补肾方调节肝巨噬细胞来源外泌体发挥抗HBV作用机制研究

When hepatitis B virus (HBV) is infected, exosomes in the liver microenvironment can mediate cell-to-cell communication and participate in immune regulation. Studies have shown that IFN-I can induce intrahepatic macrophages (M) to secrete APOBEC3A-containing exosomes to act on hepatocytes infected with HBV and play an antiviral role. Previous studies have shown that Bushen Recipe can promote the secretion of interferon I, promote the secretion of exosomes from CHB patients, and up-regulate the expression of key proteins secreted by hepatic and extrahepatic secretions of HBV transgenic mice. The hypothesis was put forward that "Bushen Recipe can exert anti-HBV effect by inducing macrophages to secrete exosomes through type I interferon". The purpose of this study is to construct a mouse model of HBV infection by using pAAV-HBV 1.3 plasmid and observe the effects of Bushen Recipe on type I interferon, M and exosome in liver; to study the intervention targets of Bushen Recipe by using M exhaustion reconstruction and shRNA interfering with M exosome secretion; and to observe the effect of Bushen Recipe on M and APOBEC3G antiviral substances in its exosome and on HepG2.2.15 intracellular and exosome. To further clarify the immunological mechanism of Bushen Decoction in treating chronic hepatitis B.

乙肝病毒(HBV)感染时，肝脏微环境中外泌体可介导细胞间沟通，参与机体免疫调节。研究表明I型干扰素可诱导肝内巨噬细胞(MΦ)分泌含APOBEC3A的外泌体作用于HBV感染的肝细胞，发挥抗病毒作用。补肾方是治疗慢乙肝的验方，前期研究表明补肾方可促进I型干扰素分泌，促进CHB患者外泌体的分泌，并上调HBV转基因小鼠肝内外泌体分泌关键蛋白的表达。由此提出假说“补肾方可通过I型干扰素诱导巨噬细胞分泌外泌体发挥抗HBV作用”。本研究拟：采用pAAV-HBV1.3质粒构建HBV感染小鼠模型，观察补肾方对肝内I型干扰素、MΦ及外泌体的影响；利用MΦ耗竭重建及shRNA干扰MΦ外泌体的分泌，研究补肾方干预靶点；采用激光共聚焦等观察补肾方对MΦ及其外泌体内APOBEC3G抗病毒物质及对HepG2.2.15细胞对外泌体内化的影响，进一步明确补肾方治疗慢乙肝的免疫学机制。

补肾方，具有抗HBV、改善肝功能作用的中药复方，经本研究证实可诱导巨噬细胞发挥抗HBV作用，主要通过I型干扰素通路调控由巨噬细胞所分泌的含APOBEC3A的外泌体所实现。本研究项目计划书所列内容已完成。目前的体内研究表明，在 pAAV-HBV1.3质粒构建HBV感染小鼠模型中，经conA诱导后，补肾方可以显著降低肝功能指标，显著降低血清HBsAg水平，提高IFN-β、IFN-γ、IL-4、IL-12的水平，增加MΦ的比例；利用MΦ耗竭重建及shRNA干扰MΦ外泌体的分泌后，补肾方失去肝脏的保护作用和抗病毒效应，同时小鼠肝组织nSMase2、GTPase和RAB27a表达下调，干扰素信号通路关键蛋白（IRF9、Jak、STAT1/2）也显著降低，提示补肾方抗乙肝作用与巨噬细胞和外泌体分泌密切相关，而I型干扰素通路是调控的关键；HepG2.2.15细胞和巨噬细胞共培养提示MΦ及其外泌体内APOBEC3G抗病毒物质是补肾方抗乙肝的干预靶点。因此，I型干扰素通路介导的巨噬细胞分泌的外泌体调控在乙肝的免疫应答机制中具有重要的临床意义。该途径为及补肾方发挥抗 HBV 效应的现代免疫学机制解析提供了一个新的视角。