神经型一氧化氮合酶在小鼠心肌缺血预处理中的作用

目的 应用神经型一氧化氮合酶(nNOS)基因敲除小鼠和nNOS抑制剂,探讨nNOS对心肌缺血预处理后心肌细胞凋亡的影响.方法 实验分为野生型缺血再灌注组(WT IR)、野生型缺血预处理组(WT IP)、野生型缺血预处理L-VNIO处理组(WT IP+ L-VNIO)、基因敲除鼠缺血再灌注组(KO IR)和基因敲除鼠缺血预处理组(KO IP).采用冠状动脉左前降支结扎法建立小鼠缺血再灌注损伤模型,缺血再灌注组缺血30 min再灌注3h,缺血预处理组分别经缺血5 min再灌注5 min连续三个循环后,再缺血30 min再灌注3h,观察TUNEL染色和Caspase-8、Caspase-9、Caspase-3的活性变化,并用Western Blot法观察Bax、Bcl-2和Fas蛋白的表达情况.结果 与WT IR组相比,WT IP组小鼠TUNEL阳性细胞数目减少,Caspase-8、Caspase-9和Caspase-3活性降低,Bax和Fas蛋白表达显著降低,Bcl-2表达显著增加(P＜0.05).而在KO IP组,与KO IR组相比,TUNEL阳性细胞数目和Caspase活性显著增加,Bax和Fas表达显著增高,Bcl-2表达显著降低(P＜0.05).结论 nNOS在心肌缺血预处理时发挥抑制心肌细胞凋亡的作用.

Effect of nNOS During Myocardial Ischemic Preconditioning in Mice

AimUsing neuronal nitric oxide synthase(nNOS) knockout mice and nNOS inhibitor to investigate the effect of nNOS during myocardial ischemic preconditioning.MethodsMice were divided into wild-type ischemia-reperfusion group(WT IR),wild-type ischemic preconditioning group(WT IP),wild-type L-VNIO treatment group(WT IP+L-VNIO),nNOS-/-mice ischemia-reperfusion group(KO IR),nNOS-/-mice ischemic preconditioning group(KO IP).They were subjected to 30 minutes of ischemia by left descending branch of coronary artery ligation followed 3 hours reperfusion.IP was induced by 3 cycles of 5 minutes ischemia and reperfusion before 30 minutes ischemia.After 3 hours reperfusion,terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) staining and activities of caspase-8,caspase-9,caspase-3,Bax,Bcl-2 and Fas expression were measured.ResultsIn WT group,compared with IR group,TUNEL positive cells,caspase-8,caspase-9,caspase-3 activities and Bax and Fas protein expression were significantly decreased,Bcl-2 expression was significantly increased(P<0.05).In KO group,compared with IR group,TUNEL positive cells,caspase-8,caspase-9,caspase-3 activities and Bax and Fas protein expression were significantly increased,Bcl-2 expression was significantly decreased(P<0.05).ConclusionnNOS was involved in myocardial ischemic preconditioning by reducing myocardial apoptosis.