纳洛酮对缺氧心肌细胞保护作用的实验研究

The protective effect of Naloxone on hypoxic cardiomyocytes

大量临床和实验研究均已证明纳洛酮在心肌缺血再灌注损伤中对心肌有保护作用．可以改善心肌能量代谢，稳定溶酶体膜，抑制中性粒细胞释放超氧阴离子、抗心律失常等，但对细胞凋亡及炎性介质的影响未见报导。本研究从凋亡和坏死后炎症两个方面观察了纳洛酮对缺氧心肌细胞的保护作用。

Backround and objective It has been reported that Naloxone may provide protective effect on ischemia/reperfusion injured myocardium. But the report about the effect on apoptosis of ischemia injured cardiomyocytes and inflammatory factors has not been found. The purpose of this study is to investigate the effect of Naloxone on mitochondrial membrane potential ΔΨm and apotosis in hypoxia/ reoxygenation cardiomyocyte and on inflammatory factors in ischemia/reperfusion cardiac tissue. Methods The study includes tow parts. Part one： second cultures of cardiomyocytes from neonate rabbits were divided into three groups： control group, hypoxia group and therapy group（hypoxia/reoxygenation combined with Naloxone therapy ） and the tow latter were further divided into three subgroups： hypoxia 2 h, hypoxia 2 h/reoxygenation 2 h, hypoxia 2 h/reoxygen 4 h. At the end of intervention, the ΔΨm and apoptosis of cardiamyocytes were detected by flow cytometry. One another subgroup of hypoxia 2 h/reoxygenation 16 h was identified as DNA fragmentation by TUNEL. Part tow： the neutrophil chemoattractants IL-8, 6-Keto- Prostaglandin F/la （6-Keto-PGFla） and Thromboxane B/2 （TXB2） generated in a model of myocardial infarction in 22 anesthetized male New Zealand white rabbits were investigated. The rabbits were divided into three groups randomly： sham-operation group（n=6）,ischemia/reperfusion group（n=8） and therapy group （ischemia/reperfusion combined with Naloxone intravenous therapy, n=8）. Coronary left anterior descend artery was occluded for 1 hour and reperfused for 4.5 hours. IL-8 from blood serum and ischemic tissue, 6-Keto-PGFla/TXB2 from blood serum were detected using radioimmunoassays. Results 1 ）After hypoxia, the ΔΨm of hypoxia and therapy group were lower than that of the control（P≤ 0.01）.The ΔΨm of therapy group was significantly higher than that of hypoxia group （P≤0.01）. 2）A remarkable losing of ΔΨm in hypoxia group occurred during the first 2 hours of rexoygenation, while in therapy group it was occurred during the second 2 h. 3 ）After reoxygenation for 4 h or 16 h, the percentage of apoptosis of hypoxia group was significantly higher than the control group and therapy group （P 〈0.01, respectively）,there was no significant difference between therapy group and control group. The same condition was observed in the ratio of necrosis. 4）Within hypoxia group, the percentage of apoptosis was obviously higher than the percentage of necrosis （P 〈0.05）. 5）In rabbits, the ratio of 6-Keto-PGFla/TXB2 were much lower than that of therapy group （P 〈0.01）, while concentration of IL-8（whatever of tissue or serum） and TXB2 of ischemia/reperfusion group was obviously higher than that in the therapy group （P 〈0.01）. Conclusion 1 ） Hypoxia/reoxygenation may cause the collapse of mitochondrial membrane potential which further led to cell＇s apoptosis as well as necrosis in cultured myocardial cell. Apoptosis is the major style of cell death. 2）The losing of ΔΨm occurred during reoxygenation period, but not hypoxia. 3）Naloxone may significantly delay the lossing of ΔΨm and then decrease the apoptosis as well as necrosis. 4）Naloxone may decrease the concentration of inflammatory factors in ischemic myocardium.