艾司氯胺酮对发育期大鼠慢性应激后学习记忆功能及海马NMDAR-CaMKⅡ-CREB信号通路的影响

Effect of esketamine on learning and memory function after chronic stress and hippocampal NMDAR-CaMK Ⅱ-CREB signaling pathway in developing rats

目的评价艾司氯胺酮对发育期大鼠慢性应激后学习记忆功能及海马N-甲基-D-天冬氨酸受体(NMDAR)-钙-钙调素依赖性蛋白激酶2型(CaMKⅡ)-环磷酸腺苷反应元件结合蛋白(CREB)信号通路的影响。方法清洁级健康SD大鼠60只,雌雄不拘,7日龄,体质量10~15 g,采用随机数字表法分为3组(n=20):对照+生理盐水组(CN组)、慢性应激+生理盐水组(NS组)和慢性应激+艾司氯胺酮组(ES组)。采用慢性不可预知性应激方法建立慢性应激模型。应激刺激结束后,ES组腹腔注射艾司氯胺酮10 mg/kg,NS组腹腔注射等容量生理盐水,连续7 d,1次/d。腹腔给药结束后,行Y迷宫实验和Morris水迷宫实验测定学习记忆功能;取静脉血样,采用ELISA法检测血清皮质醇和ROS浓度;随后麻醉断头取脑,分离海马组织,观察海马CA1区神经元病理学结果,采用Western blot法确定磷酸化NMDAR(p-NMDAR)/NMDAR、磷酸化CaMKⅡ(p-CaMKⅡ)/CaMKⅡ、磷酸化CREB(p-CREB)/CREB表达水平的比值。结果与CN组比较,NS组新臂停留时间缩短,进入新臂次数减少,逃避潜伏期延长,穿越原平台位置次数减少,血清皮质醇和ROS浓度升高,p-NMDAR/NMDAR比值、p-CaMKⅡ/CaMKⅡ比值和p-CREB/CREB比值降低(P<0.05),神经元发生明显病理学损伤;与NS组比较,ES组新臂停留时间延长,进入新臂次数增多,逃避潜伏期缩短,穿越原平台位置次数增多,血清皮质醇和ROS浓度下降,p-NMDAR/NMDAR比值、p-CaMKⅡ/CaMKⅡ比值和p-CREB/CREB比值升高(P<0.05),神经元病理学损伤减轻。结论艾司氯胺酮可改善发育期大鼠慢性应激后学习记忆功能,机制可能与降低氧化应激水平,增强海马NMDAR-CaMKⅡ-CREB信号通路活性有关。

Objective To evaluate the effect of esketamine on learning and memory function after chronic stress and the signaling pathway of N-methyl-D-aspartate receptor(NMDAR)-calmodulin-dependent protein kinase type 2(CaMKⅡ)-cAMP-responsive element-binding protein(CREB)in the hippocampus of developing rats.Methods Sixty clean-grade healthy Sprague-Dawley rats of either sex,aged 7 days,weighing 10-15 g,were divided into 3 groups(n=20 each)using a random number table method:control+normal saline group(CN group),chronic stress+normal saline group(NS group),and chronic stress+esketamine group(ES group).A chronic stress model was established using a chronic unpredictable stress method.After the end of stress stimulation,esketamine 10 mg/kg was intraperitoneally injected once a day for 7 consecutive days in ES group,and the equal volume of normal saline was given instead in NS group.Y maze test and Morris water maze test were used to assess the learning and memory function after intraperitoneal administration.Venous blood samples were obtained to measure the serum cortisol and reactive oxygen species(ROS)concentrations by enzyme-linked immunosorbent assay.The animals were then sacrificed under anesthesia,the brain was removed and the hippocampal tissue was isolated for examination of the pathological changes in the hippocampal CA1 region and for determination of the ratios of phosphorylated NMDAR(p-NMDAR)/NMDAR,phosphorylated CaMKII(p-CaMKⅡ)/CaMKⅡ,and phosphorylated CREB(p-CREB)/CREB(by Western blot).Results Compared with CN group,the time spent in the novel arm was significantly shortened,the number of entries into the novel arm was reduced,the escape latency was prolonged,the number of crossing the original platform was reduced,the serum cortisol and ROS concentrations were increased,the p-NMDAR/NMDAR ratio,p-CaMKⅡ/CaMKⅡratio and p-CREB/CREB ratio were decreased(P<0.05),and the pathological changes of neurons were marked in NS group.Compared with NS group,the time spent in the novel arm was significantly prolonged,the number of entries into the novel arm was increased,the escape latency was shortened,the number of crossing the original platform was increased,the serum cortisol and ROS concentrations were decreased,the p-NMDAR/NMDAR ratio,p-CaMKⅡ/CaMKⅡratio and p-CREB/CREB ratio were increased(P<0.05),and the pathological changes of neurons were significantly attenuated in ES group.Conclusions Esketamine can improve the learning and memory function after chronic stress in developing rats,and the mechanism may be related to reduction of oxidative stress and enhancement of the activity of hippocampal NMDAR-CaMKII-CREB signaling pathway.