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缺氧预处理激活AMPK对大鼠心肌细胞缺氧/复氧损伤的保护作用
作者:赵汝舟 余志斌 张琳 
单位:710032 西安 空军军医大学航空航天生理学教研室(赵汝舟、余志斌、张琳) 
关键词:缺氧预处理 缺氧/复氧 心肌细胞 活性氧 腺苷酸活化蛋白激酶 
分类号:R331.3
出版年,卷(期):页码:2019,44(2):120-126
摘要:

 [摘要]  目的  探讨缺氧预处理(HPC)激活腺苷酸活化蛋白激酶(AMPK)对缺氧/复氧(H/R)后心肌细胞的保护作用及其可能机制。方法  分离培养新生SD大鼠心肌细胞,建立心肌细胞H/R模型。将心肌细胞随机分为对照组、H/R组、HPC组、H/R+A-769662(AMPK激动剂)组及HPC+Compound C(AMPK抑制剂)组。采用CCK-8比色法检测各组细胞存活率;采用二氢乙啶(DHE)染色法及ATP检测试剂盒分别检测细胞内的活性氧(ROS)ATP含量;采用Western blotting检测AMPK磷酸化水平及caspase-3激活水平;采用Fluo-3AM染色法观察细胞内游离Ca2+浓度变化;采用TUNEL染色检测心肌细胞凋亡率的变化。结果  CCK-8测定结果显示,与H/R组比较,HPC组与H/R+A-769662 组心肌细胞存活率均明显升高(P<0.05);与HPC组比较,HPC+Compound C组心肌细胞存活率明显降低(P<0.01)DHE染色结果显示,与H/R组比较,HPC组及H/R+A-769662组心肌细胞ROS含量均明显减少(P<0.01);与HPC组比较,HPC+Compound C组心肌细胞ROS含量明显增加(P<0.01)。与H/R组比较,HPC组及H/R+A-769662组心肌细胞ATP含量明显增加(P<0.01);与HPC组比较,HPC+Compound C组心肌细胞ATP含量明显减少(P<0.01)Western blotting检测结果显示,与对照组比较,HPC组及H/R+A-769662组心肌细胞AMPK磷酸化水平明显提高(P<0.01), 而H/R组及HPC+Compound C组心肌细胞AMPK磷酸化水平变化不明显(P>0.05);与H/R组比较,HPC组及H/R+ A-769662组细胞AMPK磷酸化水平明显升高(P<0.05P<0.01);与对照组比较,H/R组及HPC+Compound C组心肌细胞caspase-3激活水平明显升高(P<0.01);与H/R组比较,HPC组及H/R+A-769662组心肌细胞caspase-3激活水平明显降低(P<0.01P<0.05)Fluo-3AM染色结果显示,与对照组比较,H/R组与HPC+Compound C组心肌细胞内游离Ca2+明显增加(P<0.01);与H/R组比较,HPC组及H/R+A-769662组心肌细胞内游离Ca2+明显减少(P<0.01);与HPC组比较,HPC+Compound C组细胞内游离Ca2+明显增加(P<0.01)TUNEL染色结果显示,与H/R组比较,HPC组及H/R+ A-769662组心肌细胞凋亡率明显降低(P<0.01P<0.05);与HPC组比较,HPC+Compound C组肌细胞凋亡率明显升高(P<0.05)结论  H/R可导致心肌细胞ROS生成增加,ATP产生减少,细胞内游离Ca2+增加,进而激活caspase-3,导致细胞凋亡的发生,而HPC通过激活AMPK,可以减少细胞ROS的产生,保证H/R后细胞的能量供应,防止心肌细胞凋亡,进而减轻心肌细胞H/R损伤。

[Abstract]  Objective  To investigate the protective effects of hypoxic preconditioning (HPC) on cardiomyocytes after hypoxia/reoxygenation (H/R) by activating AMP-activated protein kinase (AMPK). Methods  The primary cardiomyocytes were prepared from neonatal SD rats. The model of H/R of cardiomyocytes was established and randomly divided into control group, H/R group, HPC group, H/R+A-769662 (AMPK agonist) group and HPC+compound C (AMPK inhibitor) group. The survival rate of cardiomyocytes was detected by chromatometry with Cell Counting Kit-8 (CCK-8); the reactive oxygen species (ROS) and ATP contents in the cells were detected respectively by dihydroethidium (DHE) dyeing and ATP Detection Assay Kit measured by fluorescence microplate reader; the phosphorylation level of AMPK and activation level of caspase-3 were detected by Western blotting; the concentration of free Ca2+ in cardiomyocytes was detected by Fluo-3AM dyeing using laser scanning confocal microscopy; the apoptotic rate of cardiomyocytes was detected by TUNEL staining. Results  CCK-8 assay showed that compared with H/R group, the survival rate of cardiomyocytes in HPC group and H/R+A-769662 group increased obviously (P<0.05); compared with HPC group, the survival rate of cardiomyocytes in HPC+compound C group declined significantly (P<0.01). The results of DHE staining showed that compared with H/R group, the ROS contents in HPC group and H/R+A- 769662 group decreased markedly (P<0.01); compared with the HPC group, the ROS level in HPC+compound C group was significantly increased (P<0.01). ATP detection showed that the ATP contents in HPC group and H/R+A-769662 group were increased obviously compared with that in H/R group (P<0.01), while the ATP contents in HPC+compound C group was markedly decreased compared with that in HPC group (P<0.01). The results of Western blotting showed that compared with control group, the phosphorylation levels of AMPK in HPC group and H/R+A-769662 group elevated significantly (P<0.01), while the increase of AMPK phosphorylation levels were not obvious in H/R group and HPC+compound C group (P>0.05); compared with H/ R group, the AMPK phosphorylation levels elevated significantly in HPC group and H/R+A-769662 group respectively (P<0.05, P<0.01). Meanwhile, the activation levels of caspase-3 in H/R group and HPC+compound C group were increased obviously compared with that in control group (P<0.01), but in HPC group and H/R+A-769662 group were significantly lower than that in H/R group (P<0.01, P<0.05). The results of Fluo-3AM staining showed that compared with control group, the concentrations of intracellular free Ca2+ increased obviously in H/R group and HPC+compound C group (P<0.01); the concentrations of free Ca2+ in cardiomyocytes of HPC group and H/R+A-769662 group were less than that in H/R group (P<0.01), while the concentrations of free Ca2+ in HPC+compound C group much more increased than in HPC group (P<0.01). The results of TUNEL staining showed that compared with H/R group, the apoptotic rate of cardiomyocytes decreased obviously in HPC group and H/R+A- 769662 group (P<0.01, P<0.05); while compared with HPC group, the apoptotic rate of cardiomyocytes increased significantly in HPC+compound C group (P<0.05). Conclusions  H/R may lead to the increase of ROS production, the decrease of ATP contents, and increase the levels of free Ca2+ in cardiomyocytes, and thus activate caspase-3, lead to apoptosis eventually. While HPC may reduce the production of ROS by activating AMPK to ensure the energy supply of cardiomyocytes after H/R treatment, prevent apoptosis of cardiomyocytes, and then reduce the H/R injury to cardiomyocytes.

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