF on the active uptake of bile acids will be studied in OATP1B1- and OATP1B3-transfected HEK293 cells. The influence of SF on the efflux of bile acids mediated by human MRP2 or MRP3 will be investigated using Sf9-derived inside-out membrane vesicles. The results contribute to elucidating the role of nuclear receptor PXR-transporters OATPs/MRPs-bile acids pathways in the molecular mechanisms of SF-induced hepatotoxicity, which can provide scientific basis for clinical safety use of SF.

中药苦参临床应用广泛，前期研究表明苦参具有肝毒性，但其肝毒性机制尚未明确。课题组研究发现苦参肝毒性与胆汁酸池紊乱有关，苦参可能存在跟胆汁酸摄取转运体OATPs和外排转运体MRPs的相互作用，核受体PXR-转运体OATPs/MRPs-胆汁酸通路可能是苦参肝毒性的重要机制之一。在此基础上，本课题拟从大鼠整体采用Real-time PCR和Western Blot方法检测给药后肝脏中PXR、OATP1B1、OATP1B3、MRP2和MRP3的基因和蛋白表达；采用LC-MS/MS检测血清、肝脏和胆汁中21种胆汁酸的浓度变化；体外考察苦参对OATP1B1和OATP1B3分别转染的HEK293细胞摄取胆汁酸的影响以及对MRP2、MRP3转运体过表达的Sf9细胞膜囊外排胆汁酸的影响。以期明确核受体PXR-转运体OATPs/MRPs-胆汁酸通路在苦参肝毒性中的分子机制，为苦参的临床安全使用提供科学依据。

中药苦参临床应用广泛，前期研究表明苦参具有肝毒性，但其肝毒性机制尚未明确。课题组研究发现苦参肝毒性与胆汁酸池紊乱有关，苦参可能存在跟胆汁酸摄取转运体OATPs和外排转运体MRPs的相互作用，核受体PXR-转运体OATPs/MRPs-胆汁酸通路可能是苦参肝毒性的重要机制之一。因此，本课题以胆汁酸池内稳态为切入点，采用蛋白组学技术筛选和验证大鼠给药后肝脏中与胆汁酸转运相关核受体和转运体的基因和蛋白表达；采用代谢组学筛选肝脏中与胆汁代谢相关的差异代谢物和代谢通路；采用LC-MS/MS检测血清、肝脏和胆汁中24种胆汁酸的浓度变化。结果表明苦参显著抑制肝脏核受体Pxr、Fxr以及胆汁酸摄取转运体Oatp1、Oatp2、Oatp4、Ntcp的基因表达，显著增加胆汁酸外排转运体Bsep的蛋白表达，蛋白组学和代谢组学均筛选出Bile secretion信号通路显著富集；苦参显著增加血清、肝脏和胆汁中多种胆汁酸含量，CDCA、GLCA等多种具有潜在毒性胆汁酸的蓄积可能与苦参肝毒性有关。本课题采用组学数据挖掘和24种胆汁酸定量分析技术，系统阐释苦参对胆汁酸池内稳态的影响，明确了核受体Pxr/Fxr-转运体Oatps/Ntcp/Bsep-胆汁酸通路在苦参肝毒性的分子机制，为苦参的临床安全使用提供了科学依据。