活性氧在缺氧后处理激活大鼠心肌细胞Nrf2/ARE信号通路中的作用

Role of reactive oxygen species in hypoxia postconditioning-induced activation of Nrf2/ARE signaling pathway in rat cardiomyocytes

目的评价活性氧(ROS)在缺氧后处理激活大鼠心肌细胞核因子E2相关因子2(Nrf2)/抗氧化反应元件(ARE)信号通路中的作用。方法分离、培养成年大鼠原代心肌细胞,采用随机数字表法分成4组(n=20):正常组(N)、缺氧复氧组(HR组)、缺氧后处理组(HPO组)及HPO+ROS清除剂组(HPO+MPG)。采用缺氧45 min复氧60 min的方法制备心肌细胞缺氧复氧损伤模型;HPO组缺氧45 min后,行3个循环的氧合5 min-缺氧5 min处理,再复氧60 min;HPO+MPG组缺氧35 min时加入ROS清除剂N-(2-硫基丙酰)-甘氨酸(终浓度2 mmol/L)继续缺氧处理10 min,余同HPO组。于复氧末,检测心肌细胞内Ca^(2+)水平和Nrf2活性,观察心肌细胞超微结构并进行线粒体Flameng评分;采用Western blot法和RT-PCR法分别检测心肌细胞Nrf2、醌氧化还原酶(NQO1)、SOD1、血红素加氧酶1(HO-1)及其mRNA表达。结果与N组比较,HR组心肌细胞内Ca^(2+)水平、Nrf2活性和Flameng评分升高,Nrf2、NQO1、SOD1、HO-1及其mRNA表达下调(P<0.05);与HR组比较,HPO组心肌细胞内Ca^(2+)水平和Flameng评分降低,Nrf2活性升高,Nrf2、NQO1、SOD1、HO-1及其mRNA表达上调(P<0.05);与HPO组比较,HPO+MPG组心肌细胞内Ca^(2+)水平和Flameng评分升高,Nrf2活性降低,Nrf2、NQO1、SOD1、HO-1及其mRNA表达下调(P<0.05)。结论缺氧后处理激活大鼠心肌细胞Nrf2/ARE信号通路的机制可能与ROS有关。

Objective To evaluate the role of reactive oxygen species(ROS)in hypoxia postconditioning-induced activation of nuclear factor erythroid 2-related factor 2(Nrf2)/antioxidant response element(ARE)signaling pathway in rat cardiomyocytes.Methods Primary cardiomyocytes of adult rats were isolated and cultured and divided into 4 groups(n=20 each)using a random number table method:normal group(group N),hypoxia-reoxygenation group(group HR),hypoxia postconditioning group(group HPO)and hypoxia postconditioning plus an ROS scavenger N-(2-Amidinopropionyl)-glycine(MPG)group(group HPO+MPG).Cells were exposed to hypoxia for 45 min followed by 60 min reoxygenation to develop the cardiomyocyte hypoxia-reoxygenation injury model.In HPO group,cells were subjected to 3 cycles of 5-min hypoxia/5-min reoxygenation after 45 min hypoxia,followed by reoxygenation for 60 min.In HPO+MPG group,MPG(final concentration 2 mmol/L)was added at 35 min of hypoxia,cells were subjected to hypoxia for 10 min,and the other treatments were similar to those previously described in group HPO.At the end of reoxygenation,the intracellular calcium level and Nrf2 activity were measured,the ultrastructure of cardiomyocytes was observed,and the Flameng score of mitochondria was assessed,and the expression of Nrf2,NAD(P)H quinine oxidoreductase 1(NQO1),superoxide dismutase 1(SOD1)and heme oxygenase-1(HO-1)protein and mRNA was detected using real-time polymerase chain reaction and Western blot.Results Compared with group N,the intracellular free Ca^(2+) level,Nrf2 activity and Flameng score were significantly increased,and the expression of Nrf2,NQO1,SOD1 and HO-1 protein and mRNA was down-regulated in group HR(P<0.05).Compared with group HR,the intracellular free Ca^(2+) level and Flameng score were significantly decreased,the Nrf2 activity was increased,and the expression of Nrf2,NQO1,SOD1 and HO-1 protein and mRNA was up-regulated in group HPO(P<0.05).Compared with group HPO,the intracellular free Ca^(2+) level and Flameng score were significantly increased,the Nrf2 activity was decreased,and the expression of Nrf2,NQO1,SOD1 and HO-1 protein and mRNA was down-regulated in group HPO+MPG(P<0.05).Conclusions The mechanism by which hypoxia postconditioning activates the Nrf2/ARE signaling pathway in rat cardiomyocytes may be related to ROS.