一氧化氮供体对过氧化氢引起的心肌细胞损伤的保护作用

Protective effects of nitric oxide donor on hydrogen peroxide-induced injury of cardiomyocytes

关于一氧化氮(NO)对心肌细胞是否具有保护作用目前尚存在争议,为探讨NO对过氧化氧(H2O2)引起的心肌细胞损伤是否具有保护作用及其可能的机制,实验将体外培养的新生大鼠心肌细胞分为3组:(1)阴性对照组(Normal组);(2)H2O2组:H2O2(0.1 mmol/L)与心肌细胞共育4 h;(3)S-亚硝基-N-乙酰青霉胺(SNAP)+H2O2组:NO供体SNAP(0.5 mmol/L)处理心肌细胞10 min后,加入H2O2与心肌细胞共育4 h。用流式细胞术检测心肌细胞凋亡率,心肌细胞损伤程度以心肌细胞存活率和乳酸脱氢酶(lactate dehydrogenase,LDH)活性来表示,同时检测心肌细胞超氧化物歧化酶(superoxide dismutase,SOD)活性和丙二醛(MDA)含量。通过激光共聚焦显微术检测在不同处理条件下心肌细胞胞内钙的变化。结果表明,正常心肌细胞LDH活性和细胞存活率分别为631.4±75.6 U/L和93.1±6.2％,细胞凋亡率为0;H2O2处理细胞后可使细胞LDH活性显著增高(1580.5±186.7 U/L,P<0.01),细胞存活率明显下降(58.3±7.6％,P<0.01),流式细胞仪检测到大量心肌细胞凋亡,凋亡率为26.4±5.7％;SOD活性较正常细胞19.67±0.85 NU/ml显著下降,为14.73±1.68 NU/m(P<0.01),MDA含量较正常细胞6.95±0.83μmol/L显著增高,为15.35±3.49μmol/L(P<0.01)。SNAP预处理细胞可显著提高心肌细胞存?

To investigate the protective effects of nitric oxide (NO) on cardiomyocytes against hydrogen peroxide (H2O2)-induced injury, cultured neonatal cardiomyocytes were divided into three groups: (1) normal group; (2) H2O2 group: cells were treated with H2O2(0.1 mmol/L) for 4 h; (3) SNAP+ H2O2 group: cells were pretreated with NO donor S-nitroso-N-acetyl-1,1-penicillamine (SNAP, 0.5 mmol/L) 10 min before H2O2 treatment. Colorimetric assay was used to detect cell viability and lactate dehydrogenase (LDH) activity to evaluate cell injury. Apoptotic rate of cardiomyocytes were determined by flow cytometer. Superoxide dismutase (SOD) activity and malonaldehyde (MDA) content were measured by colorimetric assay to evaluate cell antioxidant ability. Intracellular calcium was tested by laser confocal microscopy. The results showed that after treatment with H2O2, cell viability was significantly reduced to 58.3±7.6% compared with normal group (93.1±6.2 %). LDH activity and apoptotic rates were 1580.5±186.7 U/L and 26.4±5.7% respectively, significantly higher than that of normal group (631.4±75.6 U/ L and 0). SNAP pretreatment markedly improved cell viability to 79.7±9.3% and reduced LDH activity and apoptotic rates to 957.8 ±110.9 U/L and 9.1±3.3%, respectively. Cells treated with H2O2 had a lower SOD activity of 14.73±1.68 NU/ml and a higher MDA content of (15.35±3.49) μmol/L compared with normal cells (19.67±0.85 NU/ml) and (6.95±0.83 μmol/L), respectively. Cells with SNAP pretreatment had a higher SOD activity of 21.36±3.11 NU/ml and a lower MDA content of 9.12±1.47μmol/L compared with H2O2 group. Intracellular calcium content was reduced by SNAP administration while enhanced by H2O2. Pretreatment with SNAP could antagonize the effect of H2O2 of accelerating intracellular calcium content. Based on the results observed, it is concluded that NO donor SNAP may protect cardiomyocytes from being injured by H2O2. The underlying mechanisms may include improving cell antioxidant ability and reducing intracellular calcium overload.