丙型肝炎病毒核心蛋白下调AMP激活蛋白激酶对肝细胞脂类代谢的影响被引量：3

HCV core protein down–regulates AMP-activated protein kinase induces lipid metabolism disorders in hepatocytes

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摘要目的探讨HCV核心蛋白对AMP激活蛋白激酶(AMPK)表达及肝细胞脂类代谢的影响。方法表达HCV核心蛋白的质粒转染HepG2细胞。分别应用液体闪烁计数仪、实时荧光定量-PCR(RT-PCR)、Western印迹检测AMPK活性、mRNA及蛋白的表达。试剂盒检测三酰甘油、总胆固醇(TC)的含量,液闪计数仪检测脂肪酸β氧化速度,流式细胞仪、硫代巴比妥酸比色法、黄嘌呤氧化酶法分别测定活性氧(ROS)、丙二醛(MDA)含量及超氧化物歧化酶(SOD)活性。RT-PCR检测AMPK下游调节脂类代谢的基因固醇调节元件结合蛋白(SREBP)-1c、脂肪酸合成酶(FAS)、乙酰辅酶A羧化酶(ACC)、SREBP-2、羟甲基戊二酸单酰CoA还原酶(HMGR)、羟甲基戊二酸单酰CoA合成酶(HMGS)、过氧化物酶体增殖活化受体(PPAR)α、PPARγ、肉碱脂酰转移酶1(CPT1)mRNA水平。结果与转染空载体的HepG2细胞相比,表达HCV核心蛋白HepG2细胞AMPKα2活性(0.2±0.05比1.0±0.2,t=9.505,P<0.01)、AMPKα2 mRNA(0.3±0.1比1.0±0.3,t=5.422,P<0.01)及磷酸化-AMPK蛋白的表达水平(0.25±0.05比0.6±0.2,t=4.159,P<0.01)明显下降,细胞内三酰甘油含量[(78.5±20.5)μg/mg比(40.5±11.0)μg/mg,t=4.078,P<0.01]和TC含量[(52.5±13.0)μg/mg比(32.0±8.5)μg/mg,t=3.233,P<0.01]明显升高,脂肪酸β氧化速度[(1.80±0.40)nmol·mg-1·h-1比(3.10±0.60)nmol·mg-1·h-1,t=4.416,P<0.01]明显减慢,ROS水平(3.8±0.7比1.0±0.2,t=9.421,P<0.01)明显升高,MDA含量[(8.50±2.40)nmol/mL比(3.00±0.60)nmol/mL,t=5.446,P<0.01]明显升高;SOD活性[(9.60±2.50)U/mL比(15.50±3.00)U/mL,t=3.764,P<0.01]明显降低。脂肪酸、胆固醇合成的相关基因SREBP-1c mRNA水平(1.9±0.4比1.0±0.3,t=4.409,P<0.01)、FAS mRNA水平(3.0±0.6比1.0±0.3,t=7.303,P<0.01)、ACC mRNA水平(2.6±0.5比1.0±0.3,t=6.721,P<0.01)、SREBP-2 mRNA水平(2.3±0.5比1.0±0.2,t=5.913,P<0.01)、HMGR mRNA水平(1.9±0.4比1.0±0.2,t=4.929,P<0.01)和HMGS mRNA水平(2.6±0.7比1.0±0.2,t=5.383,P<0.01)明显升高,脂肪酸β氧化的相关基因PPARα mRNA水平(0.3±0.1比1.0±0.3,t=6.971,P<0.01)和CPT1A mRNA水平(0.4±0.1比1.0±0.2,t=6.573,P<0.01)明显下降。结论 HCV核心蛋白下调AMPK活性及表达,改变其下游调节脂类代谢相关基因表达,增加三酰甘油和TC合成,减少脂肪酸β氧化,增加脂质过氧化,导致肝细胞脂类代谢紊乱。 Objective To investigate the effect of HCV core protein on expression of AMP-activated protein kinase （AMPK） and lipid metabolism of hepatocytes. Methods HCV core protein expression plasmid was transfected into HepG2 cells. The activity and expression levels of mRNA and protein of AMPK were detected by scintillation counter, real time- PCR （RT-PCR） and Western blot respectively. Intracellular levels of triacylglycerol （TG）, total cholesterol （TC） were de- tected using analysis kit. The speed of fatty acid ~ oxidation was detected by scintillation counter. The levels of reactive oxy- gen species （ROS）, malondialdehyde （MDA） and superoxide dismutase （SOD） were assayed by flow cytometry, thibabitu- ric acid method and xanthine oxidase method, respectively. The mRNA levels of AMPK downstream lipid-metabolism genes [sterol regulatory element binding protein （SREBP）-lc, fatty acid synthase （FAS）, Acetyl-CoA carboxylase （ACC）, SREBP-2, HMG-CoA reductase （HMGR）, HMG-CoA synthase （HMGS）, peroxisome proliferator-activating receptor （PPAR）~, PPAR 7, and carnitine palmitoyl transferase-l（CPT）-l] were measured by RT-PCR. Results In HepG2 cells expressing HCV core protein, the activity （0.2-± 0.05 vs. 1.0 ± 0.2, t = 9. 505, P（0.01） and expression levels of mRNA （0.3 ± 0.1 vs. 1.0 ± 0.3, t = 5. 422, P〈0.01） and protein of phospho-AMPK （0.25 ± 0.05 vs. 0.6 ± 0.2, t = 4. 159, P〈 0.01） decreased, intracellular TG contents （78.5 ± 20.5 vs 40.5 ± 11.0 tLg /mg; t = 4. 078, P〈0.01） and TC contents （52.5±13.0 vs32.0±8.5 μg /mg; t =3.233, P〈0.01） increased, fatty acid 13 oxidation speed（1.80±0.40 vs. 3.10± 0.60 nmol/mg/h, t= 4. 416, P〈0.01）decreased, ROS levels （3.8 ± 0.7 vs. 1.0 ± 0.2, t = 9. 421, P〈0.01）increased, MDA contents （8.50 ± 2.40 vs. 3.00 ± 0.60 nmol/ml, t = 5. 446, P〈0.01） increased, SOD activity （9.60 ± 2.50 vs. 15.50 ± 3.00 U/ml, t = 3. 764, P〈0.01） decreased. Fatty acid synthesis and cholesterol relevant genes SREBP-lc mRNA levels （1.9±0.4 vs 1.0±0.3; t=4.409, P〈0.01）, theFASmRNAlevels （3.0±0.6 vs 1.0±0.3, t=7.303, P〈0.01）, the ACC mRNA levels （2.6 ± 0.5 vs 1.0 ± 0.3, t = 6. 721, P〈0.01）, SREBP-2 mRNA level （2.3 ± 0.5 vs 1.0 ± 0.2, t = 5. 913, P〈0.01）, the HMGR mRNA levels （1.9 ± 0.4 vs 1.0 ± 0.2, t = 4. 929, P〈0.01） and the HMGS mRNA levels （2.6± 0.7 vs 1.0 ± 0.2, t = 5. 383, P〈0.01） increased; whereas fatty acid oxidation relevant genes the PPAR a mRNA levels （0.3 ± 0.1 vs 1.0 ± 0.3, t = 6. 971, P〈0.01） and CPT1A mRNA levels （0.4 ± 0. 1 vs 1.0 ± 0.2, t = 6. 573, P〈 0.01） decreased. Conclusion HCV core protein may down - regulate the activity and expression of AMPK, then change the expression profile of lipid-metabolism related gene, thereby increase TG and TC synthesis, lipid peroxidation and decrease fatty acid 13 oxidation, leading to lipid metabolism disorders of hepatocytes.

作者孙丽杰李树臣赵勇华于建武康鹏刘伟

机构地区哈尔滨医科大学附属第二医院感染病科哈尔滨医科大学附属第二医院科研实验中心

出处《肝脏》 2013年第3期150-154,共5页 Chinese Hepatology

基金黑龙江省教育厅科学研究资助项目

关键词丙型肝炎病毒核心蛋白 AMP激活蛋白激酶 AMPK 脂肪酸合成脂肪酸β氧化脂质过氧化 Hepatitis C virus Core protein AMP-activated protein kinase, AMPK Fatty acid synthesis Fatty acid13 oxidation Lipid peroxidation

分类号 R512.63 [医药卫生—内科学]

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