酒精调节IRF7，SOCS2和SOCS3表达对I/III型干扰素抗HCV功能的作用及机制研究

Clinical data indicate that alcohol abuse increases HCV viral load among hepatitis C patient and significantly reduces clinical therapeutic effect of IFN-α on HCV infection. Our preliminary data showed that alcohol inhibits anti-HCV activity of IFN-α and promotes HCV replication. We also found that alcohol could regulate the expression of multi-regulators of type I IFN pathway, including down-regulation of IRF7 expression and up-regulation of SOCS2 and SOCS3 expression. However, it is unclear whether alcohol inhibits IFN-α pathway and subsequently promotes HCV replication through regulation the expression of the above factors. In addition, the effect of alcohol on IFN-λ, a very promising new anti-HCV factor in the clinical application, is still unknown. Thus, this study will investigate the effect of alcohol on the expression of key factors and downstream antiviral factors of IFN-α/β and IFN-λ pathways and illuminate the effect of alcohol on anti-HCV activity of IFN-α/β and IFN-λ. Further, we will use gene over-expression and gene knockout techniques to determine the role of IRF7, SOCS2, SOCS3 in IFN-α/β- or IFN-λ-mediated anti-HCV function in the context of alcohol presence. This study will reveal the mechanisms of alcohol promoting HCV replication and decreased therapeutic effect of IFN-α on HCV infection by alcohol. This study will also clarify the effect of alcohol on antiviral activity of IFN-λ and mechanism involved. Our proposed study will provide a scientific basis for improving the efficiency of IFN-α therapy as well as development of novel HCV therapeutic factors.

临床数据表明丙肝病人嗜酒会提高HCV病毒载量并降低IFN-α疗效。我们前期研究发现酒精能抑制IFN-α的抗病毒作用，并发现酒精能下调IFN途径调节因子IRF7表达，而上调SOCS2、SOCS3表达。酒精是否通过这些因子影响IFN-α通路继而促进HCV复制尚不清楚。此外, IFN-λ是一个极有希望在临床上应用的新抗HCV因子，酒精对IFN-λ通路的影响也未知。本项目在HCV感染的条件下，用酒精处理肝细胞，观察IFN-α/β和IFN-λ通路关键因子的变化，阐明酒精对IFN抗HCV作用的影响；进一步采用基因过表达或siRNA敲除技术确定酒精调节的IRF7、SOCS2和SOCS3表达如何影响IFN-α/β和IFN-λ的抗HCV功能。本研究将从分子水平上揭示酒精增强HCV复制、降低IFN-α治疗效果的机制，并阐明酒精对IFN-λ抗HCV作用的影响，为提高IFN-α治疗效率，发展新型治疗手段提供依据。

临床数据表明丙型肝炎病人嗜酒会增高 HCV 病毒载量并降低干扰素治疗的疗效，但具体分子机制尚未阐明。本项目聚焦于酒精调节的各种宿主因子及细胞信号通路探讨酒精增强HCV复制的分子机制。本项目首先在体外研究中证实了酒精可以促进HCV JFH-1在Huh7细胞中的复制并减弱IFN-λ1的抗HCV作用。通过筛查发现酒精处理可以上调干扰素通路负调节因子SOCS2，SOCS3和PIASy的表达，并发现PIASy上调水平最为显著且可促进HCV复制。过表达PIASy能拮抗IFN-λ1的抗HCV复制作用，而敲降PIASy表达则削弱了酒精对IFN-λ1抗HCV功能的拮抗作用，同时发现PIASy抑制IFN-λ1诱导的p-STAT1，ISG56和Mx1的表达，表明PIASy具有抑制IFN-λ1通路的作用。本项目还发现酒精诱导了Huh7细胞的细胞自噬，酒精处理可以诱导LC3表达、促进了LC3转换（LC3B-II/LC3B-I）和增加细胞自噬小体（GFP-LC3）形成。酒精上调表达的PIASy在诱导细胞自噬中具有关键作用。PIASy过表达时，酒精诱导细胞自噬及增强HCV复制的作用得到加强，敲降PIASy表达则导致酒精的这些作用被抑制。机制研究表明PIASy通过上调自噬因子ATG5-12和ATG7的表达以及影响SUMO-1介导的蛋白修饰增强细胞自噬。在PIASy基因过表达或敲降表达的条件下， ATG5-12和ATG7的表达以及SUMO1介导的SUMO化修饰随之得到增强或削弱。综上，本项目研究表明酒精通过上调干扰素负调控因子PIASy的表达减弱了IFN-λ1的抗HCV作用，同时通过上调PIASy的表达促进细胞自噬、进而增强HCV复制，这从分子水平上部分揭示了酒精增强 HCV 复制、降低 IFN-λ抗 HCV 作用的机制。本项目的研究结果，一方面为临床上丙型肝炎病人嗜酒的危害性提供了分子水平的证据，另一方面也为从抑制细胞自噬角度发展抗HCV新策略提供了线索。