NADPH氧化酶亚单位nox-1在心肌细胞急性缺氧复氧损伤时的变化及心肌营养素-1的作用

Change of subunit of NADPH oxidation enzyme complex nox-1 protein in cardiocyte hypoxia-reoxygenation injury and the role of cardiotrophin-1

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。

AIM： To observe the change of subunit of NADPH oxidation enzyme complex nox - 1 protein in cardiocyte hypoxia - reoxygenation injury and the role of cardiotrophin - 1. METHODS ： Cardiomyocytes from the hearts of 1 -3 d old neonatal rats were prepared by a modified method. Five groups were included in the study： control; hypoxia/ reoxygenation; hypoxia/reoxygenation ＋ CT - 1 ; CT - 1 ＋ hypoxia/reoxygenation ＋ LY294002 （ PIK3/Akt inhibitor） ; CT - 1 ＋ hypoxia/reoxygenation ＋ PD98059 （ ERK inhibitor） ; CT - 1 ＋ hypoxia/reoxygenation ＋ DMSO. The concentration of CT -1 was 10 μg/L. The survival rate of myocytes was evaluated by MTS method. Apoptosis, mitochondrial permeability transition pore （A0m） and reactive oxygen species （ROS） were detected by flow cytometry. Nox - 1 protein was determined by Western blotting. RESULTS： Apoptosis of cardiomyocytes and the level of ROS （ 19. 7% - 1. 4% vs 2. 1% ＋ 0. 5% , 14. 07% ＋ 1.25% vs 3.54% -＋0. 86%, P 〈0. 05） increased markedly after hypoxia/reoxygenation, but cardiomyocyte survival rate and the level of △ψm （40. 55% ＋4. 25% vs 86. 28% ＋7. 15% , P 〈0. 01 ） decreased significantly. The expression of nox - 1 protein was upregnlated markedly. With CT - 1 intervention, cardiomyocyte survival rate increased markedly, apoptosis, both ROS and expression of nox -1 protein reduced significantly. The level of △ψm increased obviously. The effect of CT - 1 was inhibited by LY294002. No significant effect was observed on cells survival in DMSO group, which confirmed that LY294002 was specifically involved in blocking the protective effect of CT - 1. CONCLUSION： The expression of subunit of NADPH oxidation enzyme complex nox - 1 protein is upregulated markedly in cardio cyte hypoxia - reoxygenation injury. CT - 1 protects cardiac cells against hypoxia - reoxygenation injury by downregulating the expression of nox - 1 protein to decrease the level of ROS.