摘要
目的探讨癫痫大鼠海马齿状回(DG)酪氨酸激酶受体结合蛋白B3(Ephrin-B3)对突触后密度蛋白95(PSD95)和谷氨酸受体N-甲基-D-天冬氨酸亚基(NR2A和NR2B)的表达及海马神经元突触超微结构的影响。方法96只雄性清洁级SD大鼠按随机数字表法分为空白组、癫痫组、慢病毒对照组和Ephrin-B3过表达组,每组24只。36只雄性清洁级SD大鼠按随机数字表法分为腺病毒对照组和Ephrin-B3基因抑制组,每组18只。采用立体定位法向大鼠双侧海马DG区注射病毒;采用腹腔注射氯化锂-匹罗卡品建立癫痫大鼠模型。Racine标准分级法评估大鼠癫痫发作分级并记录发作潜伏期和持续时间;免疫荧光染色法观察大鼠海马DG区PSD95、NR2A和NR2B荧光强度;PCR和Western blot分别检测大鼠海马组织PSD95、NR2A和NR2B mRNA和蛋白表达;透射电镜观察海马神经元超微结构。采用GraphPad Prism 8对实验数据进行统计分析。结果(1)Ephrin-B3过表达实验:4组大鼠癫痫发作潜伏期和持续时间均差异有统计学意义(F=368.30,120.00,均P<0.01)。Ephrin-B3过表达组大鼠癫痫发作潜伏期长于癫痫组和慢病毒对照组[(31.32±1.10)d,(24.09±2.02)d,(21.42±0.79)d](均P<0.01),发作持续时间短于慢病毒对照组和癫痫组[(26.85±1.14)s,(40.40±1.26)s,(36.50±5.50)s](均P<0.01)。免疫荧光结果显示,Ephrin-B3过表达组PSD95、NR2A和NR2B荧光强度均高于慢病毒对照组(均P<0.01)。PCR和Western blot结果均显示,Ephrin-B3过表达组PSD95、NR2A和NR2B mRNA和蛋白表达量均高于慢病毒对照组(均P<0.05)。电镜结果显示,Ephrin-B3过表达组中突触数量多于慢病毒对照组[(9.00±1.00)个,(6.00±1.00)个](P<0.05),突触间隙窄于慢病毒对照组[(31.60±1.45)nm,(36.43±2.22)nm](P<0.05)。(2)Ephrin-B3抑制实验:Ephrin-B3基因抑制组和腺病毒对照组大鼠癫痫发作潜伏期和发作持续时间均差异有统计学意义(t=3.88,3.93,均P<0.05)。Ephrin-B3基因抑制组大鼠癫痫发作潜伏期短于腺病毒对照组[(17.10±1.88)d,(23.50±2.15)d](P<0.05),发作持续时间长于腺病毒对照组[(55.16±5.48)s,(42.06±1.83)s](P<0.05)。Western blot结果显示,Ephrin-B3基因抑制组的PSD95、NR2A和NR2B蛋白表达均低于腺病毒对照组(均P<0.01)。电镜结果显示,Ephrin-B3基因抑制组神经元突触数量少于腺病毒对照组[(3.33±0.58)个,(4.66±0.58)个](P<0.05),突触间隙宽于腺病毒对照组[(37.27±0.97)nm,(33.33±1.46)nm](P<0.05)。结论Ephrin-B3可能通过上调突触相关蛋白及谷氨酸受体表达,改善突触结构,减少癫痫发作。
ObjectiveTo investigate the effect of tyrosine kinase receptor binding protein B3(Ephrin-B3)on the expressions of postsynaptic density protein 95(PSD95),N-methyl-D-aspartic acid(NMDA)receptor subunit 2A and 2B(NR2A/NR2B)and synaptic ultrastructure of hippocampal neurons in the dentate gyrus(DG)area of epileptic rats.MethodsA total of 96 SPF-grade adult male Sprague Dawley(SD)rats were divided into blank group,epilepsy group,lentiviral vectors control group and Ephrin-B3 overexpression group(n=24 in each group)according to the random number table.Another 36 adult male SD rats were randomly divided into recombinant adeno-associated virus control group and Ephrin-B3 suppression group(n=18 in each group).The viruses were stereotaxically injected into bilateral hippocampus DG area of rats and epileptic model was established by intraperitoneal injection of lithium chloride and pilocarpine.Racine standard grading method was used to evaluate seizure rate and the duration and latency of epileptic rats were observed.Immunofluorescence staining was used to observe expression levels of PSD95,NR2A and NR2B protein in hippocampus DG area of rats.PCR and Western blot were used to detect the expression levels of PSD95,NR2A and NR2B mRNA and protein in hippocampus of rats.Transmission electron microscopy was used to observe the ultrastructure of hippocampal neurons in rats.GraphPad Prsim 8 software was used for statistical analysis.Results(1)The results of Ephrin-B3 overexpression experiment:there were statistically significant differences in the seizure latency and duration among the four groups(F=368.30,120.00,both P<0.01).The seizure latency in Ephrin-B3 overexpression group was longer than that of the epilepsy group and lentiviral vectors control group((31.32±1.10)d,(24.09±2.02)d,(21.42±0.79)d)(all P<0.01),while the seizure duration in Ephrin-B3 overexpression group was shorter than that of the epilepsy group and lentiviral vectors control group((26.85±1.14)s,(40.40±1.26)s,(36.50±5.50)s)(all P<0.05).Immunofluorescence staining showed the average fluorescence intensity of PSD95,NR2A and NR2B in Ephrin-B3 overexpression group were higher than that in the lentiviral vectors control group(all P<0.01).PCR and Western blot both showed that the Ephrin-B3 overexpression group had higher mRNA expression levels of PSD95,NR2A and NR2B than that in lentiviral vectors control group(all P<0.05).The results of transmission electron microscopy showed the number of synapses in the Ephrin-B3 overexpression group was greater than that in lentiviral vectors control group((9.00±1.00),(6.00±1.00))(P<0.05),synaptic gap was narrower than that in lentiviral vectors control group((31.60±1.45)nm,(36.43±2.22)nm)(all P<0.05).(2)The results of the Ephrin-B3 suppression experiment:the evaluation of seizures in rats showed that there were statistically significant differences in the seizure latency and duration between recombinant adeno-associated virus control group and Ephrin-B3 suppression groups(t=3.88,3.93,both P<0.05).The seizure latency in Ephrin-B3 suppression group was shorter than that of recombinant adeno-associated virus control group((17.10±1.88)d,(23.50±2.15)d)(P<0.05).The seizure duration in Ephrin-B3 suppression group was longer than that of recombinant adeno-associated virus control group((55.16±5.48)s,(42.06±1.83)s)(P<0.05).Western blot results showed that the Ephrin-B3 suppression group had lower protein expression levels of PSD95,NR2A and NR2B than those in recombinant adeno-associated virus control group(all P<0.01).The results of transmission electron microscopy showed the numbers of synapses in the Ephrin-B3 suppression group were less than recombinant adeno-associated virus control group((3.33±0.58),(4.66±0.58))(P<0.05),synaptic gap were narrower than recombinant adeno-associated virus control group((37.27±0.97)nm,(33.33±1.46)nm)(P<0.05).ConclusionEphrin-B3 can attenuate seizures and upregulate the synaptic related proteins and glutamate receptor expression,improving synaptic structure.
作者
刘田田
刘恒方
贾延劼
Liu Tiantian;Liu Hengfang;Jia Yanjie(Department of Neurology,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450000,China;Department of Neurology,the Fifth Affiliated Hospital of Zhengzhou University,Zhengzhou 450000,China)
出处
《中华行为医学与脑科学杂志》
北大核心
2025年第9期774-782,共9页
Chinese Journal of Behavioral Medicine and Brain Science
基金
河南省医学科技攻关项目(2018020066)
健康中国步长致远心脑健康公益工程基金项目(HIGHER2023102)。
