补肾方通过RIG-I信号通路调控Viperin表达抗HBV的作用机制研究

Viperin is induced expression by activation of RIG-I-MAVS-IFN signaling pathway in host cell, which can bind to viral protein and famesyl pyrophosphate synthase (FPPS) to inhibition virus assembly and release. Bushen Recipe is an effective treatment of chronic hepatitis B patients, which can significantly promote the expression of RIG-I, IFN and Viperin. Therefore, the research hypothesis is proposed that the expression of Viperin in the liver is up-regulated through RIG-I-MAVS-IFN signal pathway following Bushen Recipe treatment，which can inhibit hepatitis B virus (HBV) replication. In this study, the mouse model of Chronic HBV infection which Viperin silencing is taken as a model to investigate the effect of inhibition of HBV replication effect by Bushen recipe. Then, through HBV plasmid transient hepatocyte lines and RNA interference, to make sure that the up-regulated expression of Viperin is through RIG-I-MAVS-IFN signal pathway following Bushen Recipe treatment. Last，through cell transfection, Co-Immunoprecipitation and Confocal laser scanning microscope, to observe the effect of Bushen Recipe on Viperin and HBV virus protein binding, Viperin and FPPS interaction effects. To further clarify the partial immunological mechanism of Bushen Recipe in Chronic Hepatitis B patients.

RIG-I-MAVS-IFN信号通路活化可诱导宿主细胞表达Viperin，Viperin与病毒蛋白结合抑制病毒组装，并与法尼基焦磷酸合成酶（FPPS）结合抑制病毒释出。补肾方是治疗慢性乙型肝炎（CHB）有效验方，可显著促进RIG-I、IFN和Viperin表达。据此提出假说：补肾方通过RIG-I-MAVS-IFN信号通路上调肝脏内Viperin表达而抑制乙肝病毒（HBV）复制。本研究拟开展：1.应用沉默Viperin的HBV持续感染小鼠，探讨Viperin对补肾方抑制肝细胞HBV复制的影响；2.采用HBV质粒瞬转肝细胞和RNA干扰技术，明确补肾方通过RIG-I-MAVS-IFN信号通路上调Viperin的机制，并运用细胞转染、免疫共沉淀和激光共聚焦等技术分别观察补肾方对Viperin与HBV病毒蛋白结合、Viperin与FPPS相互作用的影响。以进一步明确补肾方治疗CHB的部分免疫学机制。

文献报道RSAD2（Viperin）可通过与病毒蛋白相互作用、以及增加脂筏流动性，影响病毒的组装和释出的功能,从而发挥抗病毒的作用。我们前期研究证实补肾方能显著促进 RIG-1（DDX58）、IRF3和RSAD2表达，抑制HBsAg和HBeAg分泌。本研究拟从三个部分进行研究:①应用HBV持续感染的小鼠模型研究补肾方通过调控RSAD2的表达而抑制HBV复制；②利用细胞模型探讨补肾方诱导RSAD2表达的信号转导机制；③采用细胞转染、免疫共沉淀和激光共聚焦等技术明确RSAD2抑制HBV组装和释出的作用机理以及补肾方对该过程的调控。结果发现：①与HBV持续感染的小鼠模型组相比，补肾方组小鼠血清中ALT、AST、HBsAg水平明显下降，肝脏内炎症程度明显改善，肝脏内HBsAg和HBcAg水平明显下降，血清中IFN、肝脏内RSAD2、DDX58、IRF3、MAVS水平明显上调；②体外研究表明，外源性表达的RSAD2能抑制HepG2.2.15细胞和pHBV瞬转HepG2细胞分泌HBsAg和HBeAg，RSAD2 siRNA干扰HepG2.2.15和pHBV瞬转HepG2细胞后HBsAg和HBeAg水平无明显变化；补肾方能明显提高IFNL1和RSAD2水平，抑制HBsAg和HBeAg表达。③RSAD2能与乙肝病毒蛋白结合，尤其与HBeAg结合能力最强。初步明确补肾方治疗CHB的部分免疫学机制，为抗HBV提供了一个新的研究靶点。