激活腺苷A2A受体对糖尿病大鼠心肌缺血再灌注损伤的影响:与自噬的关系

Effect of activating adenosine A2A receptors on myocardial ischemia-reperfusion injury in diabetic rats: the relationship with autophagy

目的评价激活腺苷A2A受体对糖尿病大鼠心肌缺血再灌注损伤的影响及其与自噬的关系。方法清洁级健康雄性SD大鼠,6周龄,体重200~250 g,采用腹腔注射1%链脲佐菌素60 mg/kg制备大鼠糖尿病模型。取糖尿病大鼠40只,采用随机数字表法分为4组(n=10):假手术组(Sham组)、缺血再灌注组(I/R组)、缺血再灌注+腺苷A2A受体激动剂CGS21680组(CGS组)、缺血再灌注+CGS21680+腺苷A2A受体拮抗剂ZM241385组(CGS+ZM组)。采用结扎左冠状动脉前降支30 min再灌注120 min的方法制备糖尿病大鼠心肌缺血再灌注损伤模型。CGS组于再灌注前10 min静脉注射CGS21680 10 μg/100 g,CGS+ZM组于再灌注前10 min依次静脉注射CGS21680 10 μg/100 g和ZM241385 0.2 mg/kg。再灌注结束时,取血标本,采用ELISA法检测血清CK-MB、LDH以cTnI浓度;处死取心肌组织,采用TTC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)和LC3Ⅱ、p62及Beclin-1的表达。结果与Sham组比较,I/R组血清CK-MB、LDH、cTnI浓度和心肌梗死体积百分比升高,p62和Beclin-1表达上调,LC3Ⅱ/LC3Ⅰ比值升高(P<0.05)。与I/R组比较,CGS组血清CK-MB、LDH、cTnI浓度和心肌梗死体积百分比降低,p62和Beclin-1表达下调,LC3Ⅱ/LC3Ⅰ比值升高(P<0.05),CGS+ZM组上述指标差异无统计学意义(P>0.05)。与CGS组比较,CGS+ZM组血清CK-MB、LDH、cTnI浓度和心肌梗死体积百分比升高,p62和Beclin-1表达下调,LC3Ⅱ/LC3Ⅰ比值降低(P<0.05)。结论激活腺苷A2A受体可减轻糖尿病大鼠心肌缺血再灌注损伤,机制可能与增强自噬水平有关。

Objective To evaluate the effect of activating adenosine A2A receptors on myocardial ischemia-reperfusion (I/R) injury in diabetic rats and the relationship with autophagy. Methods Clean-grade healthy male Sprague-Dawley rats, aged 6 weeks, weighing 200-250 g, were studied.The diabetic rat model was established by intraperitoneal injection of 1% streptozotocin 60 mg/kg.Forty diabetic rats were divided into 4 groups (n=10 each) using a random number table method: sham operation group (group Sham), I/R group (group I/R), I/R plus adenosine A2A receptor agonist CGS21680 group (group CGS), and I/R plus CGS21680 plus adenosine A2A receptor antagonist ZM241385 group (group CGS+ ZM). Myocardial I/R was produced by occlusion of the left anterior descending branch of the coronary artery for 30 min followed by 120-min reperfusion.Adenosine A2A receptor agonist CGS21680 10 μg/100g was intravenously injected at 10 min before reperfusion in group CGS.CGS21680 10 ug/100g and ZM241385 0.2 mg/kg were intravenously injected sequentially at 10 min before reperfusion in group CGS+ ZM.Blood samples were obtained at the end of reperfusion for determination of concentrations of creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH) and cardiac troponin I (cTnI) in serum (by enzyme-linked immunosorbent assay). The animals were sacrificed, and myocardial tissues were obtained for measurement of the percentage of myocardial infarct volume (by TTC staining) and for determination of the expression of microtubule-associated protein 1 light chain 3 Ⅰ(LC3Ⅰ), LC3 Ⅱ, p62 and Beclin-1 (by Western blot). LC3 Ⅱ/LC3 Ⅰ ratio was calculated. Results Compared with group Sham, the serum CK-MB, LDH and cTnI concentrations and percentage of myocardial infarct volume were significantly increased, the expression of p62 and Beclin-1 was up-regulated, and the LC3 Ⅱ/LC3 Ⅰ ratio was increased in group I/R (P<0.05). Compared with group I/R, the concentrations of serum CK-MB, LDH and cTnI and percentage of myocardial infarct volume were significantly decreased, the expression of p62 and Beclin-1 was down-regulated, and the ratio of LC3Ⅱ/LC3Ⅰ was increased in group CGS (P<0.05), and no significant change was found in the parameters mentioned above in group CGS+ ZM (P>0.05). Compared with group CGS, the concentrations of serum CK-MB, LDH and cTnI and percentage of myocardial infarct volume were significantly increased, the expression of p62 and Beclin-1 was down-regulated, and the ratio of LC3Ⅱ/LC3Ⅰ was decreased in group CGS+ ZM (P<0.05). Conclusion Activating adenosine A2A receptors can mitigate myocardial I/R injury, and the mechanism may be related to enhancing autophagy in diabetic rats.