Ca2+-CaMKⅡ-CREB信号通路在U50488H减轻CPB致大鼠围术期神经认知障碍中的作用

Role of Ca2+-CaMKⅡ-CREB signaling pathway in U50488H-induced reduction of CPB-caused perioperative neurocognitive disorders in rats

目的评价钙离子-钙调蛋白依赖性蛋白激酶Ⅱ-环腺苷酸反应元件结合蛋白(Ca2+-CaMKⅡ-CREB)信号通路在U50488H减轻CPB致大鼠围术期神经认知障碍(PND)中的作用。方法清洁级健康成年雄性SD大鼠40只,体重350~400 g,采用随机数字表法分为4组(n=10):对照组(S组)、CPB组(C组)、CPB+κ受体激动剂U50488H组(U组)和CPB+CaMKⅡ特异性抑制剂KN93+U50488H组(K组)。S组仅进行动静脉穿刺置管,其余各组建立无血预充心脏不停跳CPB模型。U组CPB前30 min时静脉注射U50488H 1.5 mg/kg;K组CPB前60 min时侧脑室注射10 μmol/L KN93 5 μl,CPB前30 min时静脉注射U50488H 1.5 mg/kg。术后第3天采用Morris水迷宫实验测试大鼠认知功能。然后处死大鼠取海马组织,采用Western blot法检测磷酸化CaMKⅡ(p-CaMKⅡ)、磷酸化CREB (p-CREB)和脑源性神经营养因子(BDNF)的表达水平,采用RT-PCR法检测CaMKⅡ、CREB、BDNF的mRNA表达水平。结果与S组比较,C组、U组和K组逃避潜伏期延长,穿越原平台次数减少,海马p-CaMKⅡ、p-CREB、CaMKⅡmRNA、CREB mRNA、BDNF及其mRNA表达下调(P<0.05);与C组比较,U组逃避潜伏期缩短,穿越原平台次数增加,海马p-CaMKⅡ、p-CREB、CaMKⅡmRNA、CREB mRNA、BDNF及其mRNA表达上调(P<0.05),K组上述指标差异无统计学意义(P>0.05);与U组比较,K组逃避潜伏期延长,穿越原平台次数减少,海马p-CaMKⅡ、p-CREB、CaMKⅡmRNA、CREB mRNA、BDNF及其mRNA表达下调(P<0.05)。结论 U50488H减轻CPB致大鼠PND的机制与激活Ca2+-CaMKⅡ-CREB信号通路有关。

Objective To evaluate the role of calcium-calmodulin-dependent protein kinaseⅡ-cyclic adenylate response element binding protein(Ca2+-CaMKⅡ-CREB)signaling pathway in U50488H-induced reduction of cardiopulmonary bypass(CPB)-caused perioperative neurocognitive disorders in rats.Methods Forty clean-grade male adult Sprague-Dawley rats,weighing 350-400 g,were divided into 4 groups(n=10 each)using a random number table method:control group(S group),CPB group(C group),CPB plus byκ-opioid receptor agonist U50488H group(U group),and CPB plus specific CaMKⅡantagonist KN93 plus U50488H group(K group).Only the arteriovenous catheter was placed in S group,and the blood-free pre-filled cardiac CPB model was established in the other groups.U50488H 1.5 mg/kg was intravenously injected at 30 min before CPB in group U.In group K,10μmol/L KN935μl was injected into left lateral cerebral ventricle at 60 min before CPB,and U50488H 1.5 mg/kg was intravenously injected at 30 min before CPB.Morris water maze test was used to assess cognitive function on 3rd day after operation.The rats were then sacrificed,and hippocampal tissues were obtained for determination of the expression of phosphorylated CaMKⅡ(p-CaMKⅡ),phosphorylated CREB(p-CREB)and brain-derived neurotrophic factor(BDNF)(by Western blot)and expression of CaMKⅡ,CREB and BDNF mRNA(by real-time polymerase chain reaction).Results Compared with S group,the escape latency was significantly prolonged,the number of crossing original platforms was decreased,and the expression of p-CaMKⅡ,p-CREB,CaMKⅡmRNA,CREB mRNA and BDNF protein and mRNA was down-regulated in C,U group and K groups(P<0.05).Compared with group C,the escape latency was significantly shortened,the number of crossing original platforms was increased,and the expression of p-CaMKⅡ,p-CREB,CaMKⅡmRNA,CREB mRNA and BDNF protein and mRNA was up-regulated in group U(P<0.05),and no significant change was found in the parameters mentioned above in group K(P>0.05).Compared with group U,the escape latency was significantly prolonged,the number of crossing original platforms was decreased,and the expression of p-CaMKⅡ,p-CREB,CaMKⅡmRNA,CREB mRNA and BDNF protein and mRNA was down-regulated in group K(P<0.05).Conclusion The mechanism by which U50488H reduces CPB-caused perioperative neurocognitive disorders is related to activating the Ca2+-CaMK II-CREB signaling pathway in rats.