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,�
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 r
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
