Wang Hai-Chang, Zhang Hai-Feng, Guo Wen-Yi, Su Hui, Zhang Kun-Ru, Li Qiu-Xia, Yan Wenli, Ma Xin L, Lopez Bernard L, Christopher Theodore A, Gao Feng
Department of Cardiology and Department of Physiology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
Apoptosis. 2006 Aug;11(8):1453-60. doi: 10.1007/s10495-006-7786-z.
Our previous study has shown that slow or "controlled" reperfusion for the ischemic heart reduces cardiomyocyte injury and myocardial infarction, while the mechanisms involved are largely unclear. In this study, we tested the hypothesis that enhancement of survival and prevention of apoptosis in hypoxic/reoxygenated cardiomyocytes by hypoxic postconditioning (HPC) are associated with the reduction in peroxynitrite (ONOO(-)) formation induced by hypoxia/reoxygenation (H/R).
Isolated adult rat cardiomyocytes were exposed to 2 h of hypoxia followed by 3 h of reoxygenation. After 2 h of hypoxia the cardiomyocytes were either abruptly reperfused with pre-oxygenized culture medium or postconditioned by two cycles of 5 min of brief reoxygenation and 5 min of re-hypoxia followed by 160 min of abrupt reoxygenation.
H/R resulted in severe injury in cardiomyocytes as evidenced by decreased cell viability, increased LDH leakage in the culture medium, increased apoptotic index (P values all less than 0.01 vs. normoxia control group) and DNA ladder formation, which could be significantly attenuated by HPC treatment applied before the abrupt reoxygenation (P < 0.05 vs. H/R group). In addition, H/R induced a significant increase in ONOO(-) formation as determined by nitrotyrosine content in cardiomyocytes (P < 0.01 vs. normoxia control). Treatment with the potent ONOO(-) scavenger uric acid (UA) at reoxygenation significantly decreased ONOO(-) production and protected myocytes against H/R injury, whereas the same treatment with UA could not further enhance myocyte survival in HPC group (P > 0.05 vs. HPC alone). Statistical analysis showed that cell viability closely correlated inversely with myocyte ONOO(-) formation (P < 0.01).
These data demonstrate that hypoxic postconditioning protects myocytes against apoptosis following reoxygenation and enhances myocytes survival, which is partly attributable to the reduced ONOO(-) formation following reoxygenation.
我们之前的研究表明，对缺血心脏进行缓慢或“控制性”再灌注可减少心肌细胞损伤和心肌梗死，但其涉及的机制尚不清楚。在本研究中，我们验证了以下假设：缺氧后处理（HPC）增强缺氧/复氧心肌细胞的存活并预防其凋亡，这与缺氧/复氧（H/R）诱导的过氧亚硝酸盐（ONOO⁻）生成减少有关。
将成年大鼠分离的心肌细胞暴露于缺氧环境2小时，随后进行3小时的复氧。在缺氧2小时后，心肌细胞要么用预氧合的培养基突然进行再灌注，要么通过两个周期的5分钟短暂复氧和5分钟再缺氧处理，随后进行160分钟的突然复氧来进行后处理。
H/R导致心肌细胞严重损伤，表现为细胞活力降低、培养基中乳酸脱氢酶泄漏增加、凋亡指数升高（与常氧对照组相比，P值均小于0.01）以及DNA梯带形成，而在突然复氧前进行HPC处理可显著减轻这些损伤（与H/R组相比，P<0.05）。此外，通过心肌细胞中硝基酪氨酸含量测定发现，H/R诱导ONOO⁻生成显著增加（与常氧对照组相比，P<0.01）。在复氧时用强效ONOO⁻清除剂尿酸（UA）处理可显著降低ONOO⁻生成，并保护心肌细胞免受H/R损伤，而在HPC组中用相同的UA处理不能进一步提高心肌细胞的存活率（与单独HPC组相比，P>0.05）。统计分析表明，细胞活力与心肌细胞ONOO⁻生成呈密切负相关（P<0.01）。
这些数据表明，缺氧后处理可保护心肌细胞在复氧后免于凋亡并提高其存活率，这部分归因于复氧后ONOO⁻生成减少。
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