肝炎病毒,丙型%沉默信息调节因子1%脂肪酸合成%脂肪酸氧化
肝炎病毒,丙型%沉默信息調節因子1%脂肪痠閤成%脂肪痠氧化
간염병독,병형%침묵신식조절인자1%지방산합성%지방산양화
Hepatitis C virus%Silent information regulator 1%Fatty acid synthesis%Fatty acid oxidation
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.
目的 用攜帶HCV複製子的Huh-7.5細胞探討HCV複製對沉默信息調節因子1(SIRT1)錶達及肝細胞脂類代謝的影響.方法 實驗分為Huh-7.5細胞、複製子細胞和榦擾素處理的複製子細胞3組.應用流式細胞儀、比色法測定細胞活性氧(ROS)變化和煙酰胺腺嘌呤二覈苷痠(NAD)+/NADH比值變化.應用液體閃爍計數儀、實時熒光定量-PCR (RT-PCR)、Western blot檢測SIRT1活性、mRNA及蛋白的錶達.RT-PCR檢測SIRT1下遊調節脂類代謝基因的mRNA水平.試劑盒檢測甘油三酯(TG)、總膽固醇(TC)的含量,液體閃爍計數儀檢測脂肪痠β氧化速度,Westem blot檢測HCV非結構蛋白5A錶達水平.計量資料兩組間比較用t檢驗,三組間比較用單因素方差分析.結果 與Huh-7.5細胞相比,複製子細胞ROS水平增加(3.8±0.5與1.0±0.2,t=12.736,P＜0.01),NAD+/NADH比值下降(0.03±0.01與0.12±0.03,t=6.971,P＜0.01).與Huh-7.5細胞和IFN處理的複製子細胞相比,複製子細胞SIRT1的活性下降(0.3±0.1與1.0±0.2、0.9±0.2,F=6.766,P＜0.01)、mRNA水平下降(0.4±0.1與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與0.8±0.2、0.9±0.2,F=5.419,P＜0.01);扠頭蛋白轉錄因子(FoxO1)燐痠化水平下降(0.2±0.1與0.5±0.1、0.6±0.2,F=4.534,P＜ 0.05),固醇調節元件結閤蛋白-1c基因錶達水平上升(1.8±0.4與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)閤成增加；過氧化物酶體增殖物活化受體α基因錶達水平下降(0.4±0.1與1.0±0.3、0.9±0.3,F=5.897,P＜ 0.01),脂肪痠β氧化速度下降(0.4±0.1與1.0±0.3、0.9±0.3,F=5.123,P＜0.01).與未處理或SIRT1激動劑處理組相比,SIRT1抑製劑處理複製子細胞72h後HCV非結構蛋白5A錶達水平明顯增加(1.90±0.45與1.0±0.15、0.20±0.06,F=13.765,P＜0.01).結論 HCV複製降低NAW/NADH比值,下調SIRT1活性及錶達,改變其下遊調節脂類代謝相關基因錶達,增加脂肪痠閤成,降低脂肪痠氧化,導緻肝細胞脂類代謝紊亂,促進HCV複製.
목적 용휴대HCV복제자적Huh-7.5세포탐토HCV복제대침묵신식조절인자1(SIRT1)표체급간세포지류대사적영향.방법 실험분위Huh-7.5세포、복제자세포화간우소처리적복제자세포3조.응용류식세포의、비색법측정세포활성양(ROS)변화화연선알선표령이핵감산(NAD)+/NADH비치변화.응용액체섬삭계수의、실시형광정량-PCR (RT-PCR)、Western blot검측SIRT1활성、mRNA급단백적표체.RT-PCR검측SIRT1하유조절지류대사기인적mRNA수평.시제합검측감유삼지(TG)、총담고순(TC)적함량,액체섬삭계수의검측지방산β양화속도,Westem blot검측HCV비결구단백5A표체수평.계량자료량조간비교용t검험,삼조간비교용단인소방차분석.결과 여Huh-7.5세포상비,복제자세포ROS수평증가(3.8±0.5여1.0±0.2,t=12.736,P＜0.01),NAD+/NADH비치하강(0.03±0.01여0.12±0.03,t=6.971,P＜0.01).여Huh-7.5세포화IFN처리적복제자세포상비,복제자세포SIRT1적활성하강(0.3±0.1여1.0±0.2、0.9±0.2,F=6.766,P＜0.01)、mRNA수평하강(0.4±0.1여1.0±0.3、0.9±0.2,F=5.864,P＜ 0.01)화단백수평하강(0.3±0.1여0.8±0.2、0.9±0.2,F=5.419,P＜0.01);차두단백전록인자(FoxO1)린산화수평하강(0.2±0.1여0.5±0.1、0.6±0.2,F=4.534,P＜ 0.05),고순조절원건결합단백-1c기인표체수평상승(1.8±0.4여1.0±0.3、0.9±0.3,F=4.091,P＜0.05),이TG(F=6.670,P＜0.01)화TC (F=5.789,P＜0.01)합성증가；과양화물매체증식물활화수체α기인표체수평하강(0.4±0.1여1.0±0.3、0.9±0.3,F=5.897,P＜ 0.01),지방산β양화속도하강(0.4±0.1여1.0±0.3、0.9±0.3,F=5.123,P＜0.01).여미처리혹SIRT1격동제처리조상비,SIRT1억제제처리복제자세포72h후HCV비결구단백5A표체수평명현증가(1.90±0.45여1.0±0.15、0.20±0.06,F=13.765,P＜0.01).결론 HCV복제강저NAW/NADH비치,하조SIRT1활성급표체,개변기하유조절지류대사상관기인표체,증가지방산합성,강저지방산양화,도치간세포지류대사문란,촉진HCV복제.
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.
