两种H9C2心肌细胞氧化损伤模型的比较

Comparison of two models of oxidative damage in H9C2 cardiomyocytes

摘要

目的 探讨低氧、低糖及血清剥夺（glucose and serum deprivation under hypoxia（1% O₂），GSDH）处理与H₂O₂处理在构建H9C2心肌细胞氧化损伤模型中的应用价值。 方法 培养 H9C2 心肌细胞，当细胞生长状态良好时，用低氧（1% O₂）、低糖（1.0 g/L）及血清剥夺联合处理或200 μmol/L的H₂O₂作用于心肌细胞24 h。采用CCK8实验检测细胞增殖能力；通过细胞凋亡试剂（annexinV-FITC/PI）、Hoechst染色检测细胞凋亡；细胞活性氧（reactive oxygen species，ROS）检测细胞氧化应激水平；BODIPY检测细胞脂质过氧化水平；过碘酸雪夫（periodic acid-schiff stain，PAS）染色检测细胞糖原合成能力；线粒体膜电位检测试剂（mitochondrial membrane potential assay kit with JC-1，JC-1）染色检测线粒体膜电位水平；Western blot检测能量代谢相关分子AMP依赖的蛋白激酶（Adenosine 5’-monophosphate（AMP）-activated protein kinase，AMPK）及氧化应激相关分子NAD（P）H醌脱氢酶1（NAD（P）H quinone dehydrogenase 1，NQO-1）、血红素氧合酶1（heme oxygenase-1，HO-1）蛋白表达水平。 结果 与空白组比较，GSDH处理组与H₂O₂处理组的心肌细胞存活率均降低，差异均有统计学意义（P<0.05）。与空白组相比，GSDH处理与H₂O₂处理都增加了心肌细胞的凋亡水平、ROS和脂滴堆积水平增高、糖原消耗量增加且线粒体膜电位降低。但相比于H₂O₂处理组，GSDH处理组的细胞糖原消耗增多更明显，脂滴堆积也更明显，并且AMPK磷酸化水平显著降低。 结论 低氧、低糖及GSDH处理与H₂O₂处理均能造成H9C2心肌细胞氧化损伤，但相比于H₂O₂处理组，GSDH处理组的效果更符合体内心肌损伤时的能量代谢转变，有望作为一种更简单便捷的心肌氧化损伤模型应用于科研。

Abstract

Objective To investigate the value of glucose and serum deprivation under hypoxia（1% O₂）（GSDH） treatment and H₂O₂ treatment in establishing a model of oxidative injury in H9C2 cardiomyocytes. Methods H9C2 cardiomyocytes were cultured，and when the cardiomyocytes were in good growth conditions，they were treated with the combination of low oxygen（1% O₂），low glucose（1.0 g/L），and serum deprivation or H₂O₂ 200 μmol/L alone for 24 hours. CCK8 assay was sued to measure the proliferation ability of cells；the apoptosis reagent（AnnexinV-FITC/PI） and Hoechst staining were used to measure cell apoptosis；reactive oxygen species（ROS） was used to measure the level of oxidative stress；BODIPY testing was used to measure the level of lipid peroxidation in cells；periodic acid-Schiff staining was used to measure the ability for glycogen synthesis；mitochondrial membrane potential assay kit with JC-1 staining was used to measure mitochondrial membrane potential；Western blot was used to measure the protein expression levels of the energy metabolism-related molecule AMP-activated protein kinase（AMPK） and the oxidative stress-related molecules NAD（P）H quinone dehydrogenase 1（NQO-1） and heme oxygenase-1（HO-1）. Results Compared with the blank group，both the GSDH treatment group and the H₂O₂ treatment group had a significant reduction in the viability of cardiomyocytes（P<0.05）. Compared with the blank group，both GSDH treatment and H₂O₂ treatment increased the levels of cardiomyocyte apoptosis，ROS and lipid droplet accumulation，and glycogen consumption，with a reduction in mitochondrial membrane potential in cardiomyocytes. However，compared with the H₂O₂ treatment group，the GSDH treatment group showed significantly greater increases in glycogen consumption and lipid droplet accumulation and a significant reduction in AMPK phosphorylation. Conclusion Both GSDH treatment and H₂O₂ treatment can cause oxidative injury to H9C2 cardiomyocytes，but compared with H₂O₂ treatment，the effect of GSDH treatment is more in line with the energy metabolism transition during myocardial injury in vivo，and therefore，it is expected to be used as a simpler and more convenient model of oxidative injury to cardiac myocardium for scientific research.

关键词

低氧低糖血清剥夺 / 过氧化氢 / 氧化应激 / 能量代谢 / 免疫印迹法

Key words

glucose and serum deprivation under hypoxia / hydrogen peroxide / oxidative stress / energy metabolism / Western blot

基金资助

国家自然科学基金面上资助项目(82270271)