尼可地尔保护大鼠缺血再灌注心肌内质网应激机制的研究

Nicorandil Protect the Myocardium from Ischemia Reperfusion Injury through Inhibiting Endoplasmic Reticulum Stress in Rats

目的观察尼可地尔对大鼠心肌缺血再灌注损伤的保护作用及其对内质网应激反应标志因子表达的影响。方法 48只SD大鼠随机分为6组:平衡灌注(Bal)组、平衡灌注+尼可地尔预处理(Bal+Nic100)组、缺血再灌注(I/R Ctrl)组、缺血再灌注+尼可地尔30μmol/L预处理(Nic 30)组、缺血再灌注+尼可地尔100μmol/L预处理(Nic 100)组、缺血再灌注+尼可地尔300μmol/L预处理(Nic 300)组。采用Langendorff灌流装置,建立离体大鼠心肌缺血再灌注损伤模型,分别给予不同剂量的尼可地尔预处理。采用多道生理信号采集系统记录分析各项心功能参数:左心室收缩峰压(LVPSP)、左心室舒张末压(LVEDP)、左心室等容期压力最大变化速率(±dp/dt max)、心率(HR)等,并计算左室发展压(LVDP=LVPSP-LVEDP);采用LDH检测试剂盒测定冠脉流出液中的乳酸脱氢酶(LDH)活性;氯化三苯基四氮唑(TTC)染色检测心肌梗死面积;Real-Time PCR检测心肌组织中GRP78及CHOP的mRNA表达水平;Western blot检测心肌组织中GRP78、CHOP的蛋白表达水平。结果与I/R Ctrl组比较,尼可地尔预处理可显著改善大鼠离体缺血再灌注心脏的各项心功能指标。与平衡灌流期比较,I/R Ctrl组再灌注120 min后冠脉流出液中的LDH活性水平显著上升,而尼可地尔预处理各组冠脉流出液LDH活性水平显著低于I/R Ctrl组。与I/R Ctrl组比较,尼可地尔预处理各组心肌梗死面积显著缩小。与Bal组比较,I/R Ctrl组心肌组织中的GRP78及CHOP mRNA及蛋白表达水平显著上调,而与I/R Ctrl组比较,尼可地尔预处理可显著降低GRP78及CHOP mRNA及蛋白表达水平,且尼可地尔作用均呈剂量依赖性。结论在大鼠离体心肌缺血再灌注损伤模型中,尼可地尔预处理可呈剂量依赖性地减轻心肌细胞损伤,促进缺血再灌注后心脏功能的恢复,还可抑制心肌缺血再灌注诱发的内质网应激反应。

Objective To observe the effect of nicorandil preconditioning on rat in vitro myocardial ischemia reperfusion（I/R）injury and the expression of the key factors in endoplasmic reticulum stress signal pathway.Methods Forty-eight Sprague-Dawley（SD）rats were randomly divided into six groups：balance group,balance＋100μmol/L nicorandil preconditioning group,I/R control group,30μmol/L nicorandil preconditioning group,100μmol/L nicorandil preconditioning group and 300μmol/L nicorandil preconditioning group.Hearts from each group were excised and subjected to 30 min global ischemia followed by 120 min reperfusion in a Langendorff apparatus.Homodynamic parameters of left ventricular（LV）were recorded and evaluated with Powerlab system,including LV developed pressure（LVDP）,LV end-diastolic pressure（LVEDP）,and the derivative of left ventricular（＋dP/dtmax and-dP/dtmax）.Cell death was reflected by lactate dehydrogenase（LDH）activity assay in coronary effluent.Cardiac infarct size was also measured after nicorandil treatment by Triphenyl Tetrazolium Chloride（TTC）staining.The mRNA levels of ER stress markers,including glucoseregulated protein（GRP）78 and C/-EBP homologous protein（CHOP）were examined by real time-PCR.The protein expressions of GRP78 and CHOP were detected by western blot.Results Nicorandil preconditioning improved post-ischemic cardiac function of isolated rat heart in a concentration dependent manner,and reduced LDH activity in coronary effluent.Consistent with the above observation,nicorandil preconditioning significantly reduced the infarct size after ischemia reperfusion.The mRNA level and protein expression of GRP78 and CHOP were upregulated in I/R heart.When the I/R hearts were treated with nicorandil,both CHOP and GRP78 levels were significantly attenuated,which emerged in an dose-dependent way.Conclusion Nicorandil preconditioning could alleviate myocardial I/R injury,ameliorate post-ischemic contractile function recovery,and inhibit endoplasmic reticulum stress induced by myocardial I/R in a dose-dependent manner in vivo.