内质网应激在大鼠心肌缺血再灌注损伤中的作用:与细胞自噬的关系

Role of endoplasmic reticulum stress in myocardial ischemia-reperfusion injury in rats:relationship with autophagy

目的评价内质网应激在大鼠心肌缺血再灌注损伤中的作用及其与细胞自噬的关系。方法清洁级健康成年雄性SD大鼠36只,体重250~300 g,采用随机数字表法分为3组(n=12):假手术组(Sham组)、心肌缺血再灌注组(IR组)和内质网应激抑制剂4-PBA组(PBA组)。采用阻断冠状动脉左前降支(LAD)缺血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。Sham组仅行开胸处理但不阻断LAD;PBA组于造模前3 d连续灌胃给予内质网应激抑制剂4-PBA 500 mg·kg^(-1)·d^(-1),Sham组和IR组给予等量生理盐水。于再灌注120 min时采集髂静脉血样,采用ELISA法测定血浆CK-MB和cTnI浓度;随后处死大鼠取心肌组织,采用Western blot法检测心肌葡萄糖调节蛋白78(GRP78)、微管相关蛋白轻链3Ⅱ(LC3Ⅱ)和自噬相关蛋白5(ATG5)的表达。结果与Sham组比较,IR组和PBA组血浆CK-MB和cTnI浓度升高,IR组心肌组织GRP78、ATG5和LC3Ⅱ表达上调(P<0.05),病理学损伤加重;与IR组比较,PBA组血浆CK-MB和cTnI浓度降低,心肌组织GRP78、ATG5和LC3Ⅱ表达下调(P<0.05),病理学损伤减轻。结论内质网应激参与了大鼠心肌缺血再灌注损伤的过程,其机制可能与促进细胞自噬有关。

Objective To evaluate the role of endoplasmic reticulum stress in myocardial ischemia-reperfusion(I/R)injury and the relationship with autophagy in rats.Methods Thirty-six healthy adult male Sprague-Dawley rats,weighing 250-300 g,were divided into 3 groups(n=12 each)using a random number table method:sham operation group(Sham group),myocardial I/R group(IR group),and endoplasmic reticulum stress inhibitor 4-PBA group(PBA group).Myocardial I/R was produced by occlusion of left anterior descending branch of coronary artery for 30 min followed by reperfusion for 120 min.Sham group only underwent thoracotomy without block of left anterior descending branch of coronary artery.Endoplasmic reticulum stress inhibitor 4-PBA 500 mg·kg^(-1)·d^(-1) was given intragastrically for 3 consecutive days before the I/R model was developed in PBA group,while the equal volume of normal saline was given instead in Sham and IR groups.The blood samples from the iliac vein were collected at 120 min of reperfusion for determination of the plasma creatine kinase isoenzymes(CK-MB)and cardiac troponin I(cTnI)concentrations(by enzyme-linked immunosorbent assay).The rats were then sacrificed,and myocardial tissues were removed for detection of myocardial glucose-regulated protein 78(GRP78)and microtubule-associated protein 1 light chain 3Ⅱ(LC3Ⅱ)and autophagy-related protein 5(ATG5)expression(by Western blot).Result Compared with Sham group,the concentrations of CK-MB and cTnI in plasma were significantly increased in IR and PBA groups,the expression of GRP78,ATG5 and LC3Ⅱ was up-regulated,and the pathological damage was aggravated in IR group(P<0.05).Compared with IR group,the concentrations of CK-MB and cTnI in plasma were significantly decreased,the expression of GRP78,ATG5 and LC3Ⅱ was down-regulated(P<0.05),and the pathological changes were significantly attenuated in PBA group.Conclusion Endoplasmic reticulum stress is involved in the process of myocardial I/R injury,and the mechanism may be related to promotion of autophagy in rats.