酪醇对糖尿病大鼠心肌缺血再灌注损伤的影响及SIRT1/AMPK/eNOS信号通路在其中的作用

Effect of tyrosol on myocardial ischemia-reperfusion injury in diabetic rats and the role of SIRT1/AMPK/eNOS signaling pathway

目的探讨酪醇对糖尿病大鼠心肌缺血再灌注损伤的影响及沉默调节蛋白1(SIRT1)/腺苷酸活化蛋白激酶(AMPK)/内皮型一氧化氮合酶(eNOS)信号通路在其中的作用。方法SPF级健康成年雄性SD大鼠,体重200~220 g,采用腹腔注射1%链脲佐菌素60 mg/kg制备糖尿病模型。取糖尿病模型制备成功的大鼠56只,采用随机数字表法分为4组(n=14):假手术组(S组)、心肌缺血再灌注组(I/R组)、心肌缺血再灌注+酪醇组(I/R+T组)和心肌缺血再灌注+酪醇+SIRT1抑制剂EX527组(I/R+T+E组)。I/R+T组和I/R+T+E组灌胃法连续45 d给予酪醇20 mg·kg^-1·d^-1,其他2组给予等体积生理盐水。I/R+T+E组于缺血前连续3 d腹腔注射EX527 5 mg·kg^-1·d^-1,并于再灌注前20 min时腹腔注射EX527 5 mg/kg。采用结扎左冠状动脉前降支30 min,再灌注2 h的方法制备心肌缺血再灌注损伤模型。采用TTC法测量心肌梗死体积,ELISA法检测血清CK-MB、LDH、15-F2t-isoprostane和心肌组织SOD水平,Western blot法检测心肌组织SIRT1、AMPK、磷酸化AMPK(p-AMPK)、eNOS和磷酸化eNOS(p-eNOS)的表达水平。结果与S组比较,I/R组血清CK-MB、LDH和15-F2t-Isoprostane的水平升高,心肌梗死体积增加,心肌组织SOD活性和SIRT1表达水平降低,I/R+T组和I/R+T+E组血清CK-MB、LDH和15-F2t-Isoprostane的水平升高,心肌梗死体积增加,心肌组织SOD活性和SIRT1表达水平降低,p-AMPK和p-eNOS表达上调(P<0.05);与I/R组比较,I/R+T组血清CK-MB、LDH和15-F2t-Isoprostane的水平降低,心肌梗死体积减少,心肌组织SOD活性和SIRT1、p-AMPK、p-eNOS的表达水平升高(P<0.05),I/R+T+E组上述指标差异无统计学意义(P>0.05);与I/R+T组比较,I/R+T+E组血清CK-MB、LDH和15-F2t-Isoprostane的水平升高,心肌梗死体积增加,心肌组织SOD活性和SIRT1、p-AMPK、p-eNOS表达水平降低(P<0.05)。结论酪醇可减轻糖尿病大鼠心肌缺血再灌注损伤,其机制与激活SIRT1/AMPK/eNOS信号通路,抑制氧化应激反应有关。

Objective To evaluate the effect of tyrosol on myocardial ischemia-reperfusion(I/R)injury in diabetic rats and the role of silent mating-type information regulation 1(SIRT1)/adenosine monophosphate-activated protein kinase(AMPK)/endothelial nitric oxide synthase(eNOS)signaling pathway.Methods SPF healthy adult male Sprague-Dawley rats,weighing 200-220 g,were intraperitoneally injected with streptozotocin 60 mg/kg to establish the model of diabetes mellitus.Fifty-six diabetic rats were divided into 4 groups(n=14 each)using a random number table method:sham operation group(S group),myocardial I/R group(I/R group),myocardial I/R plus tyrosol group(I/R+T group),and myocardial I/R plus tyrosol plus SIRT1 inhibitor EX527 group(I/R+T+E group).In I/R+T and I/R+T+E groups,tyrosol 20 mg·kg^-1·d^-1 was given by gavage for 45 consecutive days,and the equal volume of normal saline was given in the other two groups.In I/R+T+E group,EX527 5 mg·kg^-1·d^-1 was intraperitoneally injected for 3 consecutive days before ischemia,and EX527 5 mg/kg was intraperitoneally injected at 20 min before reperfusion.Myocardial I/R was induced by ligation of the left anterior descending branch of coronary artery for 30 min followed by 2-h reperfusion.The myocardial infarct volume was measured by TTC staining.The levels of creatine kinase-MB(CK-MB),lactic dehydrogenase(LDH)and 5-F2t-isoprostane in serum and superoxide dismutase(SOD)in myocardial tissues were detected by enzyme-linked immunosorbent assay.The expression of SIRT1,AMPK,phosphorylated AMPK(p-AMPK),eNOS and p-eNOS was detected by Western blot.Results Compared with S group,the levels of serum CK-MB,LDH and 15-F2t-Isoprostane and myocardial infarction volume were significantly increased,the SOD activity was decreased,and the SIRT1 expression was down-regulated in I/R group,and the levels of serum CK-MB,LDH and 15-F2t-Isoprostane and myocardial infarction volume were significantly increased,the SOD activity was decreased,the SIRT1 expression was down-regulated,and the expression of p-AMPK and p-eNOS was up-regulated in I/R+T and I/R+T+E groups(P<0.05).Compared with I/R group,the levels of serum CK-MB,LDH and 15-F2t-Isoprostane and myocardial infarction volume were significantly decreased,the SOD activity was increased,and the expression of SIRT1,p-AMPK and p-eNOS was up-regulated in I/R+T group(P<0.05),and no significant change was found in the parameters mentioned above in I/R+T+E group(P>0.05).Compared with I/R+T group,the levels of CK-MB,LDH and 15-F2t-isoprostane in serum and myocardial infarct volume were significantly increased,the SOD activity was increased,and the expression of SIRT1,p-AMPK and p-eNOS was down-regulated in I/R+T+E group(P<0.05).Conclusion Tyrosol can mitigate myocardial I/R injury,and the mechanism may be related to activating SIRT1/AMPK/eNOS signaling pathway and inhibiting oxidative stress response in diabetic rats.