舒芬太尼后处理对糖尿病大鼠心肌缺血再灌注损伤的影响及其与PI3K/Akt通路的关系

Effects of sufentanil postconditioning on myocardial ischemia-reperfusion injury in rats with diabetes effect and its correlation with PI3K/Akt pathway mechanism of myocardial cell apoptosis

目的探讨舒芬太尼后处理在糖尿病大鼠心肌缺血再灌注损伤中的作用。方法健康成年雄性SD大鼠,250~300 g,采用腹腔注射链脲佐菌素65 mg/kg,制备糖尿病大鼠模型。取造模成功大鼠24只,采用随机数字表法,将其随机分为4组（n=6）：糖尿病假手术组（DM-S组）、糖尿病缺血再灌注组（DM-IR组）、糖尿病舒芬太尼后处理组（DM-SP组）和糖尿病舒芬太尼后处理＋渥曼青霉素组（DM-SP＋W组）。另取正常大鼠24只,采用随机数字表法,将其随机分为4组（n=6）：非糖尿病假手术组（NDM-S组）、非糖尿病缺血再灌注组（NDM-IR组）、非糖尿病舒芬太尼后处理组（NDM-SP组）和非糖尿病舒芬太尼后处理＋渥曼青霉素组（NDM-SP＋W组）。采用结扎左冠状动脉前降支30 min后再灌注120 min的方法制备心肌缺血再灌注模型。S组只穿线不结扎,SP组于再灌注前5 min经舌下静脉注射舒芬太尼1.0μg/kg,SP＋W组在注射舒芬太尼前注射PI3K抑制剂Wortmannin 15μg/kg。于再灌注120 min经腹主动脉取血,测血清肌酸激酶同工酶（CK-MB）浓度。处死大鼠后,取心脏,确定心肌梗死区与缺血危险区体积的比值（IS/AAR值）,采用蛋白印迹法测定Akt、p-Akt的表达。结果在糖尿病与非糖尿病组,与S比较,IR组,SP组和SP＋W组CK-MB浓度升高,IS/AAR增大,p-Akt表达上调（P〈0.05）。非糖尿病组中,与NDM-IR组比较,NDM-SP组CK-MB浓度降低,IS/AAR减小,p-Akt表达上调（P〈0.05）,而糖尿病组中,DM-SP组与DM-IR组,上述指标差异无统计学意义,且DM-SP组CK-MB浓度和IS/AAR明显高于NDMSP组、p-Akt表达明显低于NDM-SP组。结论糖尿病因素可消除舒芬太尼后处理对心肌缺血再灌注损伤的保护作用,其机制可能与糖尿病状态下影响PI3K/Akt通路有关。

Objective To investigate the effects of Sufentanil postconditioning on myocardial ischemia-reperfusion injury in diabetic rats. Methods Healthy adult male SD rats weighing 250-300 g were included for the preparation of diabetic rat models by intraperitoneal injection of 65mg/kg streptozotocin. Twenty-four successfully established rat models were randomly divided into four groups（n=6 each）： diabetic mellitus-sham operation group（group DM-S）,diabetic mellitus-ischemia reperfusion group（group DM-IR）, diabetic mellitus-Sufentanil postconditioning group（group DM-SP）and diabetic mellitus-Sufentanil postconditioning＋Wortmannin group（group DM-SP＋W）. Another 24 normal rats were also included and randomly divided into 4 groups（n=6 each）： nondiabetic mellitus-sham operation group（group NDM-S）, nondiabetic mellitus-ischemia-reperfusion group（group NDM-IR）; nondiabetic mellitus-sufentanil postconditioning group（group NDM-SP） and nondiabetic mellitus-sufentanil postconditioning ＋Wortmannin group（group NDM-SP＋W）. The myocardial I/R models were prepared by 30 min ligation of left anterior descending coronary artery followed by 120 min reperfusion. Group S underwent the operation with a stitch placed around but not ligating the artery. Group SP received 1.0 μg/kg Sufentanil by sublingual intravenous injection at 5 min before reperfusion.Group SP＋W was injected with 15 μg/kg PI3 K inhibitor Wortmannin before injected with Sufentanil. Abdominal aortic blood samples were collected at 120 min of reperfusion for measurement of the serum creatine kinase-MB（CK-MB）concentration. The heart samples were collected after executed the rats to determine the volume ratio between myocar-dial infarct size and ischemia area at risk（IS/AAR）. The expression of Akt and phosphorylated-Akt（p-Akt） was determined by Western blotting. Results In the diabetic and nondiabetic groups, the CK-MB concentrations elevated, IS/AAR increased and the expression of p-Akt up-regulated in group IR, SP and SP＋W compared with those in group S（P〈0.05）. In the nondiabetic groups, the CK-MB concentration and IS/AAR decreased, and the expression of p-Akt upregulated in group NDM-SP compared with those in group NDM-IR（P〈0.05）. However, in diabetic rats, there was no statistically significant difference in the above indicators between group DM-SP and DM-IR. Besides, group DM-SP had significantly higher CK-MB concentration and IS/AAR than group NDM-SP, and lower expression of p-Akt than group NDM-SP. Conclusion Diabetic factor can remove the protective effects of Sufentanil postconditioning on myo-cardial ischemia-reperfusion injury, which may be associated with the pathway of PI3K/Akt under diabetic condition.