丙型肝炎病毒在干扰素耐药和胰岛素抵抗中的作用机制

Most hepatitis C patients do not response to interferon (IFN)-based therapy. Hepatitis C virus (HCV) infection is associated with IFN resistance and insulin resistance. Insulin resistance is one of the important factors influencing the IFN antiviral efficacy. However,the mechanism is unknown. Our previous study showed that the induction of USP18 and inhibition of AMPK activity were observed in primary human hepatocytes isolated from liver tissues of hepatitis C patients and HCV replicon cell line resistant to both IFN and insulin treatment.Inhibition of USP18 caused enhancement of AMPK activity and suppression of HCV RNA replication. Based on our findings and scientific rules, we think that the mutated HCV induces USP18, inhibits AMPK activity and causes IFN resistance and insulin resistance in viral-infected cells. By means of the full-length HCV replicon cells resistant to both IFN and insulin treatment and viral-infected primary human hepatocytes isolated from liver tissues of hepatitis C patients, we will analyze the differences of IFN JAK-STAT and insulin PI3K-AKT signaling pathway between HCV replicon cells resistant to insulin and IFN or viral-infected primary human hepatocytes and sensitive replicon cell or viral-infected primary human hepacytes through molecular biological and virological methods. Then we will examine the effect of mutated viral proteins on the signailng molecules of both pathway, the expression of USP18 and the activity of AMPK to explore the role of USP18 and AMPK in HCV-induced IFN resistance and insulin resistance. The results we obtain will be verified in primary human hepatocytes isolated from liver biopsy of hepatitis C patients. Finally we will try to reverse the IFN resistance and insulin resistance using gene therapy. The study will pave a novel way to improve the IFN antiviral efficacy.

大部分丙肝患者对干扰素耐药，HCV 感染与胰岛素抵抗及干扰素耐药密切相关，胰岛素抵抗是影响干扰素疗效的重要因素，机制未明。我们前期研究发现对胰岛素抵抗和干扰素耐药的丙肝组织或病毒复制子细胞USP18表达升高，AMPK T172磷酸化活性减弱；抑制细胞内USP18表达，则AMPK磷酸化活性增强，病毒复制被抑制。我们推测变异丙肝病毒诱导细胞表达USP18，抑制AMPK活性，导致胰岛素抵抗及干扰素耐药。本研究拟通过分子生物学、病毒学方法，比较对胰岛素抵抗且干扰素耐药的丙肝病毒复制子细胞及其来源细胞或丙肝病人肝细胞内干扰素JAK/STAT、胰岛素PI3K/AKT通路中信号分子差异，检测病毒蛋白对两种信号通路中差异分子以及USP18表达与AMPK磷酸化的影响，探讨USP18、AMPK在丙肝病毒所致干扰素耐药和胰岛素抵抗的分子机制，尝试基因治疗逆转胰岛素抵抗和干扰素耐药，为提高干扰素疗效提供新思路。

大部分丙肝患者对干扰素耐药，HCV 感染与胰岛素抵抗及干扰素耐药密切相关，胰岛素抵抗是影响干扰素疗效的重要因素，但HCV感染导致干扰素耐药与胰岛素抵抗机制未明。在国家自然科学基金项目的资助下，我们探讨了HCV感染导致干扰素耐药和胰岛素抵抗的作用机制。本项目的主要研究内容如下：阐明HCV对干扰素JAK/STAT和胰岛素PI3K/AKT信号通路的影响；揭示USP18、AMPK在HCV所致干扰素耐药与胰岛素抵抗中的作用机制；体外逆转HCV所致干扰素耐药与胰岛素抵抗。通过项目的实施，我们首先发现激活宿主细胞AMPK，可诱导ISGs且抑制miR-122的表达而发挥抗HCV作用，发现了通过影响脂肪代谢可以增强宿主细胞先天免疫反应，达到抗病毒作用的新机制，首次发现AMPK的抗病毒作用并初步揭示其作用机制。其次发现HCV感染通过促进USP18的表达，阻断干扰素JAK/STAT通路和胰岛素PI3K/AKT信号通路，导致病毒感染细胞对干扰素耐药和胰岛素抵抗。再次发现HCV感染通过促进长非编码RNA LncITPRIP的表达，抑制JAK/STAT1通路，进而导致病毒感染细胞对干扰素耐药。最后发现沉默病毒感染细胞内的USP18表达可增强AMPK磷酸化活性，病毒复制被抑制，逆转对干扰素耐药和胰岛素抵抗。基于此，我们认为HCV通过诱导细胞表达USP18，抑制AMPK活性，阻断干扰素JAK/STAT、胰岛素PI3K/AKT信号通路，导致干扰素耐药和胰岛素抵抗。探讨丙肝病毒在干扰素耐药和胰岛素抵抗中的作用，有助于揭示丙肝病毒导致干扰素耐药和胰岛素抵抗的分子机制，具有重要的科学意义和潜在的临床应用价值。相关研究成果发表在PNAS、Journal of Virology、Antimicrobial Agents and Chemotherapy、Virology、Antiviral Research等国际学术期刊上。