电针对慢性痛负性情绪大鼠杏仁核神经突触可塑性相关蛋白/基因表达的影响

Effect of Electroacupuncture Intervention on Expression of Synaptic Plasticity-related Molecules in Amygdala in Chronic Pain-negative Affection Rats

目的:观察电针对慢性神经痛负性情绪大鼠杏仁核神经突触可塑性相关谷氨酸(Glu)和γ-氨基丁酸(GABA)等受体蛋白/基因表达变化的影响,探讨电针镇痛的作用机制。方法:Wistar大鼠随机分为正常组、模型组、电针组、麻醉电针组,每组14只(6只用于免疫荧光染色,8只用于基因检测)。结扎坐骨神经结合足底反复电刺激造成慢性神经痛负性情绪模型。电针双侧"足三里""阳陵泉",每日1次,共7d。测量大鼠双侧足底热缩足反应潜伏期(PWL),计算其差值(PWLD)及条件控制箱停留时间。采用荧光定量PCR法检测右侧杏仁核GABA受体亚型Aβ2、B 1,Glu N-甲基-D-天门冬氨酸受体亚型NR 1(NMDA-NR 1),突触后密度蛋白(PSD)-95,突触蛋白Piccolo等基因的表达;用免疫荧光法检测杏仁基底外侧核(BLA)代谢型Glu受体亚型1(mGluR 1)及GABAB 2的表达。结果:与正常组比较,模型组PWLD显著增加(P<0.001),条件控制箱停留时间显著减少(P<0.001);与模型组比较,电针3d和7d后,电针组和麻醉电针组PWLD显著降低(P<0.05),条件控制箱停留时间显著增加(P<0.05);与电针组比较,电针3d后,麻醉电针组大鼠在条件控制箱停留时间明显缩短(P<0.01),说明电针改善了痛反应和负性情绪。与正常组比较,模型组大鼠杏仁核GABAAβ2、GABAB 1、PSD-95、Piccolo基因表达及BLA内GABAB 2阳性细胞数显著降低(P<0.05,P<0.001)。电针7d后,与模型组比较,电针组上述5个分子以及杏仁核NMDA-NR 1基因表达水平及麻醉电针组除了Piccolo外的4个基因表达水平均显著升高(P<0.05,P<0.001),电针组BLA内mGluR 1及GABAB 2阳性细胞数量显著增多(P<0.001);麻醉电针组Piccolo基因、mGluR 1及GABAB 2免疫阳性细胞数量显著低于电针组(P<0.001)。结论:重复电针可明显改善慢性痛大鼠感觉成分和情绪成分,其改善感觉成分的效应可能与上调大鼠杏仁核NMDA-NR 1、GABAAβ2、GABAB 1、PSD-95、Piccolo基因及mGluR 1、GABAB 2蛋白表达有关,其改善痛情绪成分的效应可能与其上调mGluR 1、GABAB 2蛋白和Piccolo基因的表达有关。

Objective To observe the effect of electroacupuncture（EA）on expression of synaptic plasticity-related glutamate N-methyl-D-aspartate（NMDA）receptor subunit NR 1,gamma-aminobutyric acid（GABA）receptor subunits（Aβ2,B 1）,etc.in the amygdala in chronic neuropathic pain negative affection（CNPPNA）rats,so as to reveal its mechanism underlying pain relief.Methods Male Wistar rats were randomized into normal control,CNPPNA model,EA,and anesthesia＋EA（AEA）groups（n=14in each group,8for quantitative RT-PCR and 6for immunofluorescence staining）.The CNPPNA model was established by ligation of the left sciatic nerve and repeated electrical stimulation of the hindpaw plantar skin in the pain-paired compartment.EA was applied to bilateral＂Zusanli＂（ST 36）and＂Yanglingquan＂（GB 34）for 30 min,once daily for 7days.Thermal pain threshold（paw withdrawal latency,PWL）of the bilateral paws was measured by using a Tail-Flick Unit.The conditioned place aversion（CPA）was determined by using a CPA-paired compartment.The expression levels of GABAAβ2,GABAB 1,NMDA receptor subunit NR 1,postsynaptic density-95protein（PSD-95）,Piccolo genes in the right amygdala area were determined using quantitative RT-PCR,and the immunoactivity of metabotropic glutamate receptor subunit 1（mGluR 1）and GABAB 2in the basolateral amygdala（BLA）nucleus was detected using immunofluorescence staining.Results After modeling,PWL difference（PWLD）values of the model group were significantly increased（P0.001）,and the time spent in the CPA-paired compartment was considerably decreased compared with the control group（P0.001）.After EA intervention for 3and 7days,the PWLD levels of both EA and AEA groups were apparently decreased（P0.05）,and the time spent in the CPA-paired compartment was apparently increased in the EA and AEA groups（P0.05）,suggesting apain relief and an improvement of the negative affection after EA intervention.Additionally,following EA,the apparently-decreased expression levels of GABAAβ2,GABAB 1,PSD-95,Piccolo genes and the reduced numbers of GABAB 2positive cells and NMDA-NR 1 mRNA as well as mGluR 1positive fiber numbers were remarkably increased in the EA group（P0.05,P0.001）.The expression levels of Piccolo gene,GABAB 2and mGluR 1positive cells/fiber numbers were apparently lower in the AEA group than in the EA group（P0.001）.No significant differences were found between the EA and AEA groups in the PWLD,time spent in the CPA-paired compartment,and the expression levels of NMDA-NR 1,GABAAβ2,GABAB 1and PSD-95genes（P0.05）.Conclusion Repeated EA stimulation of ST 36-GB 34 has a role in relieving both sensory and affection dimensions of chronic pain in CNPPNA rats,which may be respectively related to its effects in up-regulating the expression of GABAAβ2,GABAB 1,NMDA-NR 1,PSD-95 and Piccolo genes,and in promoting the expression of mGluR 1and GABAB 2proteins and Piccolo gene in the amygdala.