自噬与糖尿病因素影响缺血预处理对大鼠心肌保护作用的关系

Relationship between autophagy and diabetes mellitus-caused influence on ischemic preconditioninginduced cardioprotection in rats

目的评价自噬与糖尿病因素影响缺血预处理对大鼠心肌保护作用的关系。方法清洁级健康雄性SD大鼠,12周龄,体重290~320g,在高脂高糖饮食喂养1周后,腹腔注射链脲佐菌素50mg/kg,连续2d,制备糖尿病模型。取糖尿病模型制备成功的大鼠30只,体重350-450g,采用随机数字表法分为3组(n=10):假手术组(DM-S组)、心肌缺血再灌注组(DM-IR组)和缺血预处理组(DM-IP组)。另取非糖尿病大鼠30只,采用随机数字表法分为3组(n=10):假手术组(S组)、心肌缺血再灌注组(IR组)和缺血预处理组(IP组)。采用结扎左冠状动脉前降支30min,再灌注120min的方法制备心肌缺血再灌注损伤模型。IP组和DM—IP组缺血5min,再灌注5min,共3个循环,行缺血预处理,然后制备心肌缺血再灌注损伤模型。再灌注120min时,经颈内静脉采集血样,采用ELISA检测血清cTnI和CK-MB的浓度,然后处死大鼠,取心肌组织,测定心肌梗死体积,采用Westem blot法检测微管相关蛋白1轻链3(LC3)Ⅱ、Beclin-1、磷脂酰肌醇-3-激酶(PI3K)、蛋白激酶B(Akt)、磷酸化Akt(p-Akt)和哺乳动物雷帕霉素靶蛋白(mTOR)的表达水平,计算p-Akt/Akt比值。结果与S组比较,IR组血清cTnI和cK—MB的浓度升高,心肌梗死体积百分比升高,心肌组织LC3Ⅱ和Beclin-1表达上调,PI3K和mTOR表达下调,p-Akt/Akt比值降低(P<0.05)。与IR组比较,IP组血清cTnI和CK—MB的浓度降低,心肌梗死体积百分比降低,心肌组织LC3Ⅱ和Beclin-1表达下调,PI3K和InTOR表达上调,p-Akt/Akt比值升高(P<0.05)。与DM—S组比较,DM-IR组血清cTnI和CK—MB的浓度升高,心肌梗死体积百分比升高,心肌组织LC3 Ⅱ和Beclin-1表达上调,PI3K和mTOR表达下调,p-Akt/Akt比值降低(P<0.05)。与DM-IR组比较,DM—IP组上述各指标差异无统计学意义(P>0.05)。结论糖尿病因素取消缺血预处理对大鼠心肌保护作用的机制可能与抑制PI3K—Akt—mTOR信号通路激活,导致自噬增强有关。

Objective To evaluate the relationship between autophagy and diabetes mellitus-caused influence on ischemic preconditioning (IP)-induced cardioproteetion in rats.Methods Clean-grade healthy male Sprague-Dawley rats,aged 12 weeks,weighing 290-320g,were used in this study.Diabetes mellitus was induced by high-fat and high-sucrose diet (lasting for 1 week)and intraperitoneal streptozotocin 50mg/kg (for 2 consecutive days)and confirmed by fasting blood glucose level≥16.65mmol/L (for 1 week).Thirty rats with diabetes mellitns,weighing 350-450g,were divided into 3 groups (n =10 each)using a random number table method:sham operation group (DM-S group),myocardial ischemiareperfusion (I/R)group (DM-IR group)and IP group (DM-IP group).Another 30 non-diabetic rats were selected and divided into 3groups (n =I0each)using a random number table method:sham operation group (S group),myocardial I/R group (IR group)and IP group.Myocardial ischemia was induced by ligation of the anterior descending branch of left coronary artery for 30 min followed by 120 min reperfusion. IP was produced by 3 cycles of 5-min ischemia followed by 5-min reperfusion prior to establishment of myocardial I/R injury model in IP and DM-IP groups.Blood samples were collected from the internal jugular vein at the end of reperfusion for measuring serum concentrations of cardiac troponin I (cTnI)and creatine kinase-MB (CK-MB).The rats were then sacrificed and myocardial tissues were obtained for determination of myocardial infarct size and expression of microtubule-associated protein 1 light chain 3 Ⅱ(LC3Ⅱ), Beclin-1,phosphatidyl-inositol 3-kinase (PI3K),protein kinase B (Akt),phosphorylated Akt (p-Akt) and mammalian target of rapamycin (roTOR)(by Western blot),p-Akt/Akt ratio was calculated.Results Compared with S group,the serum cTnI and CK-MB concentrations were significantly increased,the percentage of myocardial infarct size was increased,the expression of LC3]I and Beclin-1in myocardial tissues was up-regulated,the expression of PI3K and roTOR was down-regulated,and p-Akt/Akt ratio was decreased in IR group (P<0.05).Compared with IR group,the serum cTnI and CK-MB concentrations were significantly decreased,the percentage of myocardial infarct size was decreased,the expression of LC3 Ⅱ and Beclin-1 in myocardial tissues was down-regulated,the expression of PI3K and roTOR was upregulated,and p-Akt/Akt ratio was increased in IP group (P<0.05).Compared with DM-S group,the serum cTnI and CK-MB concentrations were significantly increased,the percentage of myocardial infarct size was increased,the expression of LC3Ⅱ and Beclin-1in myocardial tissues was up-regulated,the expression of PI3K and mTOR was down-regulated,and p-Akt/Akt ratio was decreased in DM-IR group (P< 0.05).There was no significant difference in the parameters mentioned above between DM-IP group and DM-IR group (P>0.05).Conclusion The mechanism by which diabetes mellitus abolishes IP-induced cardioproteetion may be related to inhibiting activation of PI3K-Akt-mTOR signaling pathway and enhanced autophagy in rats.