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目的 探讨氧化应激损伤心肌细胞的分子机制。方法 采用0.5mmol/L过氧化氢(hydrogenperoxide,H2O2)作用于原代培养的新生大鼠心肌细胞;末端标记检测细胞凋亡;Westernblot检测蛋白质含量;免疫组化检测NF-κB在细胞内的分布。结果 ①H2O2损伤3h,心肌细胞死亡率和乳酸脱氢酶(LDH)释放率均较对照组明显升高(P<0.01);②H2O2损伤24h,末端标记发现大量凋亡细胞;Westemblot示NF-κB内源性抑制蛋白Ⅰ-KB(inhibitorKB,I-KB)在H2O2损伤5min即减少,15min时减至最低,而后逐渐恢复;免疫组化显示H2O2损伤0.5h可引起心肌细胞中NF-κB从胞浆向胞核移位;与单纯损伤组比,NF-κB抑制剂吡咯烷二硫代氨基甲酸盐(pyrrolidinedithiocarbamate,PDTC)能明显降低心肌细胞LDH释放率(P<0.01)。结论 在H2O2所致心肌细胞损伤中,既有坏死,又有凋亡的发生,而NF-κB/Ⅰ-κB信号通路的激活可能介导了H2O2所致的心肌细胞损伤。
Objective To investigate the molecular mechanism of oxidative stress injury in myocardial cells. Methods Primary cultured neonatal rat cardiomyocytes were treated with 0.5mmol / L hydrogen peroxide (H2O2). Apoptosis was detected by end-labeling. Protein content was detected by Western blot. The distribution of NF-κB in the cells was detected by immunohistochemistry . Results ①H2O2 injury 3h, myocardial cell death rate and lactate dehydrogenase (LDH) release rate were significantly higher than the control group (P <0.01); ② H2O2 injury 24h, terminal markers found a large number of apoptotic cells; Westemblot showed NF- The inhibitor of IKB (inhibitor KB, I-KB) decreased in 5min after H2O2 injury and decreased to a minimum at 15min, then gradually recovered. Immunohistochemistry showed that H2O2 injury for 0.5h caused NF-κB in cytoplasm (P <0.01). Compared with simple injury group, pyrrolidine dithiocarbamate (NF-κB) inhibitor pyrrolidine dithiocarbamate (PDTC) significantly reduced the LDH release rate of cardiomyocytes (P <0.01). Conclusion H2O2-induced cardiomyocyte injury has both necrosis and apoptosis. The activation of NF-κB / Ⅰ-κB signaling pathway may be mediated by H2O2-induced cardiomyocyte injury.