Objective To investigate the effects of adenosine monophosphate activated protein kinase(AMPK) regulated autophagy on restoring the sensitivity of diabetic myocardium to ischemic postconditioning(IPO). Methods Healthy male Sprague-Dawley rats(weighed 220-280 g) were used in this study. Thirty healthy rats were randomly divided into:sham group (N+S group), ischemia reperfusion group (N+IR group) and ischemia postconditionig group(N+IPO group);type 1 diabetic rat models were induced by a single intraperitoneal injection of streptozotocin at the dose of 60 mg/kg and confirmed by blood glucose concentration≥16.7 mmol/L;all rats were raised for 8 weeks. Fifty-four diabetic rats were randomly assigned into diabetic sham group (D + S group), diabetic ischemia group(D + IR group), diabetic ischemia postconditionig group(D+IPO group), diabetic ischemia+AMPK activtor A-769662 group(D+IR+A group), diabetic ischemia postconditionig+A-769662 group(D+IPO+A group). There were 6 rats in N+S and D+S group and 12 rats each in the other groups. Myocardial ischemia reperfusion model was completed by occlusion of the anterior descending branch of left coronary artery for 30 min followed by 120 min of reperfusion. IPO was induced by 3 cycles of 10 second of reperfusion and ischemia at the onset of reperfusion. AMPK agonist A-769662(6 mg/kg) was given as intraperitoneal injection 30 min before ischemia. Myocardium infarct size (IS) was detected by triphenyltetrazolium chloride staining. Transmission electron microscopeserum was used to observe autophagosomes. Arterial blood was collected to detect creatine kinase-MB(CK-MB) and lactate dehydrogenase(LDH) release. The phosphorylation of AMPK (p-AMPK) and mammalian target of rapamycin (p-mTOR), the ratio of microtubule-associated protein 1 light chain 3B/A(LC3B/A) and ubiquitin binding protein p62 in heart tissues were examined by Western blotting. The t test was adopted to detect the differences between two groups, one-way analysis of variance was used to analyze the differences between groups. Results Diabetic rats characterized with lower body weight, p-AMPK expression, autophagosomes number and LC3B/A ratio and higher CK-MB, LDH, p-mTOR, p62 and blood glucose when compared with nondiabetic rats(t=4.15-34.65, all P<0.05). Compared with N+IR group, the level of IS, CK-MB, LDH in D+IR group increased significantly(t=3.55, 4.70, 7.20, all P<0.05). Compared with N+S group, p-AMPK level, autophagosomes number and LC3B/A ratio significantly increased, p-mTOR and p62 expressions remarkably deceased in N+IR group(t=6.56, 5.51, 3.76, 3.59, 2.98, all P<0.05);while those changes were not found between D+S and D+IR group (all P>0.05). Compared with D+IR group, the IS(28%±5%vs 45%±6%), CK-MB((1 047±178) vs (2 050±325) U/L), LDH((1 303±365) vs (2 558±189) U/L), p-mTOR(0.48±0.24 vs 1.29±0.25), p62(0.74±0.12 vs 1.04±0.24) decreased significantly, and the p-AMPK level(1.81±0.19 vs 0.61±0.15), autophagosomes number(9.3±1.4 vs 3.8±0.9) and LC3B/A ratio (1.43 ± 0.23 vs 0.65 ± 0.13) increased significantly in D+IPO+A group (t=5.26-11.82, all P<0.05). Conclusion AMPK/mTOR activation by A-769662 can restore the sensitivity of diabetic hearts to IPO-induced cardioprotection through autophagy promotion.%目的：评价一磷酸腺苷活化蛋白激酶(AMPK)调控的自噬在恢复糖尿病缺血再灌注损伤心肌对缺血后处理（IPO）敏感性中的作用。方法取健康雄性SD大鼠(体重220～250 g)30只，随机数字表法分为假手术组(N+S组)、缺血再灌注组(N+IR组)、缺血后处理组(N+IPO组)；糖尿病大鼠模型采用腹腔注射1%链脲佐菌素60 mg/kg制备，取糖尿病诱导成功大鼠54只，随机数字表法分为糖尿病假手术组(D+S组)、糖尿病缺血再灌组(D+IR组)、糖尿病缺血后处理组(D+IPO)、AMPK激动剂A-769662干预的糖尿病缺血再灌注组(D+IR+A组)和A-769662干预的糖尿病缺血后处理组(D+IPO+A组)。其中N+S组与D+S组各6只，其余组各12只。缺血再灌注模型采用结扎左冠状动脉前降支30 min/开放120 min的方法建立；假手术只穿线，不结扎血管；缺血后处理组于再灌注前行3个循环的再灌注10 s/缺血10 s；A-769662于缺血前30 min腹腔注射6 mg/kg。再灌注结束后，氯化三苯基四氮唑染色测定心肌梗死面积(IS)，检测血清肌酸激酶同工酶MB(CK-MB)和乳酸脱氢酶(LDH)水平，电镜观察自噬小体数量，Western blotting法检测心肌组织磷酸化AMPK蛋白(p-AMPK)和哺乳动物雷帕霉素靶蛋白(p-mTOR)的水平，微管相关蛋白轻链3B/A(LC3B/A)的比值，泛素结合蛋白p62的表达。多组间数据比较采用单因素方差分析，两组间比较采用t检验。结果与非糖尿病大鼠相比，糖尿病大鼠体重减轻，血糖升高，心肌p-mTOR和p62明显上调，p-AMPK、自噬体数量和LC3B/A比值显著下降(t=4.15～34.65，均P<0.05)。与N+IR组相比，D+IR组IS、CK-MB、LDH显著增高(t=3.55、4.70、7.20，均P<0.05)；与N+S组相比，N+IR组p-AMPK，自噬体数量和LC3B/A比值显著上升，p-mTOR和p62表达显著下调(t=6.56、5.51、3.76、3.59、2.98，均P<0.05)，D+S组与D+IR组比较上述指标变化无明显差异。与D+IR相比，D+IPO+A组IS(28%±5%比45%±6%)、CK-MB[(1047±178)比(2050±325)U/L]、LDH[(1303±365)比(2558±189)U/L]、p-mTOR(0.48±0.24比1.29±0.25)和p62(0.74±0.12比1.04±0.24)明显下降， p-AMPK(1.81±0.19比0.61±0.15)、自噬体数量[(9.3±1.4)比(3.8±0.9)个]和LC3B/A比值(1.43±0.23比0.65±0.13)显著增加(t=5.26～11.82，均P<0.05)。结论 A-769662通过激活AMPK/mTOR通路增强心肌自噬，从而恢复糖尿病缺血再灌注心肌对IPO的敏感性。
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