CAJ | 학술논문

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1～3 d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+LY294002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3 h,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(JC1)检测心肌细胞线粒体膜电位(△ψm),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.心肌细胞磷酸化糖原合成酶激酶-3β(GSK-3β)和PI3K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(△ψm)下降[(40.55±4.25)vs.(86.28±7.15),P<0.01];磷酸化的GSK-3β和PI3K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,△ψm水平增加,磷酸化GSK-3β及PI3K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PI3K/GSK-3β信号通路的激活.
목적 연구심기영양소-1(CT-1)대심기세포급성결양복양손상적작용급신호통로.방법 본연구재강서성분자의학중점실험실완성.용개량적방법배양출생1～3 d적Sprague-Dawley(SD)유서심기세포,근거실험요구분위5조:(1)대조조;(2)결양복양조;(3)결양복양조+CT-1조;(4)결양복양조+CT-1+LY294002조(PIK3/Akt조단제);(5)결양복양조+CT-1+조용제DMSO조.CT-1적질량농도위10ng/mL,결양3 h,복양3 h.MTS법측정심기세포적존활솔,사록사을기분병미서기탄화청전화물(JC1)검측심기세포선립체막전위(△ψm),이록형광황쌍을산염(DCFH CA)검측세포활성양(ROS),류식세포의검측심기세포조망솔.심기세포린산화당원합성매격매-3β(GSK-3β)화PI3K단백검측용western blotting.이방차분석급q검험진행통계학분석.결과 결양복양배양후심기세포조망솔급세포내ROS교대조조명현증가,이심기세포존활솔현저강저,선립체막전위(△ψm)하강[(40.55±4.25)vs.(86.28±7.15),P<0.01];린산화적GSK-3β화PI3K단백초유증가.이CT-1처리적심기세포,교결양복양조심기세포존활솔명현상승(87%),심기세포조망솔급세포내ROS현저감소,△ψm수평증가,린산화GSK-3β급PI3K단백수평명현증가.CT-1적작용능피PIK3/Akt조단제LY294002억제,이조용제조칙미관찰도유사작용.결론 CT-1능보호결양복양손상적심기세포,차기작용의뢰PI3K/GSK-3β신호통로적격활.
Objective To study the effect of Cardiotrophin-1 (CT-1) on cardiocyte hypoxia-reoxygenation injury,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/reoxygeuation, 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 rote was evaluated by MTS method, apopto-sis, mitochondrial permeability transition pore (△ψm) and reactive oxygen species(ROS) were detected by flow cy-tometer, 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 hy-poxia/reoxygenation,but cardiomyocyte survival rate and the level of △ψm [(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 △ψm 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 ceils survival in DMSO group, which confirmed that LY294002 specifically in-volved blocking the protective effect of CT-1. Conclusions CT-1 can protect cardiac cells against hypoxia- reoxy-genation injury, these effects are dependent upon its ability to activate the PI3K/GSK-3β pathway.