P13K／Akt或JAK／STAT-3信号通路在肢体缺血联合吗啡后处理减轻大鼠心肌缺血再灌注损伤中的作用

Role of PI3K/Akt or JAK/STAT-3 signaling pathways in reduction of myocardial ischemia-reperfu- sion injury by combination of limb ischemic and morphine postconditioning in rats

目的评价磷脂酰肌醇-3-激酶（P13K）／蛋白激酶B（Akt）或Janus蛋白酪氨酸激酶／信号转导子与转录激活因子3（JAK／STAT-3）信号通路在肢体缺血联合吗啡后处理减轻大鼠心肌缺血再灌注损伤中的作用。方法SPF级健康雄性SD大鼠80只，8周龄，体重280～320g，采用结扎左冠状动脉前降支30min恢复灌注的方法制备大鼠心肌缺血再灌注模型。按随机数字表法将其分为8组（n=10）：心肌缺血再灌注组（I／R组）、肢体缺血后处理组（LIP组）、吗啡后处理组（MP组）、肢体缺血联合吗啡后处理组（LIP＋MP组）和分别给予信号通路阻断剂组（I／Rb组、LIPb组、MPb组和LIP＋MPb组）。I／R组、LIP组、MP组和LIP＋MP组于再灌注120min时处死大鼠取缺血区和非缺血区心肌，采用Westernblot法检测p-STAT-3、STAT-3、p-Akt和Akt的表达，PCR法检测STAT一3和Akt的mRNA表达；I／Rb组、LIPb组、MPb组和LIP＋MPb组于缺血10min时每组各5只大鼠分别静脉注射特异性P13K／Akt或JAK／STAT-3信号通路阻断剂LY2940020．3mg／kg、AG4905mg／kg。于再灌注120min时处死大鼠取缺血区，测定心肌梗死面积。结果与I／R组比较，LIP组、MP组和LIP＋MP组p-STAT-3／STAT-3比值、LIP＋MP组p-Akt／Akt比值升高，LIP＋MP组STAT-3和AktmRNA表达上调（P〈0．05）；与LIP组和MP组比较，LIP＋MP组p-STAT-3／STAT-3比值和p-Akt／Akt比值升高，STAT-3和AktmRNA表达上调（P〈0．05）。应用P13K抑制剂时4组心肌梗死面积差异无统计学意义（P〉0.05）。应用JAK2抑制剂AG490时，LIP＋MPb组心肌梗死面积较I／Rb组、LIPb组和MPb组减小（P〈0．05），I／Rb组、LIPb组和MPb组心肌梗死面积比较差异无统计学意义（P〉0．05）。结论肢体缺血联合吗啡后处理可增强P13K／Akt和JAK／STAT-3信号通路的激活，并且联合应用时的心肌保护作用依赖于P13K／Akt信号通路的完整，部分依赖于JAK／STAT-3信号通路的完整。

Objective To evaluate the role of the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） or Janus kinase （JAK）/signal transducer and activator of transcription 3 （STAT-3） signaling pathways in reduction of myocardial ischemia-reperfusion （I/R） injury by combination of limb ischemic and morphine postconditioning in rats. Methods Eighty SPF healthy male Sprague-Dawley rats, aged 8 weeks, weighing 280-320 g, were used in the study. Myocardial I/R was induced by occlusion of the left anterior descending branch of the coronary artery for 30 min followed by 120 rain reperfusion. The rats were divided into 8 groups （n= 10 each） using a random number table： I/R group, limb ischemic postconditioning group （LIP group） , morphine postconditioning group （group MP） , combination of limb ischemic and morphine postconditioning group （LIP ＋ MP group） and signaling pathway blocker groups （I/Rb group, LIPb group, MPb group, LIP＋MPb group）. In I/R, LIP, MP and LIP＋MP groups, the animals were sacrificed at the end of repcrfusion, and myocardial specimens in ischemic and non-ischemic regions were obtained for determination of phosphorylated STAT-3 （p-STAT-3） , STAT-3, phosphorylated Akt （p- Akt） and Akt expression （by Western blot） and STAT-3 and Akt mRNA expression （by polymerase chain reaction）. In I/Rb, LIPb, MPb and LIP＋MPb groups, PI3K/Akt signaling pathway blocker LY294002 0.3 mg/kg was intravenously injected in 5 rats of each group, and JAK/STAT-3 signaling pathway blocker AG490 5 mg/kg was intravenously injected in the other 5 rats of each group. The animals were sacrificed at the end of reperfusion, and myocardial specimens in the ischemic region were obtained for determination of myocardial infarct size. Results Compared with I/R group, the p-STAT-3/STAT-3 ratio in LIP, MP and LIP＋MP groups and p-Akt/Akt ratio in LIP＋MP group were significantly increased, and the expression of STAT-3 and Akt mRNA was up-regulated in LIP ＋ MP group （P〈0.05）. Compared with LIP and MP groups, the p-STAT-3/STAT-3 and p-Akt/Akt ratios were significantly increased, and the expression of STAT-3 and Akt mRNA was up-regulated in LIP＋MP group （P〈0.05）. There was no significant difference in myocardial infarct size between the four groups when PI3K inhibitor LY294002 was applied （P〉0. 05 ）. When JAK2 inhibitor AG490 was applied, the myocardial infarct size was significantly smaller in LIP＋MPb group than in I/Rb, LIPb and MPb groups （P〈0.05） , and there was no significant difference in myocar- dial infarct size between I/Rb, LIPb and MPb groups （P〉0.05）. Conclusion Combination of limb is- chemic and morphine postconditioning can enhance the activation of PI3K/Akt or JAK/STAT-3 signaling pathways, and the cardioprotection is dependent on the integrity of the PI3K/Akt signaling pathway and partially dependent on the integrity of the JAK/STAT-3 signaling pathway when applied in combination in rats.