HCV NS5A通过抑制p-AMPK并上调固醇调节元件结合蛋白2及HMG-CoA还原酶促进小鼠肝细胞胆固醇合成

HCV NS5A Promotes Cholesterol Synthesis in the Mouse Hepatocytes by Inhibiting AMPK Phosphorylation and Up-regulating SREBP-2 and HMG-CoA Reductase

已有研究证明,丙型肝炎病毒(HCV)非结构蛋白5A(NS5A)可诱导肝细胞脂肪变性。本文报告,HCV NS5A刺激肝细胞胆固醇合成,引起脂代谢失调,促进肝脂肪变性。首先,我们构建了由小鼠甲胎蛋白增强子和小鼠白蛋白启动子驱动的NS5A及NS5A domainⅠ、Ⅱ和Ⅲ的慢病毒表达载体,包装成病毒颗粒后通过尾静脉注射感染小鼠。小鼠血清总胆固醇测定及肝组织切片苏木精-伊红染色揭示,与模拟注射对照及增强绿色荧光蛋白(EGFP)慢病毒颗粒处理小鼠比较,NS5A慢病毒颗粒处理小鼠血清总胆固醇水平明显升高;肝细胞内脂滴明显增多。免疫组化和RTq PCR分析显示,胆固醇合成的关键调节酶HMG-CoA还原酶(HMGCR)在NS5A慢病毒处理的小鼠肝内表达显著升高。蛋白质印迹结果证明,与模拟注射及EGFP慢病毒颗粒处理的小鼠比较,NS5A慢病毒颗粒处理的小鼠肝细胞磷酸化的腺苷一磷酸活化蛋白激酶(p-AMPK)水平明显降低,而固醇调节元件结合蛋白2(SREBP-2)及其靶基因HMGCR的水平显著升高。进一步研究发现,NS5A domainⅡ慢病毒颗粒处理的小鼠肝细胞p-AMPK、SREBP-2和HMGCR表达水平与全长NS5A慢病毒处理的小鼠相似。上述结果提示,HCV NS5A蛋白可通过抑制AMPK磷酸化激活,上调SREBP-2而促进胆固醇合成的限速酶HMGCR的表达,从而促进小鼠肝细胞胆固醇合成。上述结果还提示,NS5A domainⅡ可能是全长NS5A蛋白调节HMGCR基因表达的有效片段。总之,本研究证明,HCV NS5A可引起胆固醇代谢紊乱,这可能是慢性HCV感染引起肝脂肪变性的机制之一。很遗憾,在本研究中,尚未测定SREBP-1的靶基因乙酰CoA羧化酶(脂肪酸合成的限速酶)的表达,相关实验正在进行中。

It has been shown that the non-structural protein 5 A（NS5A） of the hepatitis C virus（HCV）induces hepatic steatosis.In this report we describe that HCV NS5A stimulates cholesterol synthesis andinduces deregulation of lipid metabolism during chronic HCV infection-induced hepatic steatosis.Firstly,we constructed full-length,domain Ⅰ,Ⅱ and Ⅲ of NS5A in lentivirus expression plasmids,driven by the mouse albumin promoter and AFP enhancer.Then we injected the packaged virions into C57BL/6 J mice via tail intravenous to induce hepatic steatosis.Compared with control mice（Mock and EGFPlentivirus virion injection）,the levels of total serum cholesterol in the NS5A virion-injected mice were significantly elevated,and lipid accumulation in the liver was markedly increased detected by hematoxylin-eosin（HE） staining.Immunohistochemical and RT-qPCR assays showed that the expression of 3-hydroxy-3-methylglutaryl CoA reductase（HMGCR）,a key regulatory enzyme for cholesterol synthesis,was up-regulated in the liver of NS5A virion-injected mice.Western blotting demonstrated that the level of phospho-adenosine monophosphate-activated protein kinase（p-AMPK） was down-regulated,while sterol regulatory element binding protein 2（SREBP-2） and its target HMGCR were up-regulated in the liver of NS5A virion-injected mice,compared to controls.Moreover,similar expression levels of p-AMPK,SREBP-2 and HMGCR were observed in the liver of NS5A domain Ⅱ virion-injected mice.These data indicate that HCV NS5A may stimulate the synthesis of cholesterol by inhibiting AMPK phosphorylation and up-regulating SREBP-2 and HMGCR.NS5A domain Ⅱ,as an essential component of NS5A may play a key role in transcriptional activation of HMGCR.Taken together,HCV NS5A may induce deregulation of lipid metabolism,which may contribute to chronic HCV infection-induced hepatic steatosis.