ROS/GADD153蛋白在血管紧张素Ⅱ诱导心肌细胞凋亡中的作用

目的 探讨活性氧簇(ROS)/GADD153蛋白在血管紧张素Ⅱ(AngⅡ)诱导心肌细胞凋亡中的作用.方法 体外培养乳鼠心肌细胞,随机分为9组(n=5),Ⅰ组(C组)常规心肌细胞培养,不予其他处理;Ⅱ组(AngⅡ6组)给予100 nmol/L AngⅡ,孵育6 h;Ⅲ组(AnsⅡ12组)给予100 nmol/L AngⅡ,孵育12 h;Ⅳ组(ArtsⅡ24组)给予100 nmol/L Ang Ⅱ,孵育24 h;V组[N-乙酰-L半胱氨酸(NAC)+AngⅡ组]预先给予抗氧化剂NAC 5 mmol/L,2 h后给予100 nmol/L Ang Ⅱ,孵育24 h;Ⅵ组(NAC组)仅给予NAC 5 mmol/L,孵育24 h;Ⅶ组(anti-ODN组)GADD153反义寡核苷酸10 μmol/L转染24 h后,加入100nmoFL AngⅡ,孵育24 h;Ⅷ组(mis-ODN组)GADDl53错义寡核苷酸10 μmol/L转染24 h后,加入100nmol/L Ang Ⅱ,孵育24 h;Ⅸ组(脂质体对照组)加入等容量转染试剂,孵育24 h.检测细胞活力、细胞内ROS产量、细胞凋亡率及GADD153蛋白表达水平.结果 与C组相比,AngⅡ6组、AngⅡ12组、AngⅡ24组细胞活力降低,细胞凋亡率、ROS产量及GADD153蛋白表达升高,且呈时间依赖性(P＜0.05);与AngⅡ24组比较,NAC+AngⅡ组和anti-ODN组细胞活力升高,细胞凋亡率、ROS产量及GADD153蛋白表达降低(P＜0.05).结论 AugⅡ通过增加ROS产量,诱导GADD153蛋白表达上调,从而导致心肌细胞凋亡的发生.

The role of reactive oxygen species and GADD153 protein in angiotension Ⅱ-induced eardiomyocyte apoptosis in vitro

Objective To examine the role of the reactive oxygen species (ROS) and GADD 153 protein in angiotensin Ⅱ (Ang Ⅱ)-induced cardiomyocyta apeptosis in vitro.Methods Cardiomyocytes were isolated from ventricles of healthy 1-2 day old Wistar mrs of both sexes weighing 5-10 g and cultured in high glucose culture medium in an incubator filled with 5% CO2 at 37℃.The cardiomyocytes were randamiy allocated to one of 9 groups (n=5 each): group Ⅰ control (C); group Ⅱ ,Ⅲ,Ⅳ: cardiomyocytes were exposed to Ang Ⅱ 100 nmol/L for 6 h,12 h or 24 h respectively ( Ang Ⅱ 6,Ang Ⅱ12,Ang Ⅱ24) ; group Ⅴ:cardiomyocytes were preincubated with N-acetyl-L-cysteine (NAC) 5 mmol/L for 2 h before being exposed to Aag Ⅱ 100 nmol/L for 24 h (NAC + Ang Ⅱ); group Ⅵ:cardiomyocytes were exposed to NAC 5 mmol/L for 24 h (NAG); group Ⅶ:cardiomyocytes were tranfected with GADD153 antisense oligo-denxynueleotida (onti-ODN) 10 μmol/L for 24 h before being exposed to Ang Ⅱ 100 nmol/L for 24 h; group Ⅷ: cardiomyocytes were transfected with missense oligo-deoxynucleotide (mis-ODN) 10 μmol/L for 24 h before being exposed to Ang Ⅱ 100 nmol/L for 24 h and group Ⅸ: cardiomyocytes were exposed to transfection agent of same concentration for 24 h.Viability of myocytes was measured by MTT assay; ROS production was determined by spectropbotometry; apoptosis in cardiomyocytes was detected by staining with Hoechst33342; the percentage of Annexin V positive and PI negative myocytes was measured by flow cytometry as apoptosis rote and GADD153 protein expression was determined by Western blotting.Remits The viability of myocytes was significantly lower,while the ROS production,apoptosis rate and GADD153 protein expression in myocytes were significantly higher in group Ⅱ,Ⅲ,and Ⅳ(Ang Ⅱ6,Ang Ⅱ12,AngⅡ24) than in control group (P＜0.05).The cell viability was significantly higher and the ROS production,apoptosis rate and GADD153 protein expression in myocytes were significantly lower in groupⅤ(NAC+AngⅡ)and Ⅶ(anti-ODN + Ang Ⅱ) than in group Ang Ⅱ24(Ⅳ).Conclusion Angiotensin Ⅱ induces cardiomyocyte apoptosis by increasing ROS production and up-regulating GADD153 protein expression.