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ORIGINAL ARTICLE
Year : 2018  |  Volume : 131  |  Issue : 14  |  Page : 1702-1709

Effect of Autophagy Inhibition on the Protection of Ischemia Preconditioning against Myocardial Ischemia/Reperfusion Injury in Diabetic Rats


Department of Anesthesiology, Plastic Surgery Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100041, China

Correspondence Address:
Prof. Fu-Shan Xue
Department of Anesthesiology, Plastic Surgery Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100041
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0366-6999.235867

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Background: Ischemia preconditioning (IPC) remains the most powerful intervention of protection against myocardial ischemia/reperfusion injury (IRI), but diabetes can weaken or eliminate its cardioprotective effect and detailed mechanisms remain unclear. In this study, we aimed to explore whether changes of autophagy in the diabetic condition are attributable to the decreased cardioprotective effect of IPC. Methods: Sixty diabetic male Sprague-Dawley rats were randomly divided into the control (C), IRI, rapamycin (R), wortmannin (W), rapamycin + IPC (R + IPC), and wortmannin + IPC (W + IPC) groups. The in vivo rat model of myocardial IRI was established by ligaturing and opening the left anterior descending coronary artery via the left thoracotomy. Durations of ischemia and reperfusion are 30 min and 120 min, respectively. Blood samples were taken at 120 min of reperfusion for measuring serum concentrations of troponin I (TnI) and creatine kinase isoenzyme MB (CK-MB) using the enzyme-linked immunosorbent assay. The infarct size was assessed by Evans blue and triphenyltetrazolium chloride staining. The expressions of LC3-II, beclin-1, phosphoinositide 3-kinase (PI3K), mammalian target of rapamycin (mTOR), and P-Akt/Akt ratio in the ischemic myocardium were assessed by Western blotting. Results: Compared to the IRI group, infarct size (56.1% ± 6.1% vs. 75.4 ± 7.1%, P < 0.05), serum cTnI (0.61 ± 0.21 vs. 0.95 ± 0.26 ng/ml, P < 0.05), and CK-MB levels (6.70 ± 1.25 vs. 11.51 ± 2.35 ng/ml, P < 0.05) obviously decreased in the W + IPC group. Compared with the C group, myocardial expressions of LC3-II (0.46 ± 0.04 and 0.56 ± 0.04 vs. 0.36 ± 0.04, P < 0.05) and beclin-1 (0.34 ± 0.08 and 0.38 ± 0.07 vs. 0.24 ± 0.03, P < 0.05) evidently increased, and myocardial expressions of mTOR (0.26 ± 0.08 and 0.25 ± 0.07 vs. 0.38 ± 0.06, P < 0.05), PI3K (0.29 ± 0.04 and 0.30 ± 0.03 vs. 0.38 ± 0.02, P < 0.05), and P-Akt/Akt ratio (0.49 ± 0.10 and 0.48 ± 0.06 vs. 0.72 ± 0.07, P < 0.05) markedly decreased in the IRI and R groups, indicating an increased autophagy. Compared with the IRI group, myocardial expression of beclin-1 (0.26 ± 0.03 vs. 0.34 ± 0.08, P < 0.05) significantly decreased, and myocardial expressions of mTOR (0.36 ± 0.04 vs. 0.26 ± 0.08, P < 0.05), PI3K (0.37 ± 0.03 vs. 0.29 ± 0.04, P < 0.05), and P-Akt/Akt ratio (0.68 ± 0.05 vs. 0.49 ± 0.10, P < 0.05) increased obviously in the W + IPC group, indicating a decreased autophagy. Conclusions: Increased autophagy in the diabetic myocardium is attributable to decreased cardioprotection of IPC, and autophagy inhibited by activating the PI3K-Akt-mTOR signaling pathway can result in an improved protection of IPC against diabetic myocardial IRI.

 

 Abstract in Chinese

自噬抑制对缺血预处理保护糖尿病大鼠心肌缺血/再灌注损伤的影响

摘要

背景:缺血预处理(Ischemic preconditioning, IPC)仍然是心肌缺血/再灌注损伤(Ischemia/reperfusion injury,IRI)最强效的保护干预措施,但糖尿病可减弱甚至消除其心肌保护作用,并且具体机制尚不明确。本研究旨在探讨糖尿病条件下自噬改变是否参与了降低的IPC心肌保护作用。

方法:将60只雄性糖尿病Sprague-Dawley大鼠随机分为对照组(C组)、IRI组、雷帕霉素组(R组)、渥曼青霉素组(W组)、雷帕霉素+IPC组(R+IPC组)和渥曼青霉素+IPC组(W+IPC组)。通过左侧开胸结扎和开放左冠状动脉前降支建立大鼠在体心肌IRI模型,心肌缺血和再灌注的时间分别是30分钟和120分钟。再灌注120 min时采集血样,采用酶联免疫吸附法(enzyme-linked immunosorbent essay, ELISA)检测血清心肌肌钙蛋白I(cardiac troponin I,cTnI)和肌酸激酶同工酶MB(creatine kinase isoenzyme MB,CK-MB);采用伊文氏蓝和2,3,5-三苯基氯化四氮唑双染色法测定心肌梗死面积;采用Western-blotting检测LC3-Ⅱ、Beclin-1、mTOR、PI3K心肌表达情况和P-Akt/Akt比率。

结果:与IRI组相比,W+IPC组的心肌梗死面积(56.1%±6.1%比75.4±7.1%,P <0.05),血清cTnI浓度(0.61±0.21比0.95±0.26 ng/ml,P <0.05)、血清CK-MB浓度(6.70±1.25比11.51±2.35 ng/ml,P <0.05)均明显降低;与C组相比,IRI和R组LC3-Ⅱ(0.46±0.04和0.56±0.04比0.36±0.04,P <0.05)和beclin-1(0.34±0.08和0.38±0.07比0.24±0.03,P <0.05)心肌表达明显增强,mTOR(0.26±0.08和0.25 ± 0.07 比 0.38 ± 0.06, P < 0.05)和PI3K(0.29±0.04和0.30±0.03比0.38±0.02,P <0.05)心肌表达以及P-Akt/Akt比率(0.49±0.10和0.48 ± 0.06比0.72 ± 0.07, P < 0.05)明显降低,表明自噬表达增强。与IRI组相比,W+IPC组beclin-1(0.26±0.03比0.34±0.08,P <0.05)心肌表达明显降低,mTOR(0.36±0.04比0.26±0.08,P <0.05)和PI3K(0.37±0.03比0.29±0.04,P <0.05)心肌表达以及P-Akt/Akt比率(0.68±0.05比0.49±0.10,P <0.05)明显增加,表明自噬表达降低。

结论糖尿病心肌自噬表达增强可减弱IPC的心肌保护作用,激活PI3K-Akt-mTOR信号转导通路抑制自噬可改善IPC对糖尿病心肌IRI的保护作用。



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