Role of ROS in attenuation of hypoxia-reoxygenation injury in rat cardiomyocytes by pinacidil postconditioning: relationship with Nrf2-ARE signaling pathway
Role of ROS in attenuation of hypoxia-reoxygenation injury in rat cardiomyocytes by pinacidil postconditioning: relationship with Nrf2-ARE signaling pathway
目的 评价ROS在吡那地尔后处理减轻大鼠心肌细胞缺氧复氧损伤中的作用及其与核因子E2相关因子2(Nrf2)-抗氧化反应元件(ARE)信号通路的关系。 方法 分离、培养成年大鼠心肌细胞，采用随机数字表法分为4组(n＝20)：对照组(C组)、缺氧复氧组(H/R组)、吡那地尔后处理组(P组)和活性氧清除剂N-(2-巯基丙酰基)-甘氨酸(MPG)＋吡那地尔后处理组(MPG＋P组)。C组持续于37 ℃ 95%O2＋5%CO2培养箱中持续培养105 min；缺氧复氧损伤模型制备：5%CO2＋1%O2＋94%N2条件下缺氧45 min，复氧60 min；P组缺氧45 min，吡那地尔50 μmol/L处理5 min，复氧60 min；MPG＋P组缺氧45 min，MPG 2 mmol/L处理10 min，吡那地尔处理5 min，复氧60 min。于复氧末检测心肌细胞Ca2＋含量和Nrf2活性；观察心肌细胞超微结构，并行线粒体Flameng评分；采用Western blot法和RT-PCR法分别检测Nrf2、超氧化物歧化酶1(SOD1)、醌氧化还原酶1(NQO1)和血红素加氧酶1(HO-1)及其mRNA的表达。 结果 与C组比较，H/R组心肌细胞Ca2＋含量、Nrf2活性和Flameng评分升高，Nrf2、SOD1、NQO1和HO-1及其mRNA表达下调(P<0.05)，心肌细胞超微结构损伤加重；与H/R组比较，P组心肌细胞Ca2＋含量和Flameng评分降低，Nrf2活性升高，Nrf2、SOD1、NQO1和HO-1及其mRNA表达上调(P<0.05)，心肌细胞超微结构损伤减轻；与P组比较，MPG＋P组心肌细胞Ca2＋含量和Flameng评分升高，Nrf2活性降低，Nrf2、SOD1、NQO1和HO-1及其mRNA表达下调(P<0.05)，心肌细胞超微结构损伤加重。 结论 ROS参与了吡那地尔后处理减轻大鼠心肌细胞缺氧复氧损伤的过程，与激活Nrf2-ARE信号通路有关。 Objective To evaluate the role of reactive oxygen species (ROS) in attenuation of hypoxia-reoxygenation (H/R) injury in rat cardiomyocytes by pinacidil postconditioning and the relationship with nuclear factor erythrid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway. Methods Adult rat cardiomyocytes were isolated and cultured and then divided into 4 groups (n=20 each) by a random number table method: control group (group C), H/R group, pinacidil postconditioning group (group P) and reactive oxygen scavenger N-(2-mercaptopropionyl)-glycine(MPG)+ pinacidil postconditioning group (group MPG+ P). Group C was continuously exposed to 95%O2+ 5%CO2 in an incubator at 37 ℃ for 105 min. The cells were exposed to 5%CO2+ 1%O2+ 94%N2 in an incubator at 37 ℃ for 45 min followed by reoxygenation for 60 min to prepare H/R injury model. The cells were exposed to hypoxia for 45 min and then treated with pinacidil 50 μmol/L for 5 min followed by reoxygenation for 60 min in group P. The cells were exposed to hypoxia for 45 min, treated with MPG 2 mmol/L for 10 min, and then treated with pinacidil for 5 min followed by reoxygenation for 60 min in group MPG+ P. The content of Ca 2+ and activity of Nrf2 in cardiomyocytes were measured at the end of reoxygenation. The ultrastructure of cardiomyocytes was observed, and mitochondrial ultrastructure was evaluated using mitochondrial Flameng score. The expression of Nrf2, superoxide dismutase (SOD1), quinone oxidoreductase 1 (NQO1), and heme oxygenase 1 (HO-1) protein and mRNA was detected using Western blot and real-time polymerase chain reaction. Results Compared with group C, the Ca2+ content, Nrf2 activity and mitochondrial Flameng score were significantly increased, the expression of Nrf2, SOD1, NQO1 and HO-1 protein and mRNA was down-regulated (P<0.05), and the damage to the ultrastructure of cardiomyocytes was aggravated in group H/R. Compared with H/R group, the Ca2+ content and mitochondrial Flameng score were significantly decreased, the Nrf2 activity was increased, the expression of Nrf2, SOD1, NQO1 and HO-1 protein and mRNA was up-regulated (P<0.05), and the damage to the ultrastructure of cardiomyocytes was attenuated in P group. Compared with P group, the Ca2+ content and mitochondrial Flameng score were significantly increased, the Nrf2 activity was decreased, the expression of Nrf2, SOD1, NQO1 and HO-1 protein and mRNA was down-regulated (P<0.05), and the damage to the ultrastructure of cardiomyocytes was aggravated in MPG+ P group. Conclusions ROS is involved in attenuation of H/R injury by pinacidil postconditioning, which is associated with activation of the Nrf2-ARE signaling pathway in rat cardiomyocytes.
Abstract
Objective To evaluate the role of reactive oxygen species (ROS) in attenuation of hypoxia-reoxygenation (H/R) injury in rat cardiomyocytes by pinacidil postconditioning and the relationship with nuclear factor erythrid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway. Methods Adult rat cardiomyocytes were isolated and cultured and then divided into 4 groups (n=20 each) by a random number table method: control group (group C), H/R group, pinacidil postconditioning group (group P) and reactive oxygen scavenger N-(2-mercaptopropionyl)-glycine(MPG)+ pinacidil postconditioning group (group MPG+ P). Group C was continuously exposed to 95%O2+ 5%CO2 in an incubator at 37 ℃ for 105 min. The cells were exposed to 5%CO2+ 1%O2+ 94%N2 in an incubator at 37 ℃ for 45 min followed by reoxygenation for 60 min to prepare H/R injury model. The cells were exposed to hypoxia for 45 min and then treated with pinacidil 50 μmol/L for 5 min followed by reoxygenation for 60 min in group P. The cells were exposed to hypoxia for 45 min, treated with MPG 2 mmol/L for 10 min, and then treated with pinacidil for 5 min followed by reoxygenation for 60 min in group MPG+ P. The content of Ca 2+ and activity of Nrf2 in cardiomyocytes were measured at the end of reoxygenation. The ultrastructure of cardiomyocytes was observed, and mitochondrial ultrastructure was evaluated using mitochondrial Flameng score. The expression of Nrf2, superoxide dismutase (SOD1), quinone oxidoreductase 1 (NQO1), and heme oxygenase 1 (HO-1) protein and mRNA was detected using Western blot and real-time polymerase chain reaction. Results Compared with group C, the Ca2+ content, Nrf2 activity and mitochondrial Flameng score were significantly increased, the expression of Nrf2, SOD1, NQO1 and HO-1 protein and mRNA was down-regulated (P<0.05), and the damage to the ultrastructure of cardiomyocytes was aggravated in group H/R. Compared with H/R group, the Ca2+ content and mitochondrial Flameng score were significantly decreased, the Nrf2 activity was increased, the expression of Nrf2, SOD1, NQO1 and HO-1 protein and mRNA was up-regulated (P<0.05), and the damage to the ultrastructure of cardiomyocytes was attenuated in P group. Compared with P group, the Ca2+ content and mitochondrial Flameng score were significantly increased, the Nrf2 activity was decreased, the expression of Nrf2, SOD1, NQO1 and HO-1 protein and mRNA was down-regulated (P<0.05), and the damage to the ultrastructure of cardiomyocytes was aggravated in MPG+ P group. Conclusions ROS is involved in attenuation of H/R injury by pinacidil postconditioning, which is associated with activation of the Nrf2-ARE signaling pathway in rat cardiomyocytes.
关键词
活性氧/吡那地尔/缺血后处理/肌细胞,心脏/低氧/NF-E2相关因子2/抗氧化反应元件
Key words
Reactive oxygen species/Pinacidil/Ischemic postconditioning/Myocytes, cardiac/Hypoxia/NF-E2-related factor 2/Antioxidant response elements
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