基于胆汁酸转运体/代谢酶的黄芪汤改善肝内胆汁淤积分子机制研究

Intrahepatic cholestasis is a common syndrome in various liver diseases. It is also an important factor to promote the liver disease progress. The abnormal expression and function of bile acid transporters and metabolic enzymes is the key molecular mechanism of intrahepatic cholestasis. In our previous clinic and animal experiments, we found that Huangqi decoction has an obvious protective effect on intrahepatic cholestasis. Huangqi decoction treatment could upregulate the mRNA expression of CYP3a11, UGT1a1, MRP2, and their upstream regulators, nuclear receptor FXR and nuclear transcription factor Nrf2. Accordingly, we propose the hypothesis that Huangqi decoction could improve the of intrahepatic cholestasis recovery by inducing bile acid transporters and metabolic enzymes expression and function. To test this hypothesis, using intrahepatic cholestasis animal models, this study would investigate the effects of Huangqi decoction on liver function, liver histopathology, bile acid transporters and metabolic enzymes, and their regulatory factors. We will also investigate the absorbed ingredients of Huangqi decoction into the animals by liquid chromatography-mass spectrometry methods. In addition, the effects of the absorbed ingredients of Huangqi decoction on the liver function, bile acids level, transporters and metabolic enzymes are also investigated in a sandwich-cultured rat hepatocytes model. This research would provide a basis for elucidating the molecular mechanism of Huangqi decoction on intrahepatic cholestasis and provide guidance for the clinical application of Huangqi decoction. Moreover, the present study could also provide new ideas for the research of traditional Chinese herb formula to treat the intrahepatic cholestasis.

肝内胆汁淤积广泛存在于各种肝病，是肝病加重的重要因素，其发生机制与胆汁酸转运体/代谢酶的表达及功能异常密切相关。我们前期临床及动物实验发现经典方黄芪汤具有改善肝内胆汁淤积作用，并可上调小鼠肝脏CYP3a11、UGT1a1、MRP2及其上游调控因子核受体FXR、核转录因子Nrf2的表达。据此，我们提出黄芪汤通过诱导或恢复胆汁酸转运体/代谢酶的表达及功能以改善肝内胆汁淤积的假说。本项目采用肝内胆汁淤积动物模型，研究黄芪汤对肝脏功能、肝脏胆汁酸水平、肝组织病理学、胆汁酸转运体/代谢酶及其上游调控因子的影响，并采用液质联用技术分析黄芪汤吸收进入体内的成分，利用“三明治”培养动物原代肝细胞模型，研究黄芪汤吸收成分对肝细胞功能、胆汁酸水平及相应转运体/代谢酶的影响。通过本研究阐明黄芪汤改善肝内胆汁淤积的分子机制，为黄芪汤临床治疗胆汁淤积性肝病提供药理学基础，并为中药复方治疗肝内胆汁淤积的研究提供新思路

黄芪汤是中医经典方剂，前期研究发现其具有改善肝内胆汁淤积作用，但具体机制尚不清楚。本研究采用α-萘异硫氰酸酯（ANIT）诱导肝内胆汁淤积小鼠模型，发现黄芪汤可改善胆汁淤积及肝损伤。采用LC-LTQ-OBITRAP高分辨质谱方法检测了黄芪汤对小鼠血清代谢组学的影响，黄芪汤可影响胆汁酸合成及谷胱甘肽代谢；采用UPLC-MS/MS方法检测了小鼠血清、肝脏及胆汁多种胆汁酸水平，发现黄芪汤可降低疏水性胆汁酸水平，升高亲水性胆汁酸水平，改善胆汁酸组成。采用real-time PCR、western blot以及免疫组化方法，发现黄芪汤可上调胆汁淤积小鼠胆汁酸外排转运体Mrp2、Mrp3以及Mrp4表达，上调代谢酶Cyp2b10及Ugt1a1表达，上调小鼠肾脏外排转运体Mrp2及Mrp4表达，表明黄芪汤可诱导胆汁酸转运体及代谢酶的表达，促进胆汁酸外排及代谢，黄芪汤还可上调转运体及代谢酶上游调控因子Nfr2的表达。蛋白质组学研究发现黄芪汤可抑制NF-κB/IL-6-STAT3通路改善胆汁酸引起的炎症反应。采用DDC诱导慢性肝内胆汁淤积小鼠模型，发现黄芪汤给药4周可改善小鼠胆汁淤积及肝损伤，给药8周可改善小鼠肝纤维化，降低血清及肝脏胆酸水平。采用分子生物学方法发现黄芪汤给药4周可上调小鼠外排转运体Mrp2、Mrp3及Mrp4的表达，上调代谢酶Cyp2b10及Ugt1a1的表达，给药8周可上调Mrp4表达，抑制摄取转运体NTCP表达，表明黄芪汤可诱导转运体及代谢酶的表达改善胆汁淤积。此外，黄芪汤给药8周可下调α-SMA及Collagen Ⅰ及CK-19的表达，而这与其抑制NF-κB/IL-6-STAT3通路有关。采用LC-MS/MS高分辨质谱对正常大鼠或肝内胆汁淤积小鼠黄芪汤给药后血清及肝组织多成分分析，阐明了黄芪汤给药后在动物体内多成分药动学规律。采用ANIT 诱导的“三明治”培养大鼠原代肝细胞，诱导肝内胆汁淤积体外模型，观察了黄芪汤吸收成分对胆汁酸转运体及代谢酶表达的影响，发现环黄芪醇、黄芪甲苷、甘草次酸等成分具有上调胆汁酸外排转运体Mrp2、Mrp3及Mrp4表达、上调代谢酶Cyp2b10及Ugt1a1表达的作用，提示这些成分可能是黄芪汤抗肝内胆汁淤积的效应成分。本研究结果诠释了经典方剂黄芪汤的作用机制，解析了方剂抗胆汁淤积性肝病的科学内涵。