SPAK参与药物抗性癫痫小鼠海马神经元GABA信号的功能重塑  

SPAK on Functional Remodeling of GABA Signal in Hippocampal Neurons in Mice with Drug-resistant Epilepsy

作　　者：杨丽白 蔡晓冬 周列民[3] 牛争平 李新毅 

机构地区：[1]山西大医院/山西医学科学院,太原030012 [2]中山大学附属六院 [3]中山大学附属第一医院 [4]山西医科大学第一医院

出　　处：《中西医结合心脑血管病杂志》2017年第23期2980-2986,共7页Chinese Journal of Integrative Medicine on Cardio-Cerebrovascular Disease

摘　　要：目的研究氯化锂-匹罗卡品诱导的癫痫(PISE)小鼠海马神经元生理生化的变化,探讨药物抗性癫痫的γ-氨基丁酸(GABA)信号功能的重塑机制。方法将4周~6周健康雄性Balb/c小鼠制作成PISE癫痫模型,动物随机分为3组:癫痫模型组、假模型组和空白组,造模后观察1 d(静息期)、14 d(亚急性期)、45 d(慢性自发癫痫发作期)三个时间点,每组每个时间点各6只动物。采用免疫双标观察201S1相关的氨酸/丙氨酸激酶/Na+-K+-2Cl-共转运体1(NKCC1)、SPAK/K+–2Cl-共转运体2(KCC2)在海马组织共表达情况。Western Blot检测各组小鼠海马组织SPAK蛋白表达的差异。用Cl-荧光探针MQAE检测各组神经元Cl-分布情况。免疫共沉淀法检测SPAK与NKCC1、KCC2是否存在相互作用。结果 SPAK/NKCC1、SPAK/KCC2在小鼠海马组织共表达。与空白对照组及假模型组比较,模型组在PISE后1 d、14 d、45 d海马组织SPAK表达均增高,差异有统计学意义(P<0.05);模型组3个时间点比较,14 d高于1 d、45 d,与后者比较差异有统计学意义(P<0.05)。模型组小鼠海马[Cl-]i下降(P<0.05)。慢性自发性癫痫小鼠SPAK与NKCC1、KCC2分别存在相互作用。结论小鼠海马神经元SPAK通过与NKCC1、KCC2相互作用调控[Cl-],参与药物抗性癫痫GABA信号功能的重塑。Objective To explore the possible mechanism of the remodeling of GABA signaling function in drug resistant epilepsy withpilocarpine induced status epilepticus （ PISE）mice.Methods The healthy male Balb/c mice （ 4 6 weeks）were used in the study.The animals were randomly divided into three groups： a PISE group,sham control group, and blank control group.Three main pha -ses were observed following PISE：the acute stage, the subacute stage, and the chronic stage with spontaneous recurrent seizures,represented as day 1, day 14 and day 45, respectively, after PISE.Double labeling was used for investigating SPAK/NeuN co expres -sion in mice hippocampus.Western Blot were used for testing SPAK protein expressional level differences between groups in micehippocampus.Intracellular chloride concentration [ Cl-] i of mice hippocampus neurons was detected by N （ 6 methoxyquinolyl）acetoethyl ester （ MQAE） .Co immol /Lunoprecipitation were used to detect the interaction between SPAK and NKCC1 or KCC2.Re -sults SPAK expressed and co expressed with NKCC1 and KCC2 in the hippocampal neurons of mice.Significant increases inSPAK protein levels were detected during various stages of PISE in the PISE mice in comparison to levels in age matched sham（ control）and blank treatment （ control）mice （ P 〈0.05 ） .While[ Cl-] i of hippocampal neurons in the various stages PISE mice in -creased significantly （ P 〈0.05） .Using co immol /Lunoprecipitation.The intensity of interaction between SPAK and NKCC1 and be -tween SPAK and KCC2 increased markedly in the chronic stage.Conclusion SPAK is involved in remodeling of GABA signalingfunction in drug resistang epilepsy via adjusting [ Cl-] i through interacting with NKCC1, and KCC2 in mouse hippocampus neurons.

关 键 词：药物抗性癫痫 Γ氨基丁酸 SPAK 

分 类 号：R742.1[医药卫生—神经病学与精神病学] R255.2[医药卫生—临床医学]