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目的探讨心肌营养素1(CT-1)对心肌细胞缺氧复氧损伤的保护作用及信号通路所起的作用。方法用改良的方法培养出生1～3天的SD乳鼠心肌细胞,分为对照组、缺氧复氧组、阻断剂组、营养素组和助溶剂组。后4组进行缺氧3 h,加不同药物处理后,复氧3 h,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检测心肌细胞线粒体膜电位,二氯荧光黄双乙酸盐检测细胞内活性氧(ROS),用流式细胞仪检测心肌细胞凋亡率。采用RT-PCR法检测心肌细胞内皮型一氧化氮合酶(eNOS)mRNA表达,Western blot检测磷酸化磷脂酰肌醇3激酶(PI3K)蛋白水平。结果与对照组比较,缺氧复氧组心肌细胞凋亡率及ROS明显增加,而心肌细胞存活率明显降低,线粒体膜电位下降,eNOS mRNA表达明显下调(P<0.05);与缺氧复氧组比较,营养素组心肌细胞存活率明显升高,心肌细胞凋亡率及细胞内ROS明显减少,线粒体膜电位水平更高,磷酸化PI3K蛋白水平增加,eNOS mRNA表达上调。与营养素组比较,阻断剂组抑制上述指标表达。结论 CT-1能减轻缺氧复氧引起的心肌细胞损伤,其作用部分依赖PI3K/蛋白激酶B/eNOS信号通路的激活。
Objective To investigate the protective effect of myocardial nutrient 1 (CT-1) on cardiomyocyte hypoxia-reoxygenation injury and the role of signal transduction pathway. Methods Cardiomyocytes of neonatal SD rats born 1 to 3 days were cultured by modified method and divided into control group, hypoxia-reoxygenation group, blocker group, nutrient group and cosolvent group. The rats in the latter 4 groups were subjected to hypoxia for 3 hours, then treated with different drugs and reoxygenated for 3 hours. The survival rate of cardiomyocytes was determined by MTS method. The mitochondrial membrane potential of myocardial cells was detected by tetrachloro tetraethylbenzimidazolyl carbonylated iodide, Dichloro-fluorescein diacetate was used to detect intracellular reactive oxygen species (ROS), and cardiomyocyte apoptosis was detected by flow cytometry. The expression of endothelial nitric oxide synthase (eNOS) mRNA in cardiomyocytes was detected by RT-PCR and the protein level of phosphorylated phosphatidylinositol 3-kinase (PI3K) was detected by Western blot. Results Compared with the control group, the apoptosis rate and ROS of cardiomyocytes in hypoxia-reoxygenation group were significantly increased, while the survival rate of myocardial cells was significantly decreased. The mitochondrial membrane potential was decreased and the expression of eNOS mRNA was significantly down-regulated (P <0.05) Compared with the control group, the survival rate of myocardial cells in nutrient group was significantly increased, the apoptosis rate of myocardial cells and intracellular ROS were significantly decreased, mitochondrial membrane potential was higher, phosphorylated PI3K protein level was increased and eNOS mRNA expression was up-regulated. Compared with the nutrient group, the inhibitor group inhibited the expression of the above indicators. Conclusion CT-1 can attenuate cardiomyocyte injury induced by hypoxia-reoxygenation, and its effect partly depends on the activation of PI3K / protein kinase B / eNOS signaling pathway.