The role of reactive oxygen species and GADD153 protein in angiotension Ⅱ-induced eardiomyocyte apoptosis in vitro / 中华麻醉学杂志

ABSTRACT

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 Ⅴcardiomyocytes 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 Ⅵcardiomyocytes were exposed to NAC 5 mmol/L for 24 h (NAG); group Ⅶcardiomyocytes 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.