利福平所致胆汁淤积发病中肝细胞顶膜MRP2定位异常的机制研究

Rifampicin induced hepatic injury is common and the patients often have elevated conjugated bilirubin,the mechanism is unclear.As an important bile acid and bilirubin transporter located on hepatocellular apical membrane, inhibition of MRP2's（multidrug resistance associated protein 2）expression and activity can induce cholestasis.In previous study, from liver tissue of patients with rifampicin indued cholestasis, we found reduced expression of MRP2 on plasma membrane and increased expression of MRP2 in cytoplasm. The same change was also found in zoopery. Changed in localization of MRP2 from the canalicular membrane to a subapical membranous compartment of the hepatocyte is also thought to affect its function and has been linked to cholestasis.So we suppose rifampicin can induce endocytic intenalization of canalicular MRP2, then cause cholestasis. Some studies proved oxidative stress may be the hinge in the procedure of internalization,as rifampicin is inducer of many oxidase, it may infulence the redox status of hepatic cell.So we suppose rifampicin induce internalization of MRP2 protein through change the intra-hepatic cell redox status, thus caused cholestasis.Mutiple signaling pathways and cellular targets are involved in this procedure. In this study, we will focused on rifampicin' influence on localization and function of MRP2 in isolated rat hepatocytes, transfected HEK293 cells and MDCK cells, Rat model will also be used. Redox status, hinge PKCs signaling molecular and how Clathrin act on this process will be investgated.The study will provide the new knowledge in the mechanism of rifampicin induced cholestatic hepatopathy, and might provide the new target and theoretical insights for the anti-tuberculosis drug induced hepatic injury.

利福平所致肝损害主要以胆汁淤积型为主，其具体机制一直不清，最直接的表现是以血清直接胆红素升高。而肝细胞顶膜的转运蛋白MRP2是直接胆红素的转运蛋白，其功能降低是常见的胆汁淤积的发病基础。我们前期的研究发现利福平所致肝损害患者的肝细胞顶膜MRP2蛋白定位减少，而胞浆内蛋白含量增加，动物实验中同样存在这种现象，而且mRNA水平正常，提示可能存在MRP2蛋白的内吞。有研究表明氧化应激可能是是启动包膜蛋白内吞的关键因素，利福平一直被认为是一些氧化酶的诱导剂，能加强肝细胞内的氧化应激刺激。据此我们推导利福平可能是通过改变肝细胞内氧化状态而启动肝细胞顶膜MRP2的内吞使胆汁排泌功能受影响从而造成药物性胆汁淤积。本课题拟采用细胞模型和动物实验深入探讨氧化应激刺激在此过程中的作用，寻找启动内吞的关键信号分子并明确其对内吞蛋白的包裹和降解途径的影响，为避免和治疗利福平抗痨过程中出现的肝损害提供新的思路。

利福平（RF）是有效的抗结核药物，但是其肝毒性是制约其临床应用的最关键因素。流行病学调查和我们前期的研究均提示，利福平所致肝损害中，胆汁淤积型肝损害所占的比例较高，我们推测利福平所致肝损害可能与肝细胞顶膜的转运蛋白MRP2的功能损伤有关。本研究中，我们成功建立了利福平致肝损害的动物模型和细胞模型，在此基础上首先证实了胆汁淤积是利福平对肝细胞炎症损害的早期事件，而MRP2蛋白的膜定位表达受利福平影响，影响是发生在转录后的水平。再通过检测利福平处理前后细胞内GSH、NO、Ca(++）等浓度变化的趋势，证实利福平处理能改变HepG2细胞内的氧化状态，诱发氧化应激刺激，抗氧化剂预处理能改善这种影响，而通过激活氧化应激刺激，利福平能诱导PKCα、PKCδ、 PKCε 表达水平和 JNK、P38、和ERK磷酸化程度的显著增高，而p-PI3K的表达则为明显降低。提示利福平主要激活经典的PkC通路，并通过siRNA技术证实。进一步的研究表明，利福平能通过AP-2和Clathrin介导的内吞影响MRP2的膜定位，而其中内吞的启动受cPKC信号通路介导，不仅如此，利福平刺激后HepG2细胞内MRP2发生泛素化，而三个泛素连接关键酶中， GP78路径才是加速MRP2蛋白降解的关键途径。GP78的干扰能限制利福平对MRP2蛋白表达的影响。提示利福平可能通过GP78相关的泛素-蛋白酶体系统降解内吞的MRP2蛋白。. 因此，本项目验证了我们的假设，利福平所致肝损伤中顶膜MRP2蛋白的内吞可能是肝损害的早期事件，是RF相关胆汁淤积发病的病理基础。而在此过程中，RIF可能通过激活氧化应激刺激系统，激活PKC信号通路，活化Clathrin相关的MRP2蛋白内吞，并且激活泛素-蛋白酶体系统对内吞蛋白进行降解，本研究结果为避免和治疗利福平抗痨过程中出现的肝损害提供新的思路。