Objective To investigate the role of the host-encoded silent information regulator 1 (SIRT1) on hepatocytes' lipid metabolism under conditions of hepatitis C virus (HCV) infection and assess its potential effects on virus replication in vitro.Methods The Huh-7.5 human hepatocyte cell line was used as the control group and Huh-7.5 cells stably expressing the HCV replicon (Huh7.5-HCV) were used as the experimental group.Effects of interferon (IFN) treatment and activation of SIRT1 by resveratrol were also observed.The mRNA and protein expression levels of SIRT1 were detected by real time (q)PCR and western blotting.Effects on SIRT1 protein activity were tested by measuring the levels of reactive oxygen species (ROS) and the nicotinamide adenine dinucleotide (NAD+)/β-nlcotinamide adenine dinucleotide,reduced (NADH) by flow cytometry and chromatometry,and the levels of triacylglycerol (TG),total cholesterol (TC),and fatty acid β oxidation rate by enzymatic analysis and liquid scintillation counting.Effects on mRNA expression of SIRT1 downstream lipid-metabolism genes were measured by qPCR.Results The Huh7.5-HCV cells had a significantly higher level of ROS (3.8 ± 0.5 vs.Huh-7.5:1.0 ± 0.2; t =12.736,P＜ 0.01) but significantly lower levels of NAD+/NADH (0.03 ±0.01 vs.0.12±0.03; t=6.971,P＜ 0.01),SIRT1activity (0.3±0.1 vs.1.0±0.2,0.9±0.2,F=6.766,P＜ 0.01),SIRT1 mRNA (0.4± 0.1 vs.1.0±0.3,0.9±0.2,F=5.864,P＜ 0.01),and SIRT1 protein (0.3 ± 0.1 vs.0.8 ± 0.2,0.9 ± 0.2,F=5.419,P＜ 0.01).The lower levels of SIRT1 in Huh7.5-HCV cells accormpanied decreased phosphoryhtion of the forkhead box O 1 (FoxO1),which not only up-regulated the downstream genes of SREBP-1c,FAS,ACC,SREBP-2,HMGR and HMGS (which increased fatty acid synthesis) but also down-regulated the downstream genes of PPAR and CPT1A genes (which decreased fatty acid β oxidation).IFN treatment restored all of the aforementioned changes.Resveratrol-induced SIRT activation ved the pernbatiom in lipid metabolism pathways,as evidenced by an increase in fatty acid β oxidation and a decrease in TG and TC synthesis,as well as inhibited HCV replication.Conclusion HCV may decrease the NAD+/NADH ratio in hepatocytes,leading to a down-regulation of SIRT1 activity and expression and perturbing the downstream expression profile of lipid metabolism-related factors,ultimately causing lipid metabolism disorders and establishing a permissive intracellular environment for HCV replication.%目的 用携带HCV复制子的Huh-7.5细胞探讨HCV复制对沉默信息调节因子1(SIRT1)表达及肝细胞脂类代谢的影响.方法 实验分为Huh-7.5细胞、复制子细胞和干扰素处理的复制子细胞3组.应用流式细胞仪、比色法测定细胞活性氧(ROS)变化和烟酰胺腺嘌呤二核苷酸(NAD)+/NADH比值变化.应用液体闪烁计数仪、实时荧光定量-PCR (RT-PCR)、Western blot检测SIRT1活性、mRNA及蛋白的表达.RT-PCR检测SIRT1下游调节脂类代谢基因的mRNA水平.试剂盒检测甘油三酯(TG)、总胆固醇(TC)的含量,液体闪烁计数仪检测脂肪酸β氧化速度,Westem blot检测HCV非结构蛋白5A表达水平.计量资料两组间比较用t检验,三组间比较用单因素方差分析.结果 与Huh-7.5细胞相比,复制子细胞ROS水平增加(3.8±0.5与1.0±0.2,t=12.736,P＜0.01),NAD+/NADH比值下降(0.03±0.01与0.12±0.03,t=6.971,P＜0.01).与Huh-7.5细胞和IFN处理的复制子细胞相比,复制子细胞SIRT1的活性下降(0.3±0.1与1.0±0.2、0.9±0.2,F=6.766,P＜0.01)、mRNA水平下降(0.4±0.1与1.0±0.3、0.9±0.2,F=5.864,P＜ 0.01)和蛋白水平下降(0.3±0.1与0.8±0.2、0.9±0.2,F=5.419,P＜0.01);叉头蛋白转录因子(FoxO1)磷酸化水平下降(0.2±0.1与0.5±0.1、0.6±0.2,F=4.534,P＜ 0.05),固醇调节元件结合蛋白-1c基因表达水平上升(1.8±0.4与1.0±0.3、0.9±0.3,F=4.091,P＜0.05),而TG(F=6.670,P＜0.01)和TC (F=5.789,P＜0.01)合成增加；过氧化物酶体增殖物活化受体α基因表达水平下降(0.4±0.1与1.0±0.3、0.9±0.3,F=5.897,P＜ 0.01),脂肪酸β氧化速度下降(0.4±0.1与1.0±0.3、0.9±0.3,F=5.123,P＜0.01).与未处理或SIRT1激动剂处理组相比,SIRT1抑制剂处理复制子细胞72h后HCV非结构蛋白5A表达水平明显增加(1.90±0.45与1.0±0.15、0.20±0.06,F=13.765,P＜0.01).结论 HCV复制降低NAW/NADH比值,下调SIRT1活性及表达,改变其下游调节脂类代谢相关基因表达,增加脂肪酸合成,降低脂肪酸氧化,导致肝细胞脂类代谢紊乱,促进HCV复制.
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