吡那地尔超极化停搏对大鼠离体心脏缺血再灌注时心肌细胞凋亡的影响

Effect of pinacidil-induced hyperpolarized cardiac arrest on apoptosis in rat cardiomyocytes induced by myocardial ischemia-reperfusion

目的探讨吡那地尔超极化停搏对大鼠离体心脏缺血再灌注时心肌细胞凋亡的影响。方法健康SD大鼠108只,随机分为6组（n=18）,建立Langendorff离体心脏灌注模型,对照组（C组）持续灌注37℃含氧K-H缓冲液120min不停搏;37℃含氧的K-H缓冲液平衡灌注15min后,去极化停搏组（D组）灌注37℃St.Thomas停搏液（含K^＋ 16mmol/L）20ml/kg,超极化停搏组（H组）灌注37℃吡那地尔（50μmol/L,K^＋ 5mmol/L）超极化停搏液20ml/kg,线粒体ATP敏感性钾通道阻滞剂5-羟葵酸＋超极化停搏组（5-HD＋H组）和膜ATP敏感性钾通道阻滞剂HMR-1098＋超极化停搏组（HMR-1098＋H组）灌注100μmol/L 5-HD或HMR-1098,5min后灌注吡那地尔超极化停搏液20ml/kg;5-HD＋HMR-1098 ＋H组先灌注100μmol/L 5-HD和100μmol/L HMR-1098混合液,5min后灌注吡那地尔超极化停搏液20 ml/kg。各停搏组心脏常温停搏（缺血）40min,再用37℃含氧K-H缓冲液再灌注60min。于缺血前、缺血40min和再灌注60min时取心肌组织,测定caspase-3、caspase-9活性,观察细胞凋亡情况。结果缺血前各组间各指标差异无统计学意义（P〉0.05）。与C组比较,其余各组细胞凋亡指数（AI）、caspase-3、caspase-9活性增加;与D组比较,H组AI、caspase-3、caspase-9活性降低;与H组比较,加阻滞剂的3组AI、caspase-3、caspase-9活性增加;与5-HD＋H组、HMR-1098＋H组比较,5-HD＋HMR-1098＋ H组AI、caspase-3、caspase-9活性增加（P〈0.05）。与缺血前比较,在缺血40min、再灌注60min时,各组AI、caspase-3、caspase-9活性增加（P〈0.05）。结论吡那地尔超极化停搏可明显抑制大鼠离体心脏缺血再灌注时心肌细胞凋亡,产生心肌保护效应,其机制可能与降低caspase-3、caspase-9活性有关。

Objective To investigate the effect of pinacidil-induced hyperpolarized cardiac arrest on apoptosis in cardiomyocytes and changes in caspase-3 and caspase-9 activities following myocardial ischemiareperfusion.Methods One hundred and eight SD rats weighing 250-300 g were randomly divided into 6 groups （n =8 each）：groupⅠcontrol （C）;groupⅡdepolarized arrest （D）;groupⅢhyperpolarized arrest （H）;groupⅣ5-HD （mito-K_（ATP） channel blocker）＋hyperpolarized arrest （5-HD＋H）;groupⅤHMR-1098 （saco-K_（ATP） channel blocker）＋hyperpolarized arrest （HMR-1098＋H） and groupⅥ5-HD＋HMR-1098＋H.The animals were anesthetized and their hearts were excised.The aorta was cannulated and the heart was retrogradely perfused with an oxygenated K-H buffer at 5.8 kPa in a Langendorff apparatus.In group C the hearts were continuously perfused with oxygenated KHB at 37℃for 120 min.In group D and H after 15 min equilibration cardiac arrest was induced with St Thomas cardioplegic solution and cardioplegic solution containing pinacidil （50μmol/L） respectively for 40 min followed by 60 min reperfusion.In groupⅣ,ⅤandⅥthe hearts were perfused with 5-HD （100μmol/L）,HMR-1098 （100μmol/L） and a mixture of 5-HD （100μmol/L） and HMR-1098 （100μmol/L） respectively for 5 min before 40 min pinacidil-induced cardiac arrest and 60 min reperfusion.Myocardial specimens were obtained before ischemia （T0）,at the end of 40 min ischemia （T1） and 60 min reperfusion （T2） for determination of myocardial apoptosis and caspase-3 and caspase-9 activities.Results （1） There was no significant difference in apoptosis index （AI） and caspase-3 and caspase-9 activities before ischemia among the 6 groups.（2） The AI and the caspase-3 and easpase-9 activities were significantly increased at T1 and T2 as compared with the baseline values before ischemia （T0） in groupⅡ-Ⅵ.（3） The AI and caspase-3 and caspase-9 activities at T1 and T2 were significantly lower in group H than in groupⅡ,Ⅳ,ⅤandⅥ.（4） In the 3 groups pre-treated with KATP blocker （Ⅳ,Ⅴ,Ⅵ） the AI and caspase-3 and caspase-9 activities were significantly higher than in group H （Ⅲ）.Conclusion Hyperpolarized cardiac arrest induced with cardioplegic solution containing pinacidil may protect cardiomyocytes from I/R injury by inhibiting apoptosis and decreasing caspase-3 and caspase- 9 activities.