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目的:探讨心肌细胞急性缺氧复氧损伤时NADPH氧化酶亚单位nox-1的变化及心肌营养素-1的作用。方法:用改良的方法培养出生1-3d的乳鼠心肌细胞,分为6组:(1)对照组;(2)缺氧复氧组;(3)缺氧复氧+CT-1组;(4)缺氧复氧+CT-1+LY294002组(PIK3/Akt阻断剂);(5)缺氧复氧+CT-1+PD98059组(ERK阻断剂);(6)缺氧复氧+CT-1+助溶剂DMSO组。CT-1的浓度为10μg/L。MTS法测定心肌细胞的存活率,四氯四乙基苯丙咪唑基羰化青碘化物(JC1)检测心肌细胞线粒体膜电位(Δψm),二氯荧光黄双乙酸盐(DCFH-CA)检测细胞活性氧(ROS),流式细胞仪检测心肌细胞凋亡率。Nox-1蛋白采用Westernblotting检测。结果:缺氧复氧培养后心肌细胞凋亡率及细胞内ROS较对照组明显增加,分别是(19.7%±1.4%vs2.1%±0.5%,14.07%±1.25%vs3.54%±0.86%,P<0.05),而心肌细胞存活率显著降低,线粒体膜电位(Δψm)下降;nox-1表达明显升高。CT-1处理的心肌细胞,较缺氧复氧组心肌细胞存活率明显上升(87.0%±7.3%),而心肌细胞凋亡率及细胞内ROS显著减少,Δψm水平增加,nox-1蛋白表达下调。而CT-1的这些作用能被PIK3/Akt和ERK阻断剂抑制。结论:心肌细胞急性缺氧复氧损伤时NADPH氧化酶亚单位nox-1表达上调,而心肌营养素-1能通过下调nox-1表达,发挥对心肌细胞保护作用。
Objective: To investigate the changes of nox-1 in NADPH oxidase subunits and the role of cardiotrophin-1 during acute hypoxia-reoxygenation injury in cardiomyocytes. Methods: The cultured neonatal rat cardiomyocytes cultured for 1-3 days were divided into 6 groups: (1) control group; (2) hypoxia-reoxygenation group; (3) hypoxia-reoxygenation + CT-1 group; (4) hypoxia and reoxygenation + CT-1 + LY294002 group (PIK3 / Akt blocker); (5) hypoxia and reoxygenation + CT-1 + PD98059 group (ERK blocker); Oxygen + CT-1 + cosolvent DMSO group. The concentration of CT-1 was 10 μg / L. The survival rate of cardiomyocytes was measured by MTS method. The mitochondrial membrane potential (Δψm) was measured by tetrachloro tetraethylbenzimidazolyl carbonitride iodide (JC1), and the cell viability was measured by dichlorofluorescein diacetate (DCFH-CA) Oxygen (ROS), flow cytometry myocardial cell apoptosis rate. Nox-1 protein was detected by Western blotting. Results: After hypoxia and reoxygenation culture, the apoptotic rate of cardiomyocytes and intracellular ROS were significantly increased compared with the control group (19.7% ± 1.4% vs 2.1% ± 0.5%, 14.07% ± 1.25% vs 3.54% ± 0.86 %, P <0.05). However, the survival rate of cardiomyocytes was significantly decreased and the mitochondrial membrane potential (Δψm) was decreased. The expression of nox-1 was significantly increased. Compared with hypoxia-reoxygenation group, the survival rate of cardiomyocytes in CT-1 treated group was significantly higher than that in hypoxia-reoxygenation group (87.0% ± 7.3%), while the apoptosis rate and intracellular ROS were significantly decreased, the level of Δψm was increased and the expression of nox-1 protein Down. These effects of CT-1 are inhibited by PIK3 / Akt and ERK blockers. CONCLUSION: The expression of nox-1, an NADPH oxidase subunit, is up-regulated in acute hypoxia-reoxygenation injury of cardiomyocytes. Cardiotrophin-1 can protect cardiomyocytes by down-regulating the expression of nox-1.