细胞自噬阻断介导的霉酚酸抑制HCV复制的机制研究

Mycophenolic acid (MPA) is the activated metabolic component of the mycophenolate mofetil (MMF), a highly effective immunosuppressant commonly used in liver and kidney transplantation in clinic. Our preliminary data showed that MPA can block autophagy in liver cells, and pervious in vitro studies by others demonstrated that MPA has a potent antiviral ability against HCV. It is well known that the autophagy machinery is required for the initiation of HCV replication. However, there is not any report about whether MPA inhibits HCV replication through blocking autophagy and the mechanism involved. This study will use autophagy inducer and/or MPA to treat liver cells with or without HCV infection, to research the effects of MPA on autophagic flux and examine whether MPA blocks the autophagy in liver cells. At the same time, this study will examine whether the autophagy inducer compromises the inhibitory effect of MPA on HCV replication. On this basis, further studies will use a series of biological techniques including Human Autophagy RT- PCR array to screen and identify the autopahgy-related genes that are regulated by MPA, then use gene overexpression and siRNA knockout techniques to determine the key factors responsible for the actions of MPA to block autophagy,thereby to inhibit HCV replication. In addition to the mechanism research,this study will investigate whether the blockade of autophagy by MPA activates cellular interferon pathway that contributes to the anti-HCV activity of MPA. In summary, this study will reveal the role of autophagy in the anti-HCV effect of MPA and clarify the molecular mechanism of the anti-HCV activity mediated by MPA. Our proposed study will provide scientific basis for the development of new anti-HCV drugs from the perspective of blocking autophagy, and it is of great clinical significance to the treatment and prognosis of patient with liver transplanation due to HCV infection.

霉酚酸是肝肾移植免疫抑制剂霉酚酸酯的活性代谢成分。前期研究发现霉酚酸能阻断肝细胞的自噬，并能抑制HCV复制。自噬机器为HCV起始复制所必需。但霉酚酸是否通过阻断自噬而抑制HCV及机制尚无研究报道。本课题利用自噬诱导剂和霉酚酸先后或分别处理感染和未感染HCV的肝细胞，观察细胞自噬流的变化, 阐明霉酚酸对肝细胞自噬的阻断作用以及自噬诱导剂能否拮抗霉酚酸对HCV复制的抑制作用。在此基础上，用自噬RT-PCR阵列筛选霉酚酸调节的自噬基因，采用基因过表达或siRNA敲除技术确定霉酚酸作用的关键自噬基因，并明确这些基因的表达或敲除如何影响HCV复制。此外，本课题还将确定霉酚酸对细胞自噬的阻断是否激活细胞的干扰素途径，进一步阐明霉酚酸的抗HCV机理。研究成果将从分子水平上揭示霉酚酸抗HCV的机制，为从细胞自噬角度开发抗HCV药物提供新策略，并对临床上丙型肝炎肝移植的治疗和预后具有指导作用。

霉酚酸酯（mycophenolate mofetil，MMF）是目前临床上肝肾移植常用的免疫抑制剂，其代谢活性成分是霉酚酸（Mycophenolic acid, MPA)。既往研究发现MPA具有抗HCV活性，但具体机制未明。本研究目的在于阐明霉酚酸是否通过抑制细胞自噬，进而抑制HCV复制以及涉及其中的分子机制。本研究首先证实了MPA抑制HCV JFH-1在Huh7细胞的复制，再利用MPA处理自噬诱导剂或HCV感染的肝细胞，通过western blot, 免疫荧光和电镜检测观察细胞自噬流的变化, 发现霉酚酸降低LC3-II表达、降低LC3蛋白转化（LC3-II/LC3-I）、减少点状GFP-LC3和自噬体形成，阐明了MPA对细胞自噬的阻断作用；然后利用自噬基因PCR array，RT-PCR和western blot等技术筛查并确认了霉酚酸抑制自噬相关基因ATG3、ATG5、ATG7的表达，并利用过表达/基因沉默技术证实了霉酚酸下调ATG3、ATG5、ATG7表达在MPA抑制细胞自噬，进而抑制HCV复制中的关键作用。此外，研究还显示MPA可以诱导IFNα和IFNβ的表达，并提高了IFN效应分子OAS和ISG56的表达，将MPA和抗病毒通路IFN途径联系起来；同时发现III型干扰素IFN-λ1能抑制Huh7细胞的细胞自噬，进一步的机制研究证实IFN-λ1是通过下调自噬基因ATG5和GABARAP表达抑制细胞自噬，提示抑制细胞自噬可能是IFN-λ1的抗HCV机制之一。综上，本研究较为清晰地阐明了MPA对细胞自噬的阻断作用及其通过抑制细胞自噬抗HCV的分子机制，同时也阐明了IFN-λ1通过抑制细胞自噬进而抑制HCV复制，并将MPA和干扰素途径联系起来。本项目的研究结果在分子水平上提供了MPA抗HCV的新机制，为从细胞自噬角度开发抗HCV药物提供了新策略，并对临床上丙型肝炎肝移植的治疗和预后具有指导作用。