Effect of Autophagy Inhibition on the Protection of Ischemia Preconditioning against Myocardial Ischemia/Reperfusion Injury in Diabetic Rats.
Effect of Autophagy Inhibition on the Protection of Ischemia Preconditioning against Myocardial Ischemia/Reperfusion Injury in Diabetic Rats.
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DOI：
10.4103/0366-6999.235867
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发表时间：
2018-07-20
影响因子：
6.1
通讯作者：
Liao X
中科院分区：
文献类型：
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作者：
Liu YY;Sun C;Xue FS;Yang GZ;Li HX;Liu Q;Liao X
缺血预适应(IPC)仍然是对抗心肌缺血/再灌注损伤(IRI)最有效的干预措施，但糖尿病可削弱或消除其心脏保护作用，其具体机制尚不清楚。在这项研究中，我们旨在探索糖尿病状态下自噬的变化是否与IPC的心脏保护作用降低有关。60只糖尿病雄性SD大鼠随机分为对照组(C)、缺血再灌注组(IRI)、雷帕霉素(R)、Wortmannin(W)、雷帕霉素+IPC(R+IPC)和Wortmannin+IPC(W+IPC)组。采用左开胸结扎开放冠状动脉左前降支建立在体大鼠心肌缺血再灌注损伤模型。缺血和再灌流时间分别为30min和120min。于再灌注120min采血，用双抗体夹心法测定血清肌钙蛋白I(TnI)和肌酸激酶同工酶MB(CK-MB)浓度。伊文思蓝和氯化三苯基四氮唑染色评价心肌梗死面积。Western blotting检测缺血心肌中Lc3-II、Beclin-1、磷脂酰肌醇3-激酶(PI3K)、哺乳动物雷帕霉素靶标(MTOR)、P-Akt/Akt比值的变化。与IRI组比较，W+IPC组心肌梗死面积(56.1%±6.1%比75.4±7.1%，P<0.05)、血清cTnI(0.61±0.21比0.95±0.26 ng/ml，P<0.05)和CK-MB水平(6.70±1.25比11.51±2.35 ng/ml，P<0.05)明显降低。与C组相比，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)的表达明显增加，mTOR(0.26±0.08和0.25±0.07比0.38±0.06，P<；0.05)的表达明显增加；P-Akt/Akt比值(0.49±0.10和0.48±0.06比0.72±0.07，P<；0.05)，提示自噬增加。与IRI组比较，心肌Beclin-1表达(0.26±0.03比0.34±0.08，P<；0.05)显著降低，mTOR(0.36±0.04比0.26±0.08，P<；0.05)，PI3K(0.37±0.03比0.29±0.04，P<；0.05)，P-Akt/Akt比值(0.68±0.05比0.49±0.10，P<；0.05)显著降低。0.05)在W+IPC组明显增加，表明自噬减少。糖尿病心肌自噬增加的原因是IPC对心脏的保护作用减弱，通过激活PI3K-Akt-mTOR信号通路抑制自噬可以增强IPC对糖尿病心肌IRI的保护作用。
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. 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. 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. 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.