Liu Jia, Hou Li-Li, Zhao Cui-Ying
Department of Traditional Chinese Medicine, The Affiliated Obstetrics and Gynecology Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital, Nanjing, Jiangsu 210004, P.R. China.
Exp Ther Med. 2018 Apr;15(4):3699-3704. doi: 10.3892/etm.2018.5891. Epub 2018 Feb 26.
The herbal medicine Yin Huang Mixture (YHHJ; patent no. 200910031240.7) is an aqueous extract composed from various herbs, including Thunb, Thunb, Oliver, Baill, Ellis, Wolf and Turcz. Previous studies have indicated that YHHJ treatment has a beneficial effect on ameliorating itching and reducing serum bile acid levels in patients with intrahepatic cholestasis of pregnancy (ICP). However, the molecular mechanisms of action of YHHJ in ICP have not been fully elucidated. Therefore, the present study investigated an experimental hepatocellular cholestasis model to explore the regulatory role of YHHJ on the expression of the bile acid carriers, multidrug resistance-associated protein 2 (MRP2) and the bile salt export pump (BSEP). Initially, 5 mg/kg/day 17-α ethinylestradiol (EE) was used to induce cholestasis in rats and primary isolated rat hepatocytes. Subsequently, 9 or 36 g/kg/day YHHJ water extract was administrated. Blood samples were collected and serum biochemical parameters of total bile acids (TBA), total bilirubin (TBil), alanine transaminase and aspartate aminotransferase levels were determined. Rat livers and primary isolated rat hepatocytes were obtained and the protein and mRNA expression levels of MRP2 and BSEP were analyzed by western blot analysis and reverse transcription-quantitative polymerase chain reaction, respectively. Results revealed that EE-induced hepatocellular cholestasis was associated with a significant increase in serum TBA and TBil levels, whereas, YHHJ treatment significantly reversed this effect (P<0.01). Further experiments on the molecular mechanism revealed that EE significantly decreased the expression of MRP2 and BSEP compared with the control group, whereas YHHJ treatment significantly upregulated MRP2 and BSEP expression and compared with no YHHJ treatment (P<0.01). In addition, to establish whether upregulation of MRP2 and BSEP protein expression levels resulted from increased expression of their respective mRNA, the mRNA expression levels were determined. Results indicated that YHHJ treatment significantly increased MRP2 and BSEP mRNA expression levels in EE-induced hepatocellular cholestasis compared with no YHHJ treatment (P<0.01). In conclusion, the present findings suggest that YHHJ effects EE-induced cholestasis and this process may be mediated through regulating hepatobiliary transporters, MRP2 and BSEP.
中药茵黄合剂（YHHJ；专利号200910031240.7）是一种由多种草药制成的水提取物，这些草药包括茵陈、黄芩、茯苓、白术、泽泻、猪苓和栀子。先前的研究表明，茵黄合剂治疗对改善妊娠肝内胆汁淤积症（ICP）患者的瘙痒和降低血清胆汁酸水平具有有益作用。然而，茵黄合剂在ICP中的分子作用机制尚未完全阐明。因此，本研究通过实验性肝细胞胆汁淤积模型来探讨茵黄合剂对胆汁酸转运体多药耐药相关蛋白2（MRP2）和胆盐输出泵（BSEP）表达的调节作用。最初，使用5mg/kg/天的17-α乙炔雌二醇（EE）诱导大鼠和原代分离大鼠肝细胞发生胆汁淤积。随后，给予9或36g/kg/天的茵黄合剂水提取物。采集血样并测定血清总胆汁酸（TBA）、总胆红素（TBil）、丙氨酸转氨酶和天冬氨酸转氨酶水平的生化参数。获取大鼠肝脏和原代分离大鼠肝细胞，分别通过蛋白质印迹分析和逆转录-定量聚合酶链反应分析MRP2和BSEP的蛋白质和mRNA表达水平。结果显示，EE诱导的肝细胞胆汁淤积与血清TBA和TBil水平显著升高相关，而茵黄合剂治疗显著逆转了这种效应（P<0.01）。关于分子机制的进一步实验表明，与对照组相比，EE显著降低了MRP2和BSEP的表达，而茵黄合剂治疗与未用茵黄合剂治疗相比显著上调了MRP2和BSEP的表达（P<0.01）。此外，为确定MRP2和BSEP蛋白表达水平的上调是否源于其各自mRNA表达的增加，测定了mRNA表达水平。结果表明，与未用茵黄合剂治疗相比，茵黄合剂治疗显著增加了EE诱导的肝细胞胆汁淤积中MRP2和BSEP的mRNA表达水平（P<0.01）。总之，本研究结果表明茵黄合剂可影响EE诱导的胆汁淤积，且这一过程可能通过调节肝胆转运体MRP2和BSEP介导。
