血红素加氧酶-1对乳鼠心肌细胞缺氧复氧损伤的影响

Effect of heme oxygenase-1 on hypoxia-reoxygenation injury in cultured neonatal rat cardiomyocytes

目的 探讨血红素加氧酶-1（HO-1）对乳鼠心肌细胞缺氧复氧损伤的影响.方法 新生SD大鼠8只,日龄1～3 d,原代培养心肌细胞,随机分为4组：正常对照组（C组）常规培养8 h;缺氧复氧组（HR组）采用缺氧2 h,复氧6 h的方法制备心肌细胞缺氧复氧模型;血晶素组（Hemin组）缺氧前24 h及缺氧即刻,培养基中加入Hemin,终浓度为20 μmol/L;血晶素＋锌原卟啉组（Hemin＋ZnPP组）缺氧前24 h及缺氧即刻培养基中同时加入Hemin及ZnPP,终浓度均为20 μmol/L.各组细胞均接种于35 mm培养皿（2 ml/皿）或50 ml培养瓶（3 ml/瓶）,每组45皿和3瓶.于复氧结束后采用蛋白印迹法测定心肌细胞HO-1表达,台盼蓝染色法测定心肌细胞存活率,应用全自动生化分析仪测定细胞培养液乳酸脱氧酶（LDH）活性,超声破碎细胞离心后取上清,采用硫代巴比妥酸法测定细胞MDA水平,黄嘌呤氧化酶法测定细胞SOD活性.结果 与C组比较,其余3组培养液LDH活性、心肌细胞MDA水平及HO-1表达升高,心肌细胞存活率及SOD活性降低（P＜0.05）.与HR组比较,Hemin组培养液LDH活性、心肌细胞MDA水平降低,HO-1表达、心肌细胞存活率及SOD活性升高（P＜0.05）,Hemin＋ZnPP组上述指标差异无统计学意义（P＞0.05）.HR组和Hemin＋ZnPP组细胞缺氧复氧损伤明显,Hemin组细胞缺氧复氧损伤减轻.结论 HO-1可减轻乳鼠心肌细胞缺氧复氧损伤.

Objective To investigate the effect of heme oxygenase-1 （HO-1） on hypoxia-reoxygenation （H/R） injury in cultured neonatal rat cardiomyocytes. Methods Primary cultured neonatal rat cardiomyocytes were randomly divided into4 groups： Ⅰ control group （group C）, Ⅱ H/R group, Ⅲ hemin group and Ⅳ hemin＋zinc protoporphyrin （ZnPP） group. In group H/R, the cardiomyocytes were exposed to 2 h of hypoxia followed by 6 h of reoxygenation. Hemin was added to the culture medium with a final concentration of 20 μmol/L 24 h before hypoxia and immediately before hypoxia in group hemin. Hemin and ZnPP were added to the culture medium with final concentrations of 20 μmol/L 24 h before hypoxia and immediately before hypoxia in group Henin ＋ ZnPP. The cells were incubated in 35 mm culture dishes （2 ml in each dish） and 50 ml culture flasks （3 ml in each flask）（45 dishes and 3 flasks in each group）. The HO-1 expression, cell survival rate, LDH activity, malondialdehyde （MDA） level and superoxide dismutase （SOD） activity were detected after the end of reoxygenation. Results Compared with group C, the LDH activity, MDA level and HO-1 expression were significantly increased, while the cell survival rate and SOD activity decreased in the other three groups （ P ＜ 0.05）. Compared with group H/R,the LDH activity and MDA level were significantly decreased, while the HO-1 expression, cell survival rate and SOD activity increased in group hemin （ P ＜ 0.05 ）, but no significant change was found in group hemin ＋ ZnPP （P ＞0.05）. H/R injury was obvious in group H/R and hemin ＋ ZnPP, but was significantly attenuated in group hemin. Conclusion HO-1 can protect cardiomyocytes against H/R injury in neonatal rats.