自噬及PI3K-Akt通路在舒芬太尼后处理心肌缺血再灌注损伤保护中的作用

Effects of Autophagy and PI3K-Akt Pathway in Sufentanil Postconditioning on Myocardial Ischemia-reperfusion Injury Protection

目的探讨自噬及PI3K-Akt通路在舒芬太尼后处理心肌缺血再灌注损伤保护中的作用。方法选用雄性SD大鼠(220 g^260 g)35只,随机分为5组:假手术组(S组)、心肌缺血再灌注损伤组(I/R组)、舒芬太尼后处理组(SP组)、舒芬太尼后处理+PI3K-Akt通路的抑制剂Wortmannin组(SP+W组)、Wortmannin组(W组)。I/R组、SP组、SP+W组以及W组采用结扎冠脉左前降支(LAD)30 min、再灌注120 min的方法制备心肌缺血再灌注损伤模型。S组在冠脉左前降支(LAD)下穿线但不结扎。于再灌注前5 min,I/R组由股静脉给予生理盐水1 m L,SP组给予舒芬太尼(1μg/kg),SP+W组给予舒芬太尼(1μg/kg)及Wortmannin(15μg/kg),W组给予Wortmannin(15μg/kg)。再灌注120 min时腹主动脉取血,检测血清肌酸激酶同工酶(CK-MB)的浓度;处死大鼠后,取心尖组织,Western blot法检测Akt、磷酸化的Akt(p-Akt)、LC3-Ⅱ的表达;电镜下观察自噬体的生成情况。结果与S组比较,I/R组、SP组、SP+W组及W组CK-MB含量、LC3-Ⅱ表达增加,p-Akt/Akt升高,差异有统计学意义(P<0.05),自噬体数量增多;与I/R组比较,SP组CK-MB含量、LC3-Ⅱ表达减少,p-Akt/Akt升高,差异有统计学意义(P<0.05),自噬体数量减少;与SP组比较,SP+W组、W组CK-MB含量、LC3-Ⅱ表达增加,p-Akt/Akt降低,差异有统计学意义(P<0.05),自噬体数量增多。结论舒芬太尼后处理可能通过抑制自噬水平而在心肌缺血再灌注损伤中起到保护作用,其中PI3K-Akt通路可能是主要通路之一。

Objective To investigate the roles of autophagy and PI3K-Akt signaling pathway on sufentanil postconditioning-mediated alleviation of myocardial ischemia-reperfusion injury（ MIRI）. Methods Thirty-five male Sprague-Dawley（ SD） rats weighed 220 g to 260 g were randomly divided into five groups（n = 7 each） ： Sham operation group（ group S）,ischemia-reperfusion group（ group I / R）,Sufentanil postconditioning group（ group SP）,sufentanil postconditioning plus Wortmannin（ a specific PI3 K inhibitor） group（ group SP ＋ W） and Wortmannin group（ group W）. In groups I / R,SP,SP ＋ W and W,the myocardial I / R models were prepared by 30 min ligation of left anterior descending coronary artery（ LAD） followed by 120 min reperfusion. Group S underwent the operation with a stitch placed around but not ligating the artery. In groups I / R,SP,SP ＋ W and W rats were treated with 1 m L normal saline,1 μg / kg sufentanil,1 μg / kg sufentanil plus 15 μg / kg wortmannin and 15 μg / kg wortmannin intravenous infusion via the femoral vein for 5 min prior to reperfusion,respectively. Abdominal aortic blood samples were collected at 120 min of reperfusion for measurement of the serum creatine kinase-MB（ CK-MB） concentration. The cardiac apexes were collected after executed the rats. The expression of Akt,phosphorylated-Akt（ p-Akt） and LC3-Ⅱ were detected by Western blotting and the formation of autophagy was observed by electron microscope. Results Compared with group S,the concentration of CK-MB and the expression of LC3-Ⅱ increased in groups I / R,SP,SP ＋ W and W,the ratio of p-Akt / Akt higher in groups I / R,SP,SP ＋ W and W（P〈0.05） and the formation of autophagy was increased. Compared with group I / R,the concentration of CK-MB and the expression of LC3-Ⅱdecreased in group SP,the ratio of p-Akt / Akt higher in group SP（P〈0.05） and the formation of autophagy was decreased. Compared with group SP,the concentration of CK-MB and the expression of LC3-Ⅱ increased in groups SP ＋ W and W,the ratio of p-Akt / Akt lower in groups SP ＋ W and W（P〈0.05） and the formation of autophagy was increased. Conclusion Sufentanil postprocessing through inhibition of autophagy in myocardial ischemia reperfusion injury play a protective role,including PI3K-Akt pathway may be one of the main mechanisms.