五味子乙素结合CB2受体抗肝纤维化作用的分子机制研究

The preliminary study found that schisandrin B (Sch B) can significantly play the anti-hepatic fibrosis effect and also bind with CB2 receptor in vitro competitively, suggesting that the CB2 receptor may be a therapeutic target of Sch B attenuating hepatic fibrosis. The CB2 receptor is highly expressed in Kupffer cells and activated hepatic stellate cells, involved in inflammation, oxidative stress et al, which has been reported to be a potential target of anti-hepatic fibrosis, but it is unclear whether CB2 receptor is the target of Sch B attenuating hepatic fibrosis and what is its molecular mechanism. In this study, CB2 receptor antagonists/agonists, surface plasmon resonance, RNAi technique and gene knockout mice were used to validate CB2 receptor as the action target of Sch B for anti-hepatic fibrosis; From hepatic stellate cells, Kupffer cells and whole animal level, the biological function of CB2 receptor was clarified in the formation process of hepatic fibrosis; From the activation of hepatic stellate cells, polarization of Kupffer cells and interactions between the two kinds of cells, the influence of Sch B binding with CB2 receptor on their related signal pathways and molecular mechanism were investigated. The results will provide basis for finding new drugs of anti-hepatic fibrosis with the specific target and clear mechanism.

前期研究发现，五味子乙素具有显著的抗肝纤维化作用且在体外能与CB2受体竞争性结合，提示CB2受体可能是五味子乙素抗肝纤维化的作用靶点。CB2受体在Kupffer细胞和活化的肝星状细胞中高表达，参与炎症、氧化应激等过程，是已报道的潜在抗肝纤维化作用靶点，但其是否为五味子乙素抗肝纤维化的作用靶点还不明确，其相关分子机制也不清楚。本项目将应用CB2受体拮抗剂/激动剂、表面等离子体共振、RNAi技术及基因敲除小鼠，分别论证CB2受体是否为五味子乙素抗肝纤维化的作用靶点；从肝星状细胞、Kupffer细胞和整体动物水平，研究CB2受体在肝纤维化发展形成过程中的生物学功能；分别从五味子乙素影响肝星状细胞活化、Kupffer细胞极化和两种细胞交互作用三个方面，研究五味子乙素结合CB2受体后下游细胞相关信号通路的变化及抗肝纤维化的分子机制。研究结果将为寻找靶点明确、作用机制清楚的抗肝纤维化新药奠定基础。

前期研究发现五味子乙素具有显著的抗肝纤维化作用且在体外能与CB2受体竞争性结合，本项目首先采用虚拟结构对接、Biacore SPR体外成分蛋白结合和细胞实验，证明CB2受体能与Kuppfer细胞膜上的CB2受体结合并产生生物学效应，初步证明CB2可能是五味子乙素抗肝纤维化作用的靶点；然后通过构建体外CB2基因沉默RAW264.7细胞模型、RAW264.7/HSC细胞共培养体系、CB2基因敲除小鼠和四氯化碳诱导的小鼠肝纤维化模型，验证CB2受体是五味子乙素发挥抗纤维化作用的主要靶点；最后采用Real-time qPCR, ELISA, western blot和免疫荧光技术系统分析五味子乙素结合CB2受体后，调控NF-κB和p38信号通路，抑制Kupffer细胞极化，减少炎症因子释放，进而抑制肝星状细胞的异常增生和α-SMA，缓解疾病模型小鼠的肝纤维化。综上，五味子乙素抗肝纤维化的作用机制是靶向CB2受体，调控NF-κB和p38信号通路，通过抑制炎症激活起到抗肝纤维化作用。本项目研究为五味子乙素治疗肝脏纤维化疾病的临床应用奠定了基础，也为抗肝纤维化新药开发研究提供示范。本项目授权1项国家发明专利，发表SCI论文5篇，培养硕士研究生1名。