AMPK通路在硫化氢后处理减轻2型糖尿病大鼠心肌缺血-再灌注损伤中的作用

Role of AMP-activated protein kinase in hydrogen sulfide postconditioning protecting on type 2 diabetic rats with myocardial ischemia reperfusion injury

目的观察硫化氢后处理对2型糖尿病大鼠心肌缺血-再灌注(ischemia reperfusion,IR)损伤的效果,探讨AMP依赖的蛋白激酶(AMPK)通路在其中发挥的作用。方法雄性SD大鼠84只,1月龄,体重100~150g,采用随机数字表法分为六组:假手术组(Sham组)、IR组、AMPK抑制剂compound c组(CC组)、compound c溶剂组(DMSO组)、硫化氢后处理组(NaHS组)和CC+NaHS组,每组14只。成功建立糖尿病模型后,Sham组仅开胸但不结扎阻断血流;IR组在开胸分离左冠状动脉前降支后用止血钳夹紧圈套管缺血30min,松开圈套管再灌注4h后取心脏;CC组于术前1h腹腔注射CC 250μg/kg,其余操作同IR组;DMSO组于术前1h给予同样剂量的DMSO,其余操作同IR组;NaHS组于开放左冠状动脉1min内快速静脉注射NaHS 0.05mg/kg,再灌注4h;CC+NaHS组:于术前1h腹腔注射CC,随后于冠状动脉左前降支阻断30min,同样于开放左冠状动脉1min内快速静脉注射NaHS 0.05mg/kg,再灌注4h。之后对所有动物进行安乐死,采用TTC染色法测定心肌梗死范围,Western blot法测定AMPK、LC3、p62蛋白含量。结果与Sham组比较,其余五组再灌注4h时大鼠心肌梗死范围明显增加,AMPK、LC3、p62蛋白含量明显升高(P<0.05);与IR组比较,NaHS组大鼠再灌注4h时心肌梗死范围明显减小(P<0.05),AMPK蛋白含量明显升高,LC3、p62蛋白含量明显降低(P<0.05);与NaHS组比较,CC+NaHS组再灌注4h时心肌梗死范围明显增加,AMPK水平明显降低,LC3、p62蛋白表达含量明显升高(P<0.05)。CC组与DMSO组上述各指标差异无统计学意义。结论硫化氢后处理能减轻2型糖尿病大鼠缺血-再灌注损伤后心肌梗死,其机制与调节自噬小体清除及修复AMPK通路调控的自噬流有关。

Objective To investigate the effect of hydrogen sulfide（H2S or NaHS）on myocardial ischemia reperfusion injury induced in type 2diabetic rats in vivo and the role of AMPactivated protein kinase（AMPK）signal pathway.Methods The induced type 2diabetic rat models were anesthetized,left thoracotomy were performed.All the models were randomly divided into six groups（n=14）：group Sham;group IR：the left anterior descending artery was ligated 30 min,reperfused for 4hours;group CC：prior to thoracotomy,compound c was intraperitoneally injected250μg/kg,then received the same treatment as group IR;group DMSO received the same treatment as compound c group but DMSO was injected intraperitoneally as control;group NaHS：the left anterior descending artery was injected NaHS 0.05 mg/kg then reperfused for 4hours;group CC＋NaHS：prior to thoracotomy,compound c was intraperitoneally injected 250μg/kg,then NaHS 0.05mg/kg injected intravenously and reperfused 4hours.All the rat models euthanatized,infarcted area was detected by TTC assay.The AMPK,LC3 and p62were analyzed by Western blot.Results Compared with group Sham,the infarcted area and concentration of AMPK,LC3 and p62were increased in other groups（P〈0.05）.Compared with group IR,the infarcted area and concentration of LC3,p62 markablely decreased in group NaHS（P〈0.05）.Compared with group NaHS,the infarcted area and concentration of LC3,p62 significantly increased but AMPK down-regulated in group CC＋NaHS（P〈0.05）.Conclusion Hydrogen sulfide could alleviate myocardial infraction via AMPK signal pathway in type 2diabetic rats＇ IR models.