激活腺苷A2B受体对大鼠心肌缺血再灌注时自噬的影响:PI3K/Akt信号通路在其中的作用

Effect of activating adenosine A2B receptors on autophagy during myocardial ischemia-reperfusion in rats:role of PI3K/Akt signaling pathway

目的:评价激活腺苷A2B受体对大鼠心肌缺血再灌注时自噬的影响及磷脂酰肌醇3-激酶/蛋白质丝氨酸苏氨酸激酶(PI3K/Akt)信号通路在其中的作用。方法:清洁级健康雄性SD大鼠48只,体重220~280 g,采用随机数字表法分为4组(n=12):假手术组(Sham组)、心肌缺血再灌注组(I/R组)、腺苷A2B受体激动剂BAY 60-6583组(BAY组)和BAY 60-6583+PI3K抑制剂LY 294002组(BAY+LY组)。采用结扎左冠状动脉前降支30 min、再灌注120 min的方法制备大鼠心肌缺血再灌注损伤模型。BAY组于再灌注前5 min时腹腔注射BAY60-65831 mg/kg,BAY+LY组于再灌注前10 min时腹腔注射LY 29400210 mg/kg,再灌注前5 min时腹腔注射BAY60-65831 mg/kg。于再灌注结束时,采用ELISA法检测血清LDH和CK-MB浓度,处死取心肌组织,采用伊文氏蓝、TTC双重染色法确定心肌梗死体积百分比,采用Western blot法检测微管相关蛋白1轻链3Ⅰ(LC3Ⅰ)、LC3Ⅱ、Beclin-1和磷酸化Akt(p-Akt)的表达水平。结果:与Sham组比较,I/R组血清LDH、CK-MB浓度和心肌梗死体积百分比升高,心肌p-Akt表达下调,Beclin-1和LC3Ⅱ表达上调,LC3Ⅱ/LC3Ⅰ比值升高(P<0.05)。与I/R组比较,BAY组血清LDH、CK-MB浓度和心肌梗死面积百分比降低,心肌p-Akt表达上调,Beclin-1和LC3Ⅱ表达下调,LC3Ⅱ/LC3Ⅰ比值降低(P<0.05),BAY+LY组上述指标差异无统计学意义(P>0.05)。与BAY组比较,BAY+LY组血清LDH、CK-MB浓度和心肌梗死面积百分比升高,心肌p-Akt表达下调,Beclin-1和LC3Ⅱ表达上调,LC3Ⅱ/LC3Ⅰ比值升高(P<0.05)。结论:激活腺苷A2B受体可降低大鼠心肌缺血再灌注时心肌细胞自噬水平,机制可能与激活PI3K/Akt信号通路有关。

Objective To evaluate the effect of activating adenosine A2B receptors on autophagy during myocardial ischemia-reperfusion(I/R)and the role of phosphatidylinositol 3-kinase/serine/threonine protein kinase(PI3K/Akt)signaling pathway in rats.Methods Forty-eight clean-grade healthy male Sprague-Dawley rats,weighing 220-280 g,were divided into 4 groups(n=12 each)using a random number table method:sham operation group(group Sham),myocardial I/R group(group I/R),adenosine A2B receptor agonist BAY 60-6583 group(group BAY)and BAY 60-6583+PI3K inhibitor LY 294002 group(group BAY+LY).Myocardial I/R was induced by occlusion of the anterior descending branch of the left coronary artery for 30 min followed by 120-min reperfusion.BAY 60-65831 mg/kg was intraperitoneally injected at 5 min before reperfusion in group BAY.BAY 60-65831 mg/kg was intraperitoneally injected at 5 min before reperfusion and LY 29400210 mg/kg was intraperitoneally injected at 10 min before reperfusion in group BAY+LY.Blood samples were obtained at the end of reperfusion for determination of concentrations of lactate dehydrogenase(LDH)and creatine kinase-MB(CK-MB)in serum(by enzyme-linked immunosorbent assay).The animals were sacrificed,and myocardial tissues were obtained for measurement of the percentage of myocardial infarct size(by Evan Blue and TTC double-staining)and for determination of the expression of microtubule-associated protein 1 light chain 3(LC3Ⅰ),LC3Ⅱ,Beclin-1 and phosphorylated Akt(p-Akt)(by Western blot).The ratio of LC3Ⅱ/LC3Ⅰwas calculated.Results Compared with group Sham,the serum LDH and CK-MB concentrations and percentage of myocardial infarct size were significantly increased,the expression of p-Akt was down-regulated,the expression of Beclin-1 and LC3Ⅱwas up-regulated,and the ratio of LC3Ⅱ/LC3Ⅰwas increased in group I/R(P<0.05).Compared with group I/R,the concentrations of serum LDH,CK-MB and percentage of myocardial infarct size were significantly decreased,the expression of p-Akt was up-regulated,the expression of Beclin-1 and LC3Ⅱwas down-regulated,and the ratio of LC3Ⅱ/LC3Ⅰwas decreased in the group BAY(P<0.05),and no significant change was found in the parameters mentioned above in group BAY+LY(P>0.05).Compared with group BAY,the concentrations of serum LDH,CK-MB and percentage of myocardial infarct size were significantly increased,the expression of p-Akt was down-regulated,the expression of Beclin-1 and LC3Ⅱwas up-regulated and the ratio of LC3Ⅱ/LC3Ⅰwas increased in group BAY+LY(P<0.05).Conclusion Activating adenosine A2B receptors can decrease autophagy of myocardial cells during myocardial I/R injury,and the mechanism may be related to activating PI3K/Akt signaling pathway in rats.