心肌营养素1对心肌细胞缺氧复氧损伤的作用及信号机制的研究

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(P0.05);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。

Study on effect of cardiotrophin-1 on cardiomyocyte hypoxia-reoxygenation injury and signaling mechanism

Objectives To study cardioprotection of cardiotrophin-1 (CT-1) against cardiomyocyte hypoxia-reoxygenation(H/R) injury and investigate the signaling pathways PI3K/Akt/eNOS involved in the protective effect of CT-1.Methods Cardiomyocytes from hearts of 1-3-day-old neonatal rats were divided into five groups:group Ⅰ:control,group Ⅱ :H/R,group Ⅲ:H/R + CT-1 + LY294002,group Ⅳ:H/R + CT-1,group Ⅴ :H/R + CT-1 + DMSO.Cadiomyocytes survival rate was evaluated by MTS method,apoptosis,mitochondrial permeability transition pore and ROS were detected by flow cytometer.The expression of eNOSmRNA was measured by RT-PCR, phosphorated PI3K protein level was measured by Western blot.Results Compared with control group,cardiomyocyte apoptosis and ROS increased markedly,but cardiomyocyte survival rate decreased significantly.Expression of eNOS mRNA and phosphorated PI3K protein level decreased significantly.Compared with group Ⅱ,in groupⅣ ,cardiomyocyte survival rate increased markedly, apoptosis and ROS reduced significantly,expression of eNOS mRNA upregulated and phosphorated PI3K protein level increased.Compared with group Ⅳ ,the above indexes were inhibited in group Ⅲ ,which confirmed that LY294002 specifically involved in blocking the protective effect of CT-1.Conclusion CT-1 can protect cardiac cells against H/R injury,these effects depend upon its ability to activate the PI3K/Akt/eNOS pathway.