摘要
目的探讨沉默信息调节因子2相关酶1（SIRT1）-AMP激活的蛋白激酶（AMPK）信号通路在HCV核心蛋白所致肝细胞能量代谢紊乱中的作用。方法pcDNA3．1-core重组质粒转染HepG2细胞，流式细胞仪测定表达HCV核心蛋白的HepG2细胞的活性氧（ROS），液体闪烁计数仪测定ATP／ADP比例和AMPKa2酶活性，比色法测定烟酰胺腺嘌呤二核苷酸氧化态与还原态之比（NAD＋／NADH），荧光定量检测SIRT1酶活性，RT-PCR和Western印迹检测SIRT1、AMPKa2的表达。计量资料比较采用t检验。结果Western印迹检测证实，HepG2细胞表达相对分子质量为22000的HCV核心蛋白。与HepG2细胞相比，表达HCV核心蛋白的HepG2细胞ROS水平升高（1．0±0．1比4．0±0．5，t=14．411，P〈0．01）；ATP／ADP比例无明显变化（8．2±2．2比9．3±2．8，t=0．757，P〉0．05）；AMPKa2酶活性（0．8±0．2比0．2±0，t=7．345，P〈0．01）明显降低；NAD＋／NADH比例明显降低（0．08±0．02比0．024-0，t=7．348，P〈0．01）；SIRT1酶活性[（0．30±0．05）pmol·μg-1·min。比（0．15±0．04）pmol·μg-1·min，t=5．738，P〈0．01）]明显降低；SIRT1 mRNA（0．8±0．2比0．4±0．1，t=4．382，P〈0．01）和AMPKd2mRNA（0．9±0．3比0．2±0，t=5．715，P〈0．01）表达明显降低；SIRT1蛋白（0．8±0．2比0．3±0，t=5．941，P〈0．01）和磷酸化AMPK蛋白（0．5±0．1比0．1±0，t=9．608，P〈0．01）水平明显降低。结论HCV核心蛋白能下调SIRT1-AMPK信号通路活性，导致肝细胞能量代谢紊乱。
Objective To study the role of silent mating type information regulation 2 homolog-1 （SIRT1）-adenosine monophosphate （AMP）-activated protein kinase （AMPK） signaling pathway in hepatitis C virus core protein （HCV-core） induced energy metabolism disorders of hepatocytes. Methods HepG2 ceils were transfected with recombined expressed plasmid pcDNA3. 1-core. The level of reactive oxygen species （ROS）, value of ATP/ADP and activity of AMPK α-2, and nicotinamide adenine dinucleotide （NAD）＋/NADH in HepG2 cells expressing HCV-core weredetected by flow cytometry, liquid scintillation counter and chromatometry, respectively. The activity of SIRT1 was detected with a {luorometric assay kit. Reverse transcription polymerase chain reaction （RT-PCR） and Western blot assay were performed to examine the expression of SIRT1 and AMPK α- 2. Quantitative data were analyzed by t-test. Results It was confirmed by Western blot assay that HepG2 cells expressed HCV-core with relative molecular weight of 22 000. Compared to HepG2 cells, the level of ROS in HepG2 cells expressing HCV-core was significantly increased （1.0±0.1 vs 4.0±0.5, t=14. 411, P〈0. 01）, the values of ATP/ADP were similar （8. 2±2. 2 vs 9. 3±2. 8, t= 0. 757, P〉0.05）, AMPK α-2 （0.8＋_0.2 vs 0.2-1-0, t=7. 345, P〈0.01）, the values of NAD＋ / NADH （0. 08_＋0. 02 vs 0. 02±0, t=7. 348, P〈0. 01）, the activity of SIRT1 [（0. 30±0.05） pmol.μg-1 . min -1 vs （0. 15μ0. 04） pmol .μg-1 . min-1, t=5. 738, P〈0. 01] and the mRNA levels of SIRT1 （0.82±0.2 vs 0.4±0.1, t=4. 382, P〈0.01） and AMPK α-2 mRNA （0.9± 0.3 vs 0.2±0, t=5. 715, P〈0.01）, and the expression of SIRT1 （0.8±0.2 vs 0.3±0, t=5. 941, P〈0.01） and phosphorylated AMPK protein （0.5±0. 1 vs 0. 1±0, t=9. 608, P〈0.01） were all significantly decreased. Conclusion HCV core protein induces energy metabolism disorders of hepatocytes by down-regulation of SIRT1-AMPK signaling pathway.
出处
《中华传染病杂志》
CAS
CSCD
北大核心
2013年第2期71-76,共6页
Chinese Journal of Infectious Diseases
基金
黑龙江省教育厅科学研究资助项目
