摘要
目的观察自噬在小剂量衣霉素(TM)诱导内质网应激(ERS)预处理减轻缺血再灌注心肌损伤中的作用。方法使用数字表法将50只成年雄性SD大鼠随机分为5组:假手术组(Sham组)、缺血再灌注组(I/R组)、I/R+衣霉素处理组(TM组,模型建立前30min腹腔注射TM)、I/R+TM处理+3-甲基腺嘌呤(3-MA)处理1组(3-MA-1组,模型建立前30min腹腔注射TM、静脉注射自噬抑制剂3-MA);I/R+衣霉素处理+3-MA处理2组(3-MA-2组,模型建立前30min腹腔注射TM、再灌注时静脉注射3-MA);各组TM按0.6mg/kg体重、3-MA按1mg/kg体重给药。使用结扎左冠状动脉前降支并再灌注的方法建立I/R模型。在开胸后、再灌注2h时通过颈动脉取血,比色法检测血清肌酸激酶同工酶(CK-MB)和乳酸脱氢酶(LDH)活性;再灌注2h后处死动物摘出心脏,原位缺口末端标记法(TUNEL)法观察心肌细胞凋亡情况,蛋白质印迹法(Western blot)检测自噬蛋白Beclin-1和微管相关蛋白1轻链3(LC3)-Ⅱ/LC3-Ⅰ表达。使用方差分析比较各组CK-MB、LDH、细胞凋亡及自噬蛋白的表达。结果CK-MB、LDH活性在再灌注2h时,TM组较I/R组低,差异有统计学意义(t=2.672、2.834,P<0.05);3-MA-1组与I/R组比较差异无统计学意义(t=1.232、1.275,P>0.05)。Sham、I/R、TM、3-MA-1和3-MA-2组细胞凋亡指数依次为(2.5±0.3)%、(27.9±3.1)%、(13.4±1.6)%、(26.4±2.9)%和(9.4±0.8)%。TM、3-MA-2组较I/R组低,差异有统计学意义(t=3.695、3.587,P<0.05);3-MA-1组与I/R组比较,差异无统计学意义(t=0.673,P>0.05)。再灌注2h时I/R、TM、3-MA-1和3-MA-2组Beclin-1、LC3-Ⅱ/LC3-Ⅰ与Sham组比较均增高,差异有统计学意义(Beclin-1比较:t=4.047、3.773、3.738、2.542,P<0.05,LC3-Ⅱ/LC3-Ⅰ比较:t=3.447、3.363、3.169、2.527,P<0.05),TM、3-MA-1、3-MA-2组均低于I/R组,差异有统计学意义(Beclin-1比较:t=2.671、3.150、3.357,P<0.05;LC3-Ⅱ/LC3-Ⅰ比较:t=2.479、3.263、3.351,P<0.05)。结论心肌缺血再灌注损伤过程中自噬起重要作用,小剂量衣霉素诱导ERS预处理可在缺血期引起适度的细胞自噬,有助于维持心肌细胞的内稳态,产生一定程度的心肌保护效果。
Objective To investigate the role of autophagy in reducing myocardial ischemia reperfusion (I/R) injury by endoplasmic reticulum stress preconditioning induced by low dose of tunicamycin (TM). Methods Fifty male SD rats were randomly divided into five groups: sham group;I/R group;TM group (the rats received TM treatment 30 minutes before LAD ligation);3-MA-1 group (the rats received intraperitoneal injection of TM and vein injection of autophagy inhibition 30 minutes before LAD ligation);3-MA-2 group (the rats received intraperitoneal injection of TM and intravenous injection of 3-MA before reperfusion). I/R model was used ligation of left anterior descending coronary artery and reperfusion 2 h. Creatine kinase isozyme (CK-MB) and lactate dehydrogenase (LDH) activities were measured by colorimetry after thoracotomy and 2 hours after reperfusion. Cardiac apoptosis was observed by TdT-mediated dUTP nick end labeling (TUNEL) and the expression of autophagy protein Beclin-1 and LC3-Ⅱ/LC3-Ⅰ was detected by Western blotting. Results CK-MB and LDH activity at 2 hours after reperfusion were lower in TM group than in I/R group (t=2.672, 2.834, P<0.05), and there was no significant difference between 3-MA-1 group and I/R group (t=1.232, 1.275, P>0.05). The apoptotic index of Sham group, I/R group, TM group, 3-MA-1 group and 3-MA-2 group were (2.5±0.3)%,(27.9 ±3.1)%,(13.4±1.6)%,(26.4±2.9)% and (9.4±0.8)% respectively. TM group and 3-MA-2 group were lower than I/R group (t=3.695, 3.587, P<0.05);there was no significant difference between 3-MA-1 group and I/R group (t=0.673, P>0.05). At 2 hours after reperfusion, Beclin-1, LC3-Ⅱ/LC3-Ⅰ in I/R group, TM group, 3-MA-1 group and 3-MA-2 group were higher than those in Sham group (t=4.047, 3.773, 3.738, 2.542, P<0.05;t=3.447, 3.363, 3.169, 2.527, P<0.05), while those in TM group, 3-MA-1 group and 3-MA-2 group were lower than those in I/R group (t=2.671, 3.150, 3.357, P<0.05;LC3-Ⅱ/LC3-Ⅰ比较: t=2.479, 3.263, 3.351, P<0.05). Conclusion Autophagy plays an important role in the process of myocardial ischemia reperfusion injury. ERS preconditioning induced by low dose of TM can induce moderate autophagy during ischemia, help maintain the homeostasis of myocardial cells, and produce a certain degree of myocardial protection.
作者
高鲁方
周涛
杨列红
冯超
李遂宁
向道康
厉忠逵
Gao Lufang;Zhou Tao;Yang Liehong;Feng Chao;Li Suining;Xiang Daokang;Li Zhongkui(Department of Cardiac Surgery,Guizhou Provincial People’s Hospital,People’s Hospital of Guizhou University,Guiyang 550002,China)
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2019年第6期1076-1079,共4页
Chinese Journal of Experimental Surgery
基金
贵州省基础研究计划(黔科合基础[2019]1204号)
贵州省卫生计生委科学技术基金项目(gzwjkj2017-1-028)
贵州省人民医院青年基金项目(GZSYQN[2016]01号).
关键词
心肌缺血再灌注损伤
内质网应激
预处理
衣霉素
自噬
Myocardial ischemia reperfusion injury
Endoplasmic reticulum stress
Preconditioning
Tunicamycin
Autophagy