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兴奋性氨基酸受体拮抗剂减轻电休克诱发的大鼠学习记忆障碍和Tau蛋白的过度磷酸化

刘超, 闵苏^*, 魏珂, 刘东, 董军, 罗洁, 刘小滨

重庆医科大学附属第一医院麻醉科，重庆 400016

摘要

本研究通过观察兴奋性氨基酸受体拮抗剂对电休克(electroconvulsive therapy, ECT)后Wistar-Kyoto (WKY)抑郁大鼠模型学习记忆和Tau蛋白过度磷酸化的影响，探讨兴奋性氨基酸受体拮抗剂对Tau蛋白过度磷酸化的调节及两者对抑郁大鼠学习记忆的影响，以期为改善ECT后学习记忆障碍的神经心理学机制研究和临床干预性治疗提供实验依据。按随机单位组析因设计设置2个干预因素，即电休克干预(两水平：无处置、施行1疗程ECT)和兴奋性氨基酸受体拮抗剂干预(三水平：注射生理盐水、NMDAR拮抗剂MK-801、AMPAR拮抗剂DNQX)的所有组合(2 × 3析因设计)进行实验。将48只24周龄WKY大鼠随机分为6组(n = 8)：生理盐水组(经尾静脉注射2 mL生理盐水)、MK-801组(经尾静脉注射2 mL 5 mg/kg MK-801)、DNQX组(经尾静脉注射2 mL 5 mg/kg DNQX)、生理盐水 + ECT组(经尾静脉注射2 mL生理盐水 + 施行1疗程ECT)、MK-801 + ECT组(经尾静脉注射2 mL 5 mg/kg MK-801 + 施行1疗程ECT)、DNQX + ECT组(经尾静脉注射2 mL 5 mg/kg DNQX + 施行1疗程ECT)。全部ECT处置结束24 h内开始Morris水迷宫检测，然后留取各组大鼠海马组织。高效液相色谱法检测海马组织中神经递质Glu含量；免疫组织化学SP法和Western blot检测Tau5 (总Tau蛋白)、p-PHF1^Ser396/404、p-AT8^Ser199/202、p-12E8^Ser262在海马组织中的表达。结果显示，ECT和Glu离子型受体(NMDAR、AMPAR)均可造成大鼠学习记忆障碍，即延长逃避潜伏期，并缩短空间探索时间；ECT和Glu受体GluR拮抗剂合用之后，其造成的大鼠学习记忆障碍程度反而减轻。ECT可明显增加海马中Glu的浓度；GluR拮抗剂对海马中Glu的浓度没有明显影响。ECT和GluR拮抗剂对海马总Tau蛋白表达无明显影响。ECT可增加海马中磷酸化Tau蛋白的表达；GluR拮抗剂可减少海马中磷酸化Tau蛋白的表达；两者合用的影响呈相减效果，即GluR拮抗剂可使ECT造成的海马磷酸化Tau蛋白的表达增加的幅度降低。以上结果表明，ECT导致海马Glu浓度升高，Glu激动NMDAR和AMPAR，从而增加海马Tau蛋白的磷酸化程度，从而导致学习记忆功能障碍。

关键词： 兴奋性氨基酸受体拮抗剂; 电休克; 学习记忆能力; Tau蛋白; 过度磷酸化; 谷氨酸

分类号：R395.1；R749.054；R614.2+4

[MK-801 or DNQX reduces electroconvulsive shock-induced impairment of learning-memory and hyperphosphorylation of Tau in rats.] [Article in Chinese]

LIU Chao, MIN Su^*, WEI Ke, Liu Dong, DONG Jun, LUO Jie, LIU Xiao-Bin

Department of Anesthesiology, the First Affiliated Hospital of Chongqing Medical University, Chongqing Medical University, Chongqing 400016, China

Abstract

This study explored the effect of the excitatory amino acid receptor antagonists on the impairment of learning-memory and the hyperphosphorylation of Tau protein induced by electroconvulsive shock (ECT) in depressed rats, in order to provide experimental evidence for the study on neuropsychological mechanisms improving learning and memory impairment and the clinical intervention treatment. The analysis of variance of factorial design set up two intervention factors which were the electroconvulsive shock (two level: no disposition; a course of ECT) and the excitatory amino acid receptor antagonists (three level: iv saline; iv NMDA receptor antagonist MK-801; iv AMPA receptor antagonist DNQX). Forty-eight adult Wistar-Kyoto (WKY) rats (an animal model for depressive behavior) were randomly divided into six experimental groups (n = 8 in each group): saline (iv 2 mL saline through the tail veins of WKY rats ); MK-801 (iv 2 mL 5 mg/kg MK-801 through the tail veins of WKY rats) ; DNQX (iv 2 mL 5 mg/kg DNQX through the tail veins of WKY rats ); saline + ECT (iv 2 mL saline through the tail veins of WKY rats and giving a course of ECT); MK-801 + ECT (iv 2 mL 5 mg/kg MK-801 through the tail veins of WKY rats and giving a course of ECT); DNQX + ECT (iv 2 mL 5 mg/kg DNQX through the tail veins of WKY rats and giving a course of ECT). The Morris water maze test started within 1 day after the finish of the course of ECT to evaluate learning and memory. The hippocampus was removed from rats within 1 day after the finish of Morris water maze test. The content of glutamate in the hippocampus of rats was detected by high performance liquid chromatography. The contents of Tau protein which included Tau5 (total Tau protein), p-PHF1(Ser396/404), p-AT8(Ser199/202) and p-12E8(Ser262) in the hippocampus of rats were detected by immunohistochemistry staining (SP) and Western blot. The results showed that ECT and the glutamate ionic receptor blockers (NMDA receptor antagonist MK-801 and AMPA receptor antagonist DNQX) induced the impairment of learning and memory in depressed rats with extended evasive latency time and shortened space exploration time. And the two factors presented a subtractive effect. ECT significantly up-regulated the content of glutamate in the hippocampus of depressed rats which were not affected by the glutamate ionic receptor blockers. ECT and the glutamate ionic receptor blockers did not affect the total Tau protein in the hippocampus of rats. ECT up-regulated the hyperphosphorylation of Tau protein in the hippocampus of depressed rats, while the glutamate ionic receptor blockers down-regulated it, and combination of the two factors presented a subtractive effect. Our results indicate that ECT up-regulates the content of glutamate in the hippocampus of depressed rats, which up-regulates the hyperphosphorylation of Tau protein resulting in the impairment of learning and memory in depressed rats.

Key words: excitatory amino acids receptor antagonists; electroconvulsive therapy; learning and memory ability; Tau protein; hyperphosphorylation; glutamate

收稿日期：2011-10-18　　录用日期：2012-02-16

通讯作者：闵苏　　E-mail: minsu89011068@yahoo.com.cn

引用本文：

刘超, 闵苏, 魏珂, 刘东, 董军, 罗洁, 刘小滨. 兴奋性氨基酸受体拮抗剂减轻电休克诱发的大鼠学习记忆障碍和Tau蛋白的过度磷酸化[J]. 生理学报 2012; 64 (4): 387-402.

LIU Chao, MIN Su, WEI Ke, Liu Dong, DONG Jun, LUO Jie, LIU Xiao-Bin. [MK-801 or DNQX reduces electroconvulsive shock-induced impairment of learning-memory and hyperphosphorylation of Tau in rats.] [Article in Chinese]. Acta Physiol Sin 2012; 64 (4): 387-402 (in Chinese with English abstract).