CAJ | 학술논문

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AMPK α2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、AMPK α2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22 000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)；AMPK α2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0.02±0,t=7.348,P＜0.01)；SIRT1酶活性[(0.30±0.05) pmol·μg 1·min-1比(0.15±0.04) pmol·μtg-1·min-1,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AMPK α2 mRNA(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.
목적 탐토침묵신식조절인자2상관매1 (SIRT1)-AMP격활적단백격매(AMPK)신호통로재HCV핵심단백소치간세포능량대사문란중적작용.방법 pcDNA3.1-core중조질립전염HepG2세포,류식세포의측정표체HCV핵심단백적HepG2세포적활성양(ROS),액체섬삭계수의측정ATP/ADP비례화AMPK α2매활성,비색법측정연선알선표령이핵감산양화태여환원태지비(NAD+/NADH),형광정량검측SIRT1매활성,RT-PCR화Western인적검측SIRT1、AMPK α2적표체.계량자료비교채용t검험.결과 Western인적검측증실,HepG2세포표체상대분자질량위22 000적HCV핵심단백.여HepG2세포상비,표체HCV핵심단백적HepG2세포ROS수평승고(1.0±0.1비4.0±0.5,t=14.411,P＜0.01)；ATP/ADP비례무명현변화(8.2±2.2비9.3±2.8,t=0.757,P＞0.05)；AMPK α2매활성(0.8±0.2비0.2±0,t=7.345,P＜0.01)명현강저；NAD+/NADH비례명현강저(0.08±0.02비0.02±0,t=7.348,P＜0.01)；SIRT1매활성[(0.30±0.05) pmol·μg 1·min-1비(0.15±0.04) pmol·μtg-1·min-1,t=5.738,P＜0.01)]명현강저；SIRT1 mRNA(0.8±0.2비0.4±0.1,t=4.382,P＜0.01)화AMPK α2 mRNA(0.9±0.3비0.2±0,t=5.715,P＜0.01)표체명현강저；SIRT1단백(0.8±0.2비0.3±0,t=5.941,P＜0.01)화린산화AMPK단백(0.5±0.1비0.1±0,t=9.608,P＜0.01)수평명현강저.결론 HCV핵심단백능하조SIRT1-AMPK신호통로활성,도치간세포능량대사문란.
Objective To study the role of silent mating type information regulation 2 homolog1 (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 cells 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 were detected by flow cytometry,liquid scintillation counter and chromatometry,respectively.The activity of SIRT1 was detected with a fluorometric 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±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 (0.15±0.04) pmol · μg 1 · min 1,t=5.738,P＜0.01] and the mRNA levels of SIRT1 (0.8±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.