通督醒神针刺法对脑缺血再灌注后学习记忆障碍模型大鼠海马组织AMPA受体及其辅助蛋白表达的影响

Effect of Tongdu Xingshen Needling Method(通督醒神针刺法)on Expression of AMPA Receptors and Their Accessory Proteins in the Hippocampus of Rats with Learning Memory Impairment After Cerebral Ischaemia-Reperfusion

目的 探讨通督醒神针刺法治疗脑卒中后认知障碍的可能作用机制。方法 SD大鼠随机分为正常组、假手术组、模型组各12只,通督醒神组13只。模型组和通督醒神组大鼠均接受线栓法建立脑缺血再灌注后学习记忆障碍大鼠模型。造模成功后通督醒神组大鼠选取“神庭”“百会”穴电针干预,每日1次,每次30 min,持续14天,其余各组只抓取不进行其他干预。于干预的第9天开始进行5天的定位航行实验,于干预的第14天进行空间探索实验评价大鼠学习记忆能力。空间探索实验后,每组大鼠取材海马组织,采用TTC染色法观察脑梗死体积变化;高尔基染色法观察大鼠海马CA1区锥体神经元形态及树突棘密度改变;运用蛋白质免疫印迹法检测各组大鼠海马组织α-氨基-3-羧基-5-甲基异恶唑-4-丙酸(AMPA)受体亚基谷氨酸受体1 (GluR1)、谷氨酸受体2 (GluR2)、谷氨酸受体3 (GluR3)及辅助蛋白跨膜AMPA受体调节蛋白γ2(TARPγ2)、跨膜AMPA受体调节蛋白γ8 (TARPγ8)蛋白相对表达量;运用实时荧光定量PCR法检测大鼠海马组织GluR1、GluR2、GluR3 mRNA水平。结果 定位航行实验中,与正常组和假手术组比较,模型组大鼠在第1、2、3、4、5天时逃避潜伏期和总路程延长(P<0.05);通督醒神组大鼠在各时间点逃避潜伏期和总路程较模型组缩短(P<0.05)。在空间探索实验中,与正常组和假手术组比较,模型组大鼠穿越平台次数明显减少(P<0.05);通督醒神组大鼠穿越平台次数较模型组明显增加(P<0.05)。TTC染色结果显示,模型组大鼠脑梗死体积较假手术组明显增大,而通督醒神组大鼠脑梗死体积较模型组明显减小(P<0.05)。高尔基染色结果显示,与正常组和假手术组比较,模型组大鼠海马CA1区锥体神经元树突分支数减少,树突棘密度降低,而通督醒神组大鼠海马CA1区锥体神经元树突分支数及树突棘较模型组增多,且树突棘密度较模型组升高(P<0.05)。与正常组和假手术组比较,模型组大鼠海马组织中GluR1、GluR2、GluR3、TARPγ2及TARPγ8蛋白相对表达量及GluR1、GluR2、GluR3的mRNA水平降低(P<0.05);与模型组比较,通督醒神组大鼠海马组织中GluR1、GluR2、GluR3、TARPγ2、TARPγ8的蛋白相对表达量及GluR1、GluR2、GluR3的mRNA水平升高(P<0.05)。结论 通督醒神针刺可改善脑缺血再灌注后学习记忆障碍,其作用可能与上调海马组织中AMPA受体及其辅助蛋白TARP的表达,促进突触可塑性有关。

Objective To explore the possible mechanism of Tongdu Xingshen needling method(通督醒神针刺法)on post-stroke cognitive impairment.Methods SD rats were randomly divided into a normal group(n=12),a sham surgery group(n=12),a model group(n=12),and a electroacupuncture group(n=13).The rats in the model group and electroacupuncture group were subjected to the wire bolus method to establish the rats model with learning memory impairment after cerebral ischaemia-reperfusion.After successful modelling,the rats in the electroacupuncture group were given electroacupuncture interventions at“Shenting(GV 24)”and“Baihui(GV 20)”once a day for 30 minutes for 14 days.The other three groups did not receive other interventions but grasp.A 5-day localisation navigation experiment was conducted on the 9th day of intervention,and a spatial exploration experiment was conducted on the 14th day of intervention to evaluate the learning and memory abilities of the rats.After the spatial exploration experiment,hippocampal tissues were taken from each group of rats,and the changes in the volume of cerebral infarction were observed by TTC staining;the changes in the morphology of pyramidal neurons and the density of dendritic spines in the CA1 area of the hippocampus were observed by Golgi staining;protein immunoblotting was used to detect the relative protein expression of the subunits of theα-amino-3-carboxy-5-methylisoxazole-4-propionic acid(AMPA)receptor including glutamate receptor 1(GluR1),glutamate receptor 2(GluR2),glutamate receptor 3(GluR3)and auxiliary proteins TARPγ2,TARPγ8 in hippocampal tissues of rats in each group;the real-time fluorescence quantitative PCR was used to detect GluR1,GluR2,GluR3 mRNA levels in the hippocampal tissues of rats.Results In the localisation navigation experiment,compared with the normal group and sham surgery group,the escape latency and total distance of rats in the model group were significantly extended(P<0.05)at day 1,2,3,4,and 5;and the escape latency and total distance of rats in the electroacupuncture group tended to be significantly shorter than those in the model group(P<0.05).In the spatial exploration experiment,compared with the normal group and the sham surgery group,the number of rats crossing the platform in the model group was significantly reduced(P<0.05),and the number of crossings of the platform in the electroacupuncture group increased significantly(P<0.05).The results of TTC staining showed that the volume of cerebral infarction increased clearly in the model group compared with the sham surgery group(P<0.05),and apparently decreased in the electroacupuncture group compared with the model group(P<0.05).Golgi staining showed that the number of dendritic branches of pyramidal neurons and dendritic spines in hippocampal CA1 region significantly decreased in the model group compared with the normal group and the sham surgery group(P<0.05).The number of dendritic branches of pyramidal neurons and the density of dendritic spines in hippocampal CA1 region significantly increased in the electroacupuncture group compared with the model group(P<0.05).The protein relative expression levels of GluR1,GluR2,GluR3,TARPγ2 and TARPγ8,and the mRNA levels of GluR1,GluR2 and GluR3 in hippocampus decreased in the model group compared with the normal group and the sham surgery group(P<0.05).The protein relative expression levels of GluR1,GluR2,GluR3,TARPγ2 and TARPγ8,and the mRNA levels of GluR1,GluR2 and GluR3 in hippocampus increased in the electroacupuncture group compared with model group(P<0.05).Conclusion The Tongdu Xingshen needling method can improve learning memory impairment after cerebral ischaemia-reperfusion,which may be related to up-regulation of the expression of AMPA receptor and their auxiliary protein TARP,and promoting the synaptic plasticity of hippocampal tissues.