基于星形胶质细胞NDRG2/GLT-1通路探讨推拿对神经病理痛大鼠脊髓背角谷氨酸摄取及突触间隙的影响  

Effects of Tuina on Glutamate Uptake in Spinal Dorsal Horn and Synaptic Cleft in Rats ofNeuropathological Pain Based on Astrocyte NDRG2/GLT-1 Pathway

作　　者：张幻真 黄丽梅 林志刚 陈水金 陈乐春 江煜 陈进城 蒋晶晶 ZHANG Huanzhen;HUANG Limei;LIN Zhigang;CHEN Shuijin;CHEN Lechun;JIANG Yu;CHEN Jincheng;JIANG Jingjing(Affiliated Rehabilitation Hospital,Fujian University of Traditional Chinese Medicine,Fuzhou 350003,China;Fujian Key Laboratory of Rehabilitation Technology,Fuzhou 350003,China)

机构地区：[1]福建中医药大学附属康复医院,福州350003 [2]福建省康复技术重点实验室,福州350003

出　　处：《世界科学技术-中医药现代化》2024年第8期2125-2132,共8页Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology

基　　金：国家自然科学基金委员会青年科学基金项目(82105039):基于NDRG2/GLT-1介导星形胶质细胞调控突触可塑性探讨推拿干预腰椎间盘突出症的镇痛机制,负责人:张幻真;福建省科技厅自然科学基金面上项目(2022J01881):基于NDRG2-NMDAR介导星形胶质细胞-神经元通讯探讨委中穴推拿治疗腰椎间盘突出症疼痛的机制,负责人:张幻真;福建省卫健委医学创新课题(2020CXA052):推拿点按干预神经病理性疼痛大鼠脊髓背角AMPA/NMDA受体-支架蛋白以及突触可塑性的影响,负责人:陈水金。

摘　　要：目的 基于星形胶质细胞NDRG2/GLT-1通路探讨推拿对神经病理痛大鼠脊髓背角谷氨酸摄取及突触间隙的影响,阐释推拿的潜在镇痛机制。方法 随机将54只SD大鼠分为空白组、模型组和推拿组(每组18只),模型组建立坐骨神经慢性压迫损伤(Chronic constrictive injury,CCI)模型,推拿组在CCI模型制备成功后第4天起接受按揉“委中”穴干预,连续干预14天。观察每组大鼠造模前后不同时间点机械痛阈值的变化,评估推拿的镇痛效应。采用免疫荧光法观察脊髓背角N-myc下游调控基因2(N-myc downstream regulated gene2,NDRG2)、谷氨酸转运体1(Glutamatetransporter1,GLT-1)与星形胶质细胞标志物胶质纤维酸性蛋白(Glail fibrillary acidicprotein,GFAP)的共表达情况;荧光定量PCR法检测星形胶质细胞中NDRG2、GLT-1 mRNA的水平;液相色谱串联质谱法检测突触间隙谷氨酸的浓度;透射电镜观察突触间隙的宽度。结果 CCI造模后,模型组较空白组机械痛阈值持续下降(P<0.01),脊髓背角NDRG2与GFAP共标阳性细胞数显著增加(P<0.01),GLT-1与GFAP共标阳性细胞数显著减少(P<0.01);星形胶质细胞中NDRG2 mRNA表达显著上调(P<0.01),GLT-1 mRNA表达显著下降(P<0.01);谷氨酸浓度显著增高(P<0.01);突触间隙明显缩窄(P<0.01)。推拿干预后,显著逆转上述趋势,推拿组较模型组于CCI术后11天起机械痛阈值上升(P<0.01),脊髓背角NDRG2与GFAP共标阳性细胞数明显减少(P<0.01),GLT-1与GFAP共标阳性细胞数明显增多(P<0.01);并下调星形胶质细胞中NDRG2 mRNA表达(P<0.01),恢复GLT-1 mRNA表达(P<0.01);谷氨酸浓度降低(P<0.05);突触间隙相对增宽(P<0.05)。结论 推拿可能通过促进星形胶质细胞NDRG2/GLT-1通路摄取谷氨酸这一过程,恢复突触间隙宽度,逆转突触可塑性,在脊髓层面减轻CCI模型大鼠的疼痛。Objective To observe the effect of tuina on glutamate uptake and synaptic cleft in the spinal dorsal horn of rats with neuropathic pain through astrocyte NDRG2/GLT-1 pathway,and to explore the potential analgesic mechanism of tuina on neuropathic pain.Methods A total of 54 SD rats were randomly divided into naive group,model group and tuina group(n=18).The CCI model was established in the model group,and the tuina group was treated with acupressure at"Weizhong"(BL 40)from the 4th day after CCI model was successfully established for 14 days.The changes of paw withdrawal threshold at different time points were observed to evaluate the analgesic effect of tuina.Immunofluorescence was used to observe the co-expression of NDRG2ˎGLT-1 and astrocytes in the spinal dorsal horn.The mRNA levels of NDRG2 and GLT-1 in astrocytes were detected by quantitative real time polymerase chain reaction.The concentrations of glutamate in synaptic cleft were measured by liquid chromatography coupled to tandem mass spectrometry.The width of the synaptic cleft was observed by transmission electron microscopy.Results Compared with the naive group,the paw withdrawal threshold in the CCI group decreased continuously(P<0.01).The number of NDRG2 and GFAP colabeling positive cells in the spinal dorsal horn increased significantly(P<0.01),and the number of GLT-1 and GFAP co-labeled positive cells was significantly reduced(P<0.01).NDRG2 mRNA expression was up-regulated and GLT-1 mRNA expression decreased in astrocytes(P<0.01).The concentrations of glutamate increased significantly(P<0.01);The synaptic cleft was significantly narrowed(P<0.05).After tuina intervention,the above trend was significantly reversed.Compared with the model group,the paw withdrawal threshold of the tuina group increased from 11 days after CCI(P<0.01),the number of NDRG2 and GFAP co-labeling positive cells in the spinal dorsal horn was significantly reduced(P<0.01),and the number of GLT-1 and GFAP co-labeled positive cells increased significantly(P<0.01);down-regulated t

关 键 词：推拿 神经病理痛 星形胶质细胞 突触 镇痛机制 

分 类 号：R285.5[医药卫生—中药学]