磷酸化叉头框O4型在H9c2心肌细胞缺氧/复氧损伤中的作用  

Effects of phosphorylated forkhead box subtype O4 in H9c2 cardiomyocytes with hypoxia/reoxygenation injury

作　　者：苏忠雪 刘华跃[1] 孟晓文[1] 王辉[1] 张俊[1] 嵇富海[1] Su Zhongxue;Liu Huayue;Meng Xiaowen;Wang Hui;Zhang Jun;Ji Fuhai(Department of Anesthesiology,the First Affiliated Hospital of Soochow University,Suzhou 215021,China)

机构地区：[1]苏州大学附属第一医院麻醉科,215021

出　　处：《国际麻醉学与复苏杂志》2021年第4期337-343,共7页International Journal of Anesthesiology and Resuscitation

基　　金：国家自然科学基金面上项目(81873925,81671880);江苏省自然科学基金(BK20191171);江苏省医学创新团队(CXTDA2017043);苏州市重点病种项目(LCZX201603)。

摘　　要：目的探讨磷酸化叉头框O4型(phosphorylated forkhead box subtype O4,p‑FoxO4)在H9c2心肌细胞缺氧/复氧(hypoxia/reoxygenation,H/R)损伤过程中的作用。方法H9c2细胞正常培养24 h后,均匀接种于6孔板中,密度为4.5×105个/ml,每组≥3次重复,按照随机数字表法分为4组:对照组(Control组)、缺氧1 h复氧1 h组(H1R1组)、缺氧1 h复氧3 h组(H1R3组)和缺氧1 h复氧6 h组(H1R6组)。采用细胞增殖‑毒性检测法检测4组细胞相对存活率;实时定量聚合酶链反应(real‑time quantitative polymerase chain reaction,qPCR)法检测叉头框O4型(forkhead box subtype O4,FoxO4)、Bcl‑2细胞死亡的相互作用介质(Bcl‑2 interacting mediator of cell death,Bim)、B淋巴细胞瘤‑2基因(B‑cell lymphoma‑2,Bcl‑2)和Bcl‑2相关X蛋白(Bcl2‑Associated X,Bax)的mRNA含量,以此确定最佳H/R时间点。Control组与H1R1组分别通过Hoechst染色检测细胞凋亡程度,Western blot法检测FoxO4、p‑FoxO4、Bim、Bcl‑2和Bax蛋白含量。将6~8周C57BL/6小鼠按随机数字表法分为5组(每组3只):假手术组(sham组)、缺血30 min再灌注3 h组(R3组)、缺血30 min再灌注6 h组(R6组)、缺血30 min再灌注12 h组(R12组)、缺血30 min再灌注24 h组(R24组)。采用qPCR法检测结扎小鼠左前降支后导致缺血的左心室前壁的FoxO4、Bim、Bcl‑2和Bax的mRNA含量。结果与Control组比较,H1R1组、H1R3组和H1R6组细胞相对生存率下降,H1R1组最低(P<0.05);与H1R1组比较,H1R3组和H1R6组细胞相对存活率升高(P<0.05);与H1R3组比较,H1R6组细胞相对存活率升高(P<0.05)。与Control比较,H1R1组、H1R3组、H1R6组FoxO4 mRNA表达增加(P<0.05),H1R1组Bim mRNA表达增加(P<0.05),H1R1组Bcl‑2 mRNA表达降低(P<0.05),H1R1组Bax mRNA表达增加(P<0.05),H1R3组、H1R6组Bax mRNA表达减少(P<0.05);与H1R1组比较,H1R3组、H1R6组Bim mRNA表达和Bax mRNA表达降低(P<0.05)。与Sham组比较,R12组、R24组FoxO4 mRNA含量增加(P<0.05),R6组Bim mRNA减少(P<0.05),R24组BiObjective To investigate the effects of phosphorylated forkhead box subtype O4(p‑FoxO4)in H9c2 cells with hypoxia/reoxygenation(H/R)injury.Methods H9c2 cells were cultured in 6‑well plates for 24 h at a density of 4.5×105 cells/ml.Each group was repeated more than or equal to three times.They were divided into four groups according to the random number table method:a control group,a hypoxia 1 h/reoxygenation 1 h(H1R1)group,a hypoxia 1 h/reoxygenation 3 h(H1R3)group and a hypoxia 1 h/reoxygenation 6 h(H1R6)group.The relative survival rate of the cells was detected by the cell proliferation‑toxicity detection method.The mRNA contents of forkhead box subtype O4(FoxO4),Bcl‑2 interacting mediator of cell death(Bim),B‑cell lymphoma‑2(Bcl‑2)and Bcl‑2‑Associated X(Bax)were detected by real‑time quantitative PCR(qPCR)to determine the optimal H/R time point.The apoptosis in the control group and the H1R1 group was detected by Hoechst staining.The levels of FoxO4,p‑FoxO4,Bim,Bcl‑2 and Bax were measured by Western blot.According to the random number table method,C57BL/6 mice aged 6 to 8 weeks were divided into five groups(n=3):a sham operation(sham)group,an ischemia 30 min and reperfusion 3 h(R3)group,an ischemia 30 min and reperfusion 6 h(R6)group,an ischemia 30 min and reperfusion 12 h(R12)group,and an ischemia 30 min and reperfusion 24 h(R24)group.The mRNA contents of FoxO4,Bim,Bcl‑2 and Bax in the anterior wall of the left ventricle in mice with ischemia after ligation of the left anterior descending branch were detected by qPCR.Results Compared with the control group,the relative survival rate of cells in the H1R1,H1R3 and H1R6 groups decreased,where the HIR1 group was the lowest(P<0.05).Compared with the H1R1 group,the relative survival rates of the cells in the H1R3 and H1R6 groups increased(P<0.05).Compared with the H1R3 group,the relative survival rate of the cells in the H1R6 group increased(P<0.05).Compared with the control group,the levels of FoxO4 mRNA increased in the H1R1,H1R3,and H1

关 键 词：缺氧/复氧损伤 磷酸化叉头框O4型 细胞凋亡 

分 类 号：R542.2[医药卫生—心血管疾病]