褪黑素通过抑制挛缩减轻大鼠离体心脏的缺血再灌注损伤

Melatonin protects against myocardial ischemia-reperfusion injury by inhibiting contracture in isolated rat hearts

目的探讨褪黑素(Mel)抑制挛缩改善大鼠离体心脏缺血再灌注(IR)损伤的作用及机制。方法采用Langendorff离体心脏灌流方法模拟缺血再灌注模型,40只SD大鼠按随机数字表法分为4组(10只/组)。正常对照组(Control):正常灌注175 min;IR组:全心缺血45 min,再灌注120 min;Mel+IR组:用Mel(5μmol/L)灌注1 min,全心缺血45 min,用Mel(5μmol/L)再灌注5 min,Krebs-Henseleit液再灌注115 min;挛缩抑制剂2,3-丁二酮单肟(BDM)干预缺血再灌注组(BDM+IR):用BDM(20 mmol/L)灌注1 min,全心缺血45 min,用BDM(20 mmol/L)再灌注5 min,KH液再灌注115 min。以心肌死亡面积、caspase-3活性、细胞色素c(cytochrome c)和cleaved caspase-3蛋白表达检测各组心肌损伤程度;以HE染色、冠脉流出液中乳酸脱氢酶(LDH)活性、肌钙蛋白I(cTnI)含量、左心室舒张末压(LVEDP)和电镜观察肌原纤维收缩带的形成评价各组心脏挛缩程度;检测心肌组织中ATP含量的变化。结果与Control组相比,IR组心肌死亡面积、caspase-3活性、cytochrome c和cleaved caspase-3蛋白表达显著增加(P<0.01);同时,冠脉流出液中LDH活性和cTnI含量明显增加,再灌注末LVEDP显著抬升,HE染色显示心肌纤维发生明显断裂,ELISA检测发现ATP含量显著降低(P<0.01);另外,透射电镜结果表明IR组肌节过度收缩,形成明显的肌原纤维收缩带;而给予Mel和BDM处理后,可显著减小心肌死亡面积、caspase-3活性、cytochrome c和cleaved caspase-3蛋白表达,还可明显抑制LDH活性、cTnI含量和LVEDP,减少心肌纤维断裂,并增加胞内ATP含量;更为重要地是,Mel和BDM能减轻IR引起的肌节过度收缩,并抑制收缩带的形成。结论Mel可通过抑制挛缩明显改善心肌缺血再灌注损伤,其机制可能与增加心肌细胞内ATP含量,减轻心肌机械性损伤引起的肌膜撕裂,减少细胞内容物的漏出有关。

Objective To investigate the protective effect of melatonin against myocardial ischemia reperfusion(IR)injury in isolated rat hearts and explore the underlying mechanisms.Methods The isolated hearts from 40 male SD rats were randomly divided into 4 groups(n=10):the control group,where the hearts were perfused with KH solution for 175 min;IR group,where the hearts were subjected to global ischemia for 45 min followed by reperfusion for 120 min;IR+melatonin(Mel+IR)group,where melatonin(5μmol/L)was administered to the hearts 1 min before ischemia and during the first 5 min of reperfusion,followed by 115 min of reperfusion;and IR+2,3-butanedione monoxime(IR+BDM)group,where the hearts were treated with BDM(20 mmol/L)in the same manner as melatonin treatment.Myocardial injury in the isolated hearts was assessed based on myocardial injury area,caspase-3 activity,and expressions of cytochrome C and cleaved caspase-3 proteins.Cardiac contracture was assessed using HE staining and by detecting lactate dehydrogenase(LDH)activity and the content of cardiac troponin I(cTnI)in the coronary outflow,measurement of left ventricular end-diastolic pressure(LVEDP)and electron microscopy.The content of ATP in the cardiac tissue was also determined.Results Compared with those in the control group,the isolated hearts in IR group showed significantly larger myocardial injury area and higher caspase-3 activity and the protein expressions of cytochrome C and cleaved caspase-3 with significantly increased LDH activity and cTnI content in the coronary outflow and elevated LVEDP at the end of reperfusion;HE staining showed obvious fractures of the myocardial fibers and the content of ATP was significantly decreased in the cardiac tissue;electron microscopy revealed the development of contraction bands.In the isolated hearts with IR,treatment with Mel or BDM significantly reduced the myocardial injury area,caspase-3 activity,and protein expressions of cytochrome C and cleaved caspase-3,obviously inhibited LDH activity,lowered the content of cTnI and LVEDP,reduced myocardial fiber fracture,and increased ATP content in the cardiac tissue.Both Mel and BDM inhibited the formation of contraction bands in the isolated hearts with IR injury.Conclusion Mel can alleviate myocardial IR injury in isolated rat hearts by inhibiting cardiac contracture,the mechanism of which may involve the upregulation of ATP in the cardiac myocytes to lessen the tear of membrane and reduce cell content leakage.