糖原合成酶激酶-3β介导心肌营养素-1对心肌细胞急性缺氧复氧损伤的作用

PI3K/GSK-3β signaling pathway mediates cardiotrophin-1 cardioprotection against cardiocyte hypoxia-reoxygenation injury

目的研究心肌营养素-1（CT-1）对心肌细胞急性缺氧复氧损伤的作用及信号通路。方法本研究在江西省分子医学重点实验室完成。用改良的方法培养出生1～3d的Sprague-Dawley（SD）乳鼠心肌细胞，根据实验要求分为5组：（1）对照组；（2）缺氧复氧组；（3）缺氧复氧组＋CT-1组；（4）缺氧复氧组＋CT-1＋LY29d002组（PIK3／Akt阻断剂）；（5）缺氧复氧组＋CT-1＋助溶剂DMSO组。CT-1的质量浓度为10ng／mL，缺氧3h，复氧3h。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（DCFHCA）检测细胞活性氧（ROS），流式细胞仪检测心肌细胞凋亡率。心肌细胞磷酸化糖原合成酶激酶．3B（GSK-33）和P13K蛋白检测用western blotting。以方差分析及q检验进行统计学分析。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，而心肌细胞存活率显著降低，线粒体膜电位（Δψm）下降[（40．55±4．25）VS．（86．28±7．15），P〈0．01]；磷酸化的GSK-3β和P13K蛋白稍有增加。而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％），心肌细胞凋亡率及细胞内ROS显著减少，Δψm水平增加，磷酸化GSK-3口及P13K蛋白水平明显增加。CT-1的作用能被PIK3／Akt阻断剂LY294002抑制，而助溶剂组则未观察到类似作用。结论CT-1能保护缺氧复氧损伤的心肌细胞，且其作用依赖P13K／GSK-3β信号通路的激活。

Objective To study the effect of Cardiotrophin-1 （CT-1） on cardiocyte hypoxia-reoxygenation injmy, and to investigate the signaling pathways involved in the protective effect. Method This study was carried out in Key Lab of Molecular Medicine in Jiangxi Province. Cardiomyocytes from the hearts of 2-day-old Sprague- Dawley neonatal rats were prepared by a modified method. Five groups were included in the study. Group （i）： control, Group （ii）： hypoxia/reoxygenation, Group （iii）： hypoxia/reoxygenation ＋ CT-1, Group （iv）： CT-1 ＋ hypoxia / reoxygenation ＋ LY294002 （PIK3/Akt inhibitor）, Group （v） ： CT-1 ＋ hypoxia / reoxygenation ＋ DMSO. The concentration of CT-1 was 10 ng/mL. Myocytes survival rate was evaluated by MTS method, apoptosis, mitochondrial permeability transition pore （Δψm） and reactive oxygen species（ROS） were detected by flow cytometer, phosphorylased GSK-3β and PI3K protein by western blotting. Analysis of variance and q test as statistical methods was used to analyze the data. Results Cardiomyocyte apoptosis and ROS increased markedly after hypoxia/reoxygenation, but cardiomyocyte survival rate and the level of Δψm [ （40.55 ± 4.25 ） vs. （ 86.28 ± 7.15）, P 〈 0.01 ] decreased significantly. With CT- 1 intervention, cardiomyocyte survival rate increased markedly （87%） ,apoptosis and ROS reduced significantly. The level of A@n increased, the level of phosphorylased GSK- 3β and phosphorylased PI3K protein obviously increased. The effect of CT-1 was inhibited by LY294002, but no significant effect was observed on cells survival in DMSO group, which confirmed that LY294002 specifically involved blocking the protective effect of CT-1. Conclusions CT-1 can protect cardiac cells against hypoxia- reoxygenation injury, these effects are dependent upon its ability to activate the PI3K/GSK-3β pathway.