腺苷酸激活蛋白激酶激活参与促进慢性缺氧时心肌细胞存活

AMPK activation participates in promoting myocardial cell survival in chronic hypoxia

目的检测腺苷酸激活蛋白激酶（adenosine monophosphate-activated protein kinase,AMPK）在慢性缺氧心肌组织中的激活水平,探讨AMPK激活对慢性缺氧心肌细胞存活的影响及其意义。方法选择本科2013年行手术矫治的先心病患儿24例,其中紫绀型先心病患儿12例,非紫绀型先心病患儿12例,取手术中切除的右室流出道心肌组织作为标本;选用4-6周龄清洁级雄性SD大鼠24只,分为常氧组和缺氧组（n=12）。将缺氧组置入低压舱（模拟海拔5 000 m,气压405 mm Hg）,常氧组正常饲养。同时喂养28 d后,处死大鼠后迅速取出心脏并分离出右心室部分作为标本。采用Western blot分别检测p-AMPK在人体、大鼠缺氧心肌组织中的激活水平。选用H9c2心肌细胞株,并将其分为常氧组、低氧组、阻断组、激活组。将后3组细胞置入缺氧培养箱3 d（94%N2,5%CO2,1%O2）,建立H9c2心肌细胞株慢性缺氧模型;常氧组置入细胞培养箱3 d,对照培养。分别向阻断组和激活组中加入AMPK特异性阻断剂Compound C和激活剂AICAR,采用Western blot检测p-AMPK的表达,流式细胞仪、TUNEL法检测心肌细胞凋亡,Hoechst33528染色观察细胞核形态、LDH测定心肌细胞死亡率。结果 Western blot结果显示与常氧组相比,缺氧心肌组织中p-AMPK/AMPK比值明显增高。与低氧组比较,AMPK阻断组凋亡细胞比例升高,TUNEL-阳性细胞比例、不正常核比例、细胞死亡率均明显升高（P〈0.05,P〈0.01）;AMPK激活组与低氧组比较,凋亡细胞比例、TUNEL-阳性细胞比例、不正常核比例、细胞死亡率均有降低（P〈0.05）。结论在慢性缺氧条件下,AMPK蛋白磷酸化水平增强,p-AMPK对心肌细胞慢性缺氧适应具有一定保护作用。

Objective To detect adenosine monophosphate-activated protein kinase（ AMPK）activation level in chronic hypoxic myocardial tissues,and explore the protective effect of phosphorylated AMPK（ p-AMPK） to the currival of chronic hypoxic myocardial cells. Methods Twenty-four children with congenital heart disease,including 12 cases of cyanotic type and type 12 cases of non-cyanotic,were selected from Xinqiao Hospital of Third Military Medical University in 2013,and the myocardial tissues of right ventricular outflow tract were taken out as samples. Twenty-four SD male rats were divided into normoxic control group and hypoxia group. The hypoxia group was placed in a low-pressure tank（ simulated altitude of5 000 m,405 mm Hg）,while the normoxic control group received normal feeding. After feeding for 28 d,the rats were executed and the hearts were immediately taken out to isolate the right ventricular tissues. Western blotting was used to detect the activation level of AMPK in the hypoxic myocardial tissues of the children and rats. H9c2 myocardial cells were divided into normoxic control group,hypoxia group,blocking group and activation group. Except the normoxic control group,other three groups were placed in a hypoxia incubator（ 94% N2,5% CO2,1% O2） for 3 d to establish H9c2 chronic hypoxia cell models,while the normoxic control group was cultured in a cell culture box for 3 d in comparison. AMPK-specific blocker Compound C and activator AICAR were added to the blocking group and the activation group,respectively. Western blotting was employed to detect the expression of p-AMPK,and flow cytometry and TUNEL assay were adopted to detect myocardial cell apoptosis. The cell nucleus shape was observed by Hoechst staining,and myocardial cell death was measured with LDH test kit. Results Western blot results showed that compared with the normoxian group,the p-AMPK / AMPK ratio in the hypoxic myocardial tissue was increased significantly.Compared with the hypoxia group,the apoptosis cell percentage,TUNEL-positive cell percentage（ P〈0. 05）,abnormal nuclear scale（ P〈0. 01）,and cell mortality（ P〈0. 01） of the blocking group were increased significantly. Compared with the hypoxia group,the apoptosis cell percentage,TUNEL-positive cell percentage（ P〈0. 05）,abnormal nuclear scale（ P〈0. 05）,and cell mortality（ P〈0. 05） of the AMPK activation group were decreased. Conclusion Under the condition of chronic hypoxia,AMPK protein phosphorylation level is enhanced,and p-AMPK provides a certain protective effect to myocardial cells in chronic hypoxia adaptation.