氯通道阻滞剂对大鼠心肌缺血再灌注损伤致心肌细胞凋亡的作用

摘要

目的 本研究应用大鼠在体心肌缺血再灌注损伤(I/RI)动物模型,观察氯通道阻滞剂DIDS(4,4'-disothiocyanostibibene-2,2'-disulfonic acid)对心肌细胞凋亡的作用以及与磷脂酰肌醇3激酶蛋白激酶B(PI3K-Akt)信号通路可能存在的相互作用,明确DIDS心肌保护作用的可能机制.方法 75只大鼠随机分成5组:假手术组,I/RI组,I/RI+ DIDS(14 mg/kg),I/RI+LY294002(PI3K-Akt特异性阻断剂),I/R+DIDS+LY294002组.TTC染色检测心肌梗死范围;TUNEL法测定细胞凋亡;Caspase-3试剂盒检测caspase-3活性;免疫印迹法检测p-Akt水平.结果 与I/R组比较DIDS可以减少心肌梗死范围(各组n＝4,P<0.01),可以明显抑制心肌细胞凋亡(各组n＝5,P<0.01),降低凋亡效应子caspase-3活性(各组n＝5,P<0.01),提高I/RI心肌的p-Akt水平(各组n＝5,P<0.01),LY294002可以部分阻断这些作用,LY294002自身并无减少心肌梗死范围的作用.结论 氯离子通道阻滞剂DIDS对缺血再灌注损伤的心肌具有保护作用,其作用部分是通过 PI3K/Akt信号通路来实现的.%OBJECTIVE To investigate the effects of chloride channel blocker 4 ,4 '- disothiocyanostibibene - 2 .2 '- disulfonic acid ( DIDS ) on myocardium underwent ischemia and reperfusion injury ( I/RI ). METHODS Seventy - five male healthy SD rats were randomized into five groups ( n = 15 ) : group A, sham operation group; group B, I/RI group, ischemia 30 min, reperfusion 4 h; group C, I/RI + DIDS group: ischemia 30 min. DIDS was intravenously administered at 7 mg/kg/h exactly at the starting of reperfusion for 2 h; group D, I/RI + LY294002 group: ischemia 30 min, LY294002 ( 0. 3 mg/kg ) was infused during the second 15 min of ischemia; group E, I/R + DIDS + LY294002 group : rats were treated with combination of C and D group treatment procedure.After 4 h reperfusion, hearts were harvested. Myocardial infarct size, apoptotic index of cardiomyocytes, caspase - 3 activity and p -Akt levels were detected. RESULTS DIDS reduced myocardial infarct size ( n =4, P ＜ 0. 01 vs. I/RI group ), inhibited cardiomyocytes apoptosis ( n = 5 , P ＜0. 01 vs. I/RI group ), decreased caspase - 3 activity ( n =5 , P ＜0. 01 vs. I/RI group ) and significantly increased p - Akt levels ( n = 5 , P ＜0. 01 vs. I/RI group ); LY294002 partially blocked these effects except the levels of p - Akt.But LY294002 itself had no effect on myocardial infarct size. CONCLUSION DIDS play a protective role on myocardium underwent ischemia and reperfusion injury, and its protective effect is partially through PI3K/Akt signal pathway.