电针对痛负性情绪大鼠杏仁核细胞突触可塑性及相关蛋白表达的影响

发布时间：2018-06-25 13:20

本文选题：电针 + 慢性神经痛　；参考：《中国中医科学院》2017年硕士论文

【摘要】：研究背景疼痛是一种与组织损伤相关联的不愉快的感觉和情绪体验,含有感觉、情绪、认知等多个维度。近年来,有关痛情绪及痛认知障碍的研究受到了越来越多的关注。众所周知:边缘系统参与脑记忆、感觉和情绪活动。杏仁核是边缘系统的一个重要核团,被称作“情绪脑”,是脑内调节与焦虑、恐惧相关行为、自主活动和激素水平等生理反应的关键结构。慢性疼痛除了疼痛过敏、认知、精神心理的变化外,还伴有大脑结构和功能的改变。近年来的研究结果表明:慢性痛可导致脑结构及功能的可塑性变化,脑解剖学结构重组,脑皮层灰质密度的改变。慢性痛中杏仁核突触可塑性功能发生变化可能与突触前后蛋白变化有关,谷氨酸是神经系统中主要的兴奋性神经递质,丫-氨基丁酸(GABA)是中枢神经系统主要的抑制性神经递质,突触后密度蛋白95(PSD-95)是突触后致密物中的重要结构蛋白,可将位于细胞膜上的谷氨酸N-甲基-D-天冬氨酸受体(NMDAR)和位于细胞内的信号转导系统联系起来,突触前基质蛋白Piccolo蛋白位于突触前细胞质基质活性区,在大脑神经递质传递中有重要作用。针刺作为缓解慢性痛的有效且方便快捷的治疗方法,在临床上疗效显著。随着对痛情绪在慢性痛发展过程中重要性的研究,发现针刺可以治疗疼痛诱发的情绪改变,针刺可以缓解抑郁、焦虑、失眠等情绪障碍性疾病以及疼痛诱发的负性情绪。我们前期研究观察到:重复电针改善慢性神经痛性负性情绪大鼠疼痛的感觉成分和痛情绪成分的作用可能分别与其上调杏仁核促皮质释放激素受体(CRF)-1R、(CRF)-2R、谷氨酸N-甲基-D-门冬氨酸(NMDA)受体亚型NR2B、γ-氨基丁酸(GABA)受体亚型A和C基因表达和阿片μ受体(MOR)蛋白表达,抑制谷氨酸受体GluA1的降低有关,但其机制仍远不清楚。本研究拟在同一慢性疼痛负性情绪模型上,观察电针“足三里”-“阳陵泉”对动物痛行为的感觉成分和情感成分的影响,它与杏仁核神经细胞突触可塑性相关分子GABA受体亚型A β 2,B1,Glu-NMDA受体亚型NR1,突触蛋白PSD95及Piccolo表达变化的关系;杏仁核内微量注射GABAA及GABAB受体拮抗剂,验证它们参与电针改善动物痛感觉成分和痛情感成分效应的情况,并用透射电镜观察杏仁核细胞突触超微结构的变化,试图从结构和功能相结合的角度,探讨杏仁核介导的针刺缓解慢性痛的作用机制。材料与方法第一部分实验:健康雄性Wistar大鼠56只(230g-250g),随机分为4组:正常组,CCI+NA(chronic compressive injury+NA)模型组,CCI+NA+ 电针(EA)组,CCI+NA+麻醉电针(AEA)组,其中每组8只用于Real-timePCR,其余6只用于Western Blot检测。除正常组外,其余各组大鼠行左下肢坐骨神经结扎术,恢复三天后进行足底电刺激造成负性情绪模型。电针组和麻醉电针组电针刺激双侧“足三里”、“阳陵泉”,1mA,2/15Hz,每天1次,每次30分钟,共7天。电针干预后,即刻将放入条件控制箱20分钟,大于两小时后,放于非条件控制箱20分钟。采用痛觉测定仪测定大鼠双侧足底热辐射痛阈值,取双侧缩足潜伏期差值(PWLD),采用条件性位置偏恶系统测定痛情绪方面。动物直接取材后分别采用Real-time PCR、Western Blot检测杏仁核内突触可塑性相关分子GABAAβ2,GABAB,,Glu-NMDA-NR1,PSD95 及 Piccolo 基因及蛋白表达。第二部分实验:健康雄性Wistar大鼠50只(230g-250g),随机分成5组:正常组,CCI+NA+生理盐水组,CCI+NA+EA+GABAA抑制剂(Flumazenil)组,CCI+NA+EA+GABAB抑制剂(Phaclofen)组,CCI+NA+EA+DMSO溶剂组,每组10只(数据完整者6只)。正常组除外,将其余各组大鼠左下肢坐骨神经结扎。恢复5天后,除正常组外,余下4组在脑立体定位仪上实施双边杏仁核埋管(坐标:P:2.5mm,L,R:4.5mm,H:7.7mm),7天后造负性情绪模型,双侧分别微量注射生理盐水0.5 μL/次/侧、GABAA抑制剂 Flumazenil(2 μ g/μ L)0.5 u L/次/侧、GABAB抑制剂(Phaclofen)(2μ g/μ L)0.5 μ L/次/侧、DMS0 溶剂(5 u g/μ L)0.5 μ L/次/侧,推进速度 0.1 μL/min,注射完成之后,将大鼠固定于鼠架上电针刺激双侧“足三里-阳陵泉”参数为1mA,2/15Hz,每天1次,每次30分钟,共7天。电针结束后,测定大鼠双侧足底热辐射痛阈值,计算双侧缩足潜伏期差值(PWLD)及大鼠在条件控制箱停留时间。第三部分实验:健康雄性Wistar大鼠16只(230g-250g),实验动物分组、模型制备、电针和麻醉电针干预、热痛阈测定、条件位置偏恶检测同实验一,用4%多聚甲醛和2%戊二醛混合后灌流后取材,运用透射电镜技术观察杏仁核细胞突触界面相关参数的变化。每个样本任选15个视野拍照。每个视野任选1个突触前、后膜及突触囊泡较为清晰的突触,采用图像分析软件OPTPro3000。统计学处理:所得实验数据为计量资料,以均数±标准差(M±SD)表示,用SPSS16.0统计软件进行数据处理,采用重复性单因素方差分析(行为学数据采用重复测量方差分析),组间两两比较选用LSD检验,P0.05表示差异有统计学意义。实验结果:1电针对慢性痛大鼠痛感觉和情绪成分行为反应的影响造模后(CCI+NA),除正常组外,其余各组大鼠PWLD显著增加(P0.001),在条件控制箱停留时间显著缩短(P0.001),提示:动物痛阈降低,负性情绪产生。与模型组比较,电针和麻醉电针3天和7天后PWLD显著降低(P0.05),在条件控制箱停留时间显著延长(P0.05)。电针组与麻醉电针组相比,两组PWLD差异无统计学意义(P0.05),而麻醉电针3天后条件控制箱停留时间明显缩短(P0.05),7天后两组条件控制箱停留时间差异无统计学意义(P0.05),提示电针改善了痛反应的感觉成分和负性情绪成分。2电针对杏仁核突触可塑性相关分子GABAAβ2,GABAB1,Glu-NMDA-NR1,PSD95及Piccolo基因及蛋白表达的影响定量RT-PCR及Western Blot的结果表明,造模后模型组杏仁核内GABAAβ2,GABAB1,PSD95基因及蛋白表达显著降低(P0.05),Piccolo基因表达水平显著降低(P0.05),谷氨酸NMDA-NR1基因表达有上升趋势(P0.05),其蛋白表达明显上升(P0.05)。电针后,与模型组相比,电针组NMDA-NR1、GABAAβ2,,GABAB1,PSD95基因及蛋白表达明显升高(P0.05),Piccolo基因表达明显上升(P0.05);而麻醉电针组NMDA-NR1、GABAAβ2,GABAB1、PSD95基因表达水平较模型组显著上调(P0.05),Piccolo基因表达明显下降(P0.001)。这些结果提示:NMDA-NR1,GABAAβ2,GABAB1,PSD95,Piccolo可能参与针刺改善慢性神经痛感觉的成分,Piccolo有可能还参与电针改善慢性痛负性情绪的作用。3杏仁核内注射GABAA和GABAB受体拮抗剂验证其参与针刺改善痛感觉和/或痛情绪的作用造模后,除正常组外,其余各组大鼠PWLD值显著增加(P0.001),电针3天后,与模型组相比,DMS0溶剂对照电针组PWLD明显缩短(P0.001),提示该溶剂不影响电针改善痛感觉成分的效应,其余各组未见明显变化(P0.05);与GABAA抑制剂电针组、GABAB抑制剂电针组相比较,DMSO溶剂对照电针组PWLD明显缩短(P0.01)。电针7天后,与模型组相比,DMSO溶剂对照电针组PWLD明显缩短(P0.001),GABAA抑制剂电针组、GABAB抑制剂电针组有下降趋势,但无统计学差异。GABAA抑制剂电针组、GABAb抑制剂电针组组间无统计学差异。即说明杏仁核内注射GABAA和GABAB抑制剂,使电针的镇痛效应明显减弱。造模后,各组大鼠在条件控制箱停留时间显著减少(P0.001),电针3天后,与模型组相比,DMSO溶剂电针组在条件控制箱停留时间显著增加(P0.01),提示该溶剂不影响电针改善痛情绪成分的效应,其余各组未见明显变化(P0.05)。与GABAA抑制剂电针组、GABAB抑制剂电针组相比较,DMSO电针组在条件控制箱停留时间显著增加(P0.01)。电针7天后,与模型组相比,DMSO电针组在条件控制箱停留时间显著增加(P0.001),GABAA抑制剂电针组、GABAb抑制剂电针组有升高趋势,但无统计学差异(P0.05)。GABAA抑制剂电针组、GABAb抑制剂电针组组间无统计学差异(P0.05)。说明在杏仁核内阻断GABAA、GABAb受体后,削弱了电针改善模型大鼠痛情绪的作用。上述结果表明:GABAA和GABAB均参与电针改善慢性神经痛负性情绪大鼠的痛感觉及痛情绪成分的效应。4电针对慢性痛负性情绪大鼠杏仁核突触结构变化的影响与正常组比,模型组大鼠杏仁核突触活性带长度、突触后致密物厚度和突触界面曲率明显减少(P0.001),突触间隙明显增宽(P0.001);与模型组比,电针以及麻醉电针后,大鼠杏仁核突触活性带长度明显增加(P0.001),突触后致密物厚度明显增加(P0.001),突触间隙宽度明显减少(P0.001,P0.01),电针组突触界面曲率增大(P0.05),麻醉电针组突触界面曲率无明显变化(P0.05);电针组与麻醉电针组杏仁核突触界面参数变化无统计学差异(P0.05)。说明电针可明显改善慢性神经痛负性情绪大鼠杏仁核突触间隙的增宽,突触前活性带的长度、突触后致密物(PSD)厚度及突触界面的曲率的减低。小结1、电针“足三里-阳陵泉”可以改善神经病理性慢性痛负性情绪大鼠痛的感觉成分和情感成分的行为学反应。2、电针改善痛感觉成分的作用可能与上调大鼠杏仁核NMDA-NR1、GABAAβ2,GABAb1、PSD95基因及蛋白表达水平及Piccolo基因表达水平有关,电针改善痛情绪成分的作用可能与上调大鼠杏仁核Piccolo基因表达水平有关。3、阻断杏仁核内GABAA以及GABAB后,电针的镇痛效应以及改善痛情绪的作用明显减弱,GABAA和GABAb受体可能同时参与了痛感觉成分与情感成分的调节。4、电针可以改善慢性痛引发的杏仁核神经细胞突触可塑性变化,调节杏仁核突触界面,明显减小突触间隙,增加突触前活性带的长度、突触后致密物(PSD)厚度及突触界面的曲率。其改善突触可塑性可能与其调节突触可塑性相关蛋白的表达有关。
[Abstract]:Background pain is a kind of unpleasant sensation and emotional experience associated with tissue damage, including sensory, emotional, cognitive and other dimensions. In recent years, more and more attention has been paid to the research on pain and cognitive impairment. It is known that the marginal system is involved in brain memory, sensory and emotional activity. The amygdala is a marginal system. An important nuclear group, known as the "emotional brain", is the key structure of physiological responses such as regulation and anxiety, fear related behavior, autonomic activity, and hormone levels in the brain. Chronic pain is accompanied by changes in pain allergy, cognitive, mental and mental changes, and changes in brain structure and function. The changes in the plasticity of the brain structure and function, the reorganization of the brain anatomy, the change in the density of the gray matter in the cortex. The changes in the synaptic plasticity in the amygdala may be related to the changes in the pre and post synaptic proteins in the chronic pain. Glutamate is the main excitatory neurotransmitter in the nervous system, and GABA is the main central nervous system. The inhibitory neurotransmitter, postsynaptic density protein 95 (PSD-95) is an important structural protein in the postsynaptic densification. It can connect the glutamic acid N- methyl -D- aspartic acid receptor (NMDAR) on the cell membrane and the signal transduction system in the cell. The presynaptic protein Piccolo protein is located in the pre synaptic cytoplasmic matrix activity. Areas have an important role in the transmission of neurotransmitters in the brain. Acupuncture as an effective and convenient and quick treatment to alleviate chronic pain has a significant clinical effect. With the study of the importance of pain mood in the process of chronic pain development, acupuncture can be used to treat pain induced mood changes, and acupuncture can relieve depression, anxiety, insomnia and so on. The effect of repeated electroacupuncture on the improvement of the sensory and emotional components of pain in chronic neuropathic negative emotional rats may be compared with the up regulation of the amygdalotropin receptor (CRF) -1R, (CRF) -2R, and N- methyl -D- aspartate (NMD), respectively. A) receptor subtype NR2B, gamma aminobutyric acid (GABA) receptor subtype A and C gene expression and opioid receptor (MOR) protein expression, inhibit the decrease of glutamate receptor GluA1, but its mechanism is still far from clear. This study is to observe the feeling of "Zusanli" - "Yang Ling spring" on animal pain behavior in the same chronic negative emotional model. The effects of components and emotional components on the relationship between the synaptic plasticity related molecular GABA receptor subtype A beta 2, B1, Glu-NMDA receptor subtype NR1, the synaptic protein PSD95 and Piccolo expression in the amygdala nerve cells, and the microinjection of GABAA and GABAB receptor antagonists in the amygdala to verify their involvement in electroacupuncture in improving pain sensory components and pain in animals. The changes in the synaptic ultrastructure of the amygdala cells were observed by transmission electron microscopy. The mechanism of amygdala mediated acupuncture for alleviating chronic pain was explored from the angle of combination of structure and function. The first part of the materials and methods: 56 healthy male Wistar rats (230g-250g) were randomly divided into 4 groups: normal group, C The CI+NA (chronic compressive injury+NA) model group, the CCI+NA+ electroacupuncture (EA) group, and the CCI+NA+ anesthesia electroacupuncture (AEA) group, of which 8 were used in Real-timePCR and the other 6 were used for Western Blot detection. Except the normal group, the rest of the rats were ligation of the left lower extremities, and the electrical stimulation of the foot caused the negative emotional model for three days. The acupuncture group and the electroacupuncture group stimulated bilateral "Zusanli", "Yang Ling spring", 1mA, 2/15Hz, 1 times a day, 30 minutes each time, for a total of 7 days. The prognosis of electroacupuncture would be put into the condition control box for 20 minutes, after more than two hours, and placed in the non conditioned control box for 20 minutes. The difference in the incubation period (PWLD) and the conditioned position deviation system were used to determine the pain mood. After direct sampling, Real-time PCR and Western Blot were used to detect the synaptic plasticity related molecules GABAA beta 2, GABAB, Glu-NMDA-NR1, PSD95 and Piccolo genes and protein expression in the amygdala. The second part of the experiment: healthy male Wistar rats 50 (230g-250g) were randomly divided into 5 groups: normal group, CCI+NA+ saline group, CCI+NA+EA+GABAA inhibitor (Flumazenil) group, CCI+NA+EA+GABAB inhibitor (Phaclofen) group, CCI+NA+EA+DMSO solvent group, 10 rats in each group (6 patients with complete data). The rest of the left lower extremities were ligated in the rest of the rats. 5 days later, except the normal group, The remaining 4 groups carried out bilateral amygdala buried tube (coordinates: P:2.5mm, L, R:4.5mm, H:7.7mm) on the stereotaxis, and 7 days later, the negative emotional model was made. The bilateral injection of physiological saline was 0.5 mu L/ / side, GABAA inhibitor Flumazenil (2 mu L) 0.5 u L/ / side, GABAB inhibitor (2 mu) 0.5 micron / side, 5 solvent (5) U g/ mu L) 0.5 mu L/ sub / side, propelling speed of 0.1 mu L/min, after the injection completed, the rats were fixed on the rat frame to stimulate bilateral "Zusanli Yang spring" parameters to be 1mA, 2/15Hz, 1 times a day, 30 minutes a time for 7 days. After the end of the electroacupuncture, the threshold of the bilateral foot heat radiation pain was measured, and the difference of the bilateral foot latent period (PWLD) and the large amount were calculated. Rats in the condition control box residence time. Third part experiment: 16 healthy male Wistar rats (230g-250g), experimental animals group, model preparation, electroacupuncture and anaesthesia electroacupuncture intervention, thermal pain threshold determination, condition location deviation detection and experiment one, after mixed with 4% polyformaldehyde and 2% amyl two aldehyde, after perfusion, using transmission electron microscope technique to observe almond Changes in the parameters related to the synaptic interface of the nuclear cells. Each sample selected 15 fields of vision to take pictures. Each field of vision was selected 1 presynaptic presynaptic, posterior membrane and synaptic vesicles with a clearer synapse, using the image analysis software OPTPro3000. statistics processing: the obtained experimental data were measured with a mean number of standard deviation (M + SD), and SPSS16.0 statistical software was used. Data processing, repeated single factor analysis of variance (behavior data using repeated measurement of variance analysis), 22 comparison between groups selected LSD test, P0.05 indicated that the difference was statistically significant. Experimental results: 1 electroacupuncture on the pain sensation and emotional components of chronic pain in rats (CCI+NA), except the normal group, the rest of the other groups The PWLD of rats increased significantly (P0.001), the retention time of the condition control box was significantly shortened (P0.001), suggesting that the pain threshold of the animal decreased and the negative emotion produced. Compared with the model group, the PWLD decreased significantly (P0.05) for 3 days and 7 days after the electroacupuncture and Electroacupuncture (P0.05), and the retention time in the condition control box was significantly prolonged (P0.05). Compared with the electroacupuncture group, the two groups of PWLD were compared with the electroacupuncture group. The difference was not statistically significant (P0.05), but the retention time of the condition control box was significantly shortened (P0.05) after 3 days of anaesthetized electroacupuncture (P0.05), and there was no significant difference in the residence time between the two groups of condition control cases (P0.05) after 7 days, suggesting that electroacupuncture improved the sensory components of the pain response and the negative emotional formation of.2 electroacupuncture on the amygdala synaptic plasticity related molecule GABAA beta 2, GABA The effects of B1, Glu-NMDA-NR1, PSD95 and Piccolo gene and protein expression on the quantitative RT-PCR and Western Blot showed that the expression of GABAA beta 2, GABAB1, PSD95 genes and protein expression in the amygdala was significantly reduced (P0.05), and the expression level of Piccolo genes decreased significantly after the model group. After electroacupuncture, the expression of NMDA-NR1, GABAA beta 2, GABAB1, PSD95 gene and protein expression in the electroacupuncture group increased significantly (P0.05), and the expression of Piccolo gene increased significantly (P0.05) compared with the model group. The expression level of the gene expression in the anesthetic electroacupuncture group was significantly higher than that in the model group. P0.001. These results suggest that NMDA-NR1, GABAA beta 2, GABAB1, PSD95, Piccolo may be involved in acupuncture improvement of chronic neuralgia, and Piccolo may also be involved in the effect of electroacupuncture in improving chronic pain and negative emotions. The.3 amygdala is injected with GABAA and GABAB receptor antagonists to verify their participation in acupuncture to improve pain sensation and / or pain mood. In addition to the normal group, the PWLD value of the rest of the rats increased significantly (P0.001). After 3 days of electroacupuncture, the PWLD in the DMS0 solvent control group was significantly shorter than that in the model group (P0.001), suggesting that the solvent did not affect the effect of Electroacupuncture on the improvement of pain sensory components, and the other groups did not change significantly (P0.05); and GABAA inhibitor electroacupuncture group, GABAB suppression. Compared with the electroacupuncture group, the PWLD in the DMSO solvent control group was significantly shorter (P0.01). Compared with the model group, the PWLD in the electroacupuncture group was significantly shorter than the model group (P0.001). The GABAA inhibitor electroacupuncture group and the GABAB inhibitor electroacupuncture group had a downward trend, but there was no statistical difference between the.GABAA inhibitor electroacupuncture group and the GABAb inhibitor electroacupuncture group. The statistical difference. That means the injection of GABAA and GABAB inhibitors in the amygdala made the analgesic effect of the electroacupuncture significantly weakened. After the model, the residence time of the rats in the condition control box was significantly reduced (P0.001). After 3 days of electroacupuncture, the retention time of the DMSO solvent electroacupuncture group increased significantly (P0.01), suggesting that the solvent was not in shadow. The effect of electroacupuncture improved pain emotion components, and the rest of the other groups did not change significantly (P0.05). Compared with the GABAA inhibitor electroacupuncture group, GABAB inhibitor electroacupuncture group, the retention time of the DMSO electroacupuncture group increased significantly (P0.01). After 7 days of electroacupuncture, the retention time of the DMSO Electroacupuncture group was significantly increased (P0.001), G ABAA inhibitor electroacupuncture group, GABAb inhibitor electroacupuncture group has a rising trend, but there is no statistical difference (P0.05).GABAA inhibitor electroacupuncture group, GABAb inhibitor electroacupuncture group there is no statistical difference between groups (P0.05). Indicating that the blockage of GABAA in the amygdala, GABAb receptor weakens the effect of electroacupuncture to the model rats' pain emotion. The results show that GABAA and GA are: GABAA and GA BAB all participated in the effect of Electroacupuncture on the pain and emotional components of the chronic neuralgia negative emotional rats. The effect of.4 electroacupuncture on the synaptic structural changes in the amygdala of the chronic painful negative emotional rats was compared with the normal group. The length of the synapse active zone in the amygdala, the thickness of the postsynaptic density and the curvature of the synaptic interface decreased significantly (P0.001). The synaptic gap was significantly widened (P0.001), and the synaptic active zone length of the amygdala was significantly increased (P0.001), the thickness of the postsynaptic density increased significantly (P0.001), the synaptic gap width decreased significantly (P0.001, P0.01), the synaptic interface curvature increased (P0.05) in the electroacupuncture group (P0.05), and the synaptic interface curvature of the electroacupuncture group. There was no significant change (P0.05); there was no significant difference in the synaptic interface parameters between the electroacupuncture group and the anaesthetized electroacupuncture group (P0.05), indicating that the electroacupuncture could obviously improve the widening of the synaptic gap in the amygdala, the length of the presynaptic active band, the thickness of the postsynaptic density (PSD) and the curvature of the synapse interface in the chronic neuropathic rats. 1, Electroacupuncture "Zusanli Yang Ling spring" can improve the behavioral response of the sensory and emotional components of neuropathic chronic pain negative emotional rats. The effect of Electroacupuncture on the improvement of pain sensory components may be related to the up-regulation of NMDA-NR1, GABAA beta 2, GABAb1, PSD95 gene, the level of egg white and the level of Piccolo gene expression in the amygdala of rats. The effect of Electroacupuncture on improving the emotional components of pain may be related to the increase of the expression level of Piccolo gene in the amygdala of rats. After blocking the GABAA and GABAB in the amygdala, the analgesic effect of the electroacupuncture and the effect of improving the pain mood are obviously weakened. The GABAA and GABAb receptors may participate in the regulation of.4 in the sense of pain and emotional components at the same time, and the electroacupuncture can be changed. The synaptic plasticity changes in amygdala nerve cells induced by good chronic pain, modulate the synaptic interface of the amygdala, reduce the synaptic gap, increase the length of the presynaptic active band, the thickness of the postsynaptic density (PSD) and the curvature of the synaptic interface. The improvement of synaptic plasticity may be related to the regulation of the expression of synaptic plasticity related protein.
【学位授予单位】：中国中医科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R245

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