蛋白酶激活受体-2对缺血再灌注大鼠心肌细胞凋亡的影响

The effect of protease-activated receptor2 on rat apoptotic cardiomyocytes underwent ischemia reperfnsion injury

目的探讨蛋白酶激活受体-2（PAR-2）对缺血再灌注大鼠心肌细胞凋亡的影响。方法40只雄性SD大鼠随机分为5组：假手术组（sham组），缺血再灌注组（I／R组）和丝-亮-异亮-甘-精-亮-酰胺（SLIGRL—NH2）低剂量组（0．5mg／kg）、中剂量组（1mg／kg）、高剂量组（3mg／kg），每组8只，建立大鼠在体心肌缺血再灌注模型。采用末端标记法（TUNEL）和DNA琼脂糖凝胶电泳的方法检测心肌细胞凋亡，免疫组织化学法检测凋亡相关蛋白Bcl-2、Bax的表达，实时荧光定量PCR检测心肌细胞PAR-2 mRNA的表达情况。结果（1）SLIGRL—NH2中、高剂量组凋亡指数明显低于L／R组（SLIGRL—NH2中、高剂量组分别为23．36％±3．77％、15．56％±1．24％比I／R组35．19％±4．50％，P〈0．05～0．01）；凋亡相关蛋白Bcl-2高于I／R组（SLIGRL—NH2中、高剂量组分别为0．983±0．103、1．197±0．119比I／R组0．761±0．043，P〈0．05～0．01）；凋亡相关蛋白Bax的表达显著低于I／R组（SLIGRL—NH2中、高剂量组分别为0．646±0．041，0．578±0．029比I／R组0．759±0．035，P均〈0．01）；PAR-2mRNA的表达明显高于I／R组（SLIGRL-NH2中、高剂量组分别为3．73±0．45，7．62±0．81比I／R组1．42±0．41，P均〈0．01）。（2）DNA凝胶电泳结果显示，I／R组、SLIGRL-NH2低剂量组可见到DNA梯带，假手术组和SLIGRL—NH2中、高剂量组则无明显DNA梯带。结论PAR-2激动剂SLIGRL—NH2可上调并激活PAR-2，并通过增加Bcl-2／Bax的比值抑制大鼠缺血再灌注心肌细胞的凋亡而发挥心肌保护效应，且该作用具有一定的剂量依赖效应。

Objective To investigate the effect of protease-activated receptor2 （PAR-2） on rat apoptotic cardiomyocytes underwent ischemia reperfusion （I/R） injury. Methods Healthy male Sprague- Dawley rats were randomly divided into five groups （n = 8 each） ： sham-operation group, I/R （ligating the left coronary artery for 30 minutes and followed by 120 minutes reperfusion） group and three SLIGRL-NH2 groups treated with intravenous PAR-2 agonist SLIGRL-NH2 at different doses （0. 5,1,3 mg/kg） 5 minutes before reperfusion. Apoptic cardiomyocytes was detected by TUNEL staining and by DNA ladder on agarose gel electrophoresis. Bax and Bcl-2 expression in myocardium was analyzed by immunohistochemical technique. The mRNA expression of PAR-2 was determined by Real-time quantitative polymerase chain reaction（RT-PCR） . Results （1） The apoptosis index and the expression of Bcl-2 and Bax were significantly increased in IR group and SLIGRL-NH2 groups than those in sham group （P 〈 0. 05 -0. 01 ）. （2） Compared with I/R group, the apoptosis index and the expression of Bax were significantly reduced while the expression of Bcl-2 and PAR-2 mRNA were significantly upregulated by SLIGRL-NH2 in a dosedependent manner. （3） DNA Agarose gel electrophoresis demonstrated that DNA ladder existed in I/R and 0. 5 mg/kg SLIGRL-NH2 group, but not in 1,3 mg/kg SLIGRL-NH2 groups. Conclusions PAR-2 agonist SLIGRL-NH2 could reduce myocardial apoptosis by upregulating the Bcl-2 and PAR-2 mRNA level and downregulating Bax expression in a dose-dependent manner in this rat I/R model.