缺血预处理对小鼠心肌缺血再灌注损伤的保护作用研究

Effects of Ischemic Preconditioning on Myocardial Ischemia-reperfusion Injury in Mice

通过建立C57/B6雄性小鼠心肌缺血再灌注(ischemia-reperfusion,I/R)模型,探讨缺血预处理对小鼠心肌缺血再灌注损伤的保护作用。首先,将36只6~8周C57/B6雄性小鼠随机分为3组(n=12):假手术组(Sham组)、缺血再灌注组(I/R组)及缺血预处理组(Ipost组)。然后,利用苏木素伊红(hematoxylin and eosin,HE)染色、脱氧三磷酸尿苷缺口末端标记(Td T-mediated d UTP-biotin nick end labeling,TUNEL)染色、免疫组化及蛋白质印迹方法,对比3组小鼠的心肌病理学改变、心肌细胞凋亡情况、梗死心肌边缘区微血管密度(microvessel density,MVD)的变化,以及肿瘤相关蛋白质PTEN(phosphatase and tensin homolog deleted from chromosome 10,即人第10号染色体缺失的磷酸酶及张力蛋白同源基因的编码产物)、自噬相关蛋白质LC3I/II和腺苷酸活化蛋白激酶(5-AMP activated protein kinase,AMPK)的表达水平。结果显示,I/R组心肌组织细胞水肿、炎症细胞浸润等组织病理学变化情况较Sham组明显,而Ipost组中的情况相比I/R组有明显改善;同时,Ipost组心肌凋亡率高于Sham组,但显著低于I/R组(P<0.01);Ipost组梗死心肌边缘区域的微血管密度显著高于I/R组(P<0.01)。此外,缺血预处理后,PTEN的表达水平降低,AMPK磷酸化水平以及LC3I/II蛋白的表达水平均增强。由此可见,缺血预处理可减轻I/R损伤,减少心肌梗死面积,减轻心肌水肿,对心肌细胞有明显的保护作用,其机制可能与梗死心肌中PTEN表达下调、AMPK磷酸化水平增强、心肌细胞自噬增强和凋亡减少有关。

To explore the effects of ischemic preconditioning on myocardial ischemia-reperfusion injury, the myocardial ischemia-perfusion model was established in C57/B6 male mice aged 6-8 weeks. Thirty-six mice were randomly divided into three groups： sham group （Sham, n=12）, ischemia-reperfusion group （I/R, n=12） and ischemic preconditioning group （Ipost, n=12）. The hematoxylin-eosin staining （HE）, TUNEL, immunohistochemical staining, and Western-blot were used to detect the changes in myocardial tissues from three groups. Compared with the Sham group, the histopathological changes, including myocardial tissue edema and inflammatory cell infiltration, were obviously serious in the I/R group. However, Ipost group was im- proved significantly compared with the I/R group. TUNEL results showed that the myocardial apoptosis rate in Ipost group was significantly lower than that in I/R group （P〈0.01）, though higher than that in the Sham group. In Ipost group, the number of capillaries on the edge of myocardial infarction area was significantly higher than that in I/R group （P〈0.01）. Western-blot results indicated that the expression of PTEN protein in the my- ocardial tissue in Ipost group was down-regulated, whereas the AMPK phosphorylation and LC3I/II expres- sions were increased. Taken together, ischemic preconditioning may have protective effects on I/R injury, and could reduce the infarct area. The protective mechanism of lpost may be related to reduction of myocardial cell apoptosis, down-regulation of PTEN, enhancement of the phosphorylation level of AMPK and enhancement of myocardial cell autophagy in ischemic infarcted myocardium after pretreatment.