电针对肌萎缩侧索硬化症模型小鼠皮质小胶质细胞/神经元活性影响的机制研究  

Mechanism of electroacupuncture on microglial and cortical neurons in mice with amyotrophic lateral sclerosis model

作　　者：赵蔚佳 曾佳玮 刘隽阳 刘珊珊 李泐 李杰 冯卫星 赵颖倩 王强 ZHAO Weijia;ZENG Jiawei;LIU Junyang(The Second Clinical Medical College,Shanxi University of Chinese Medicine,Xianyang,Shaanxi,712046)

机构地区：[1]陕西中医药大学第二临床医学院,陕西省咸阳市712046 [2]陕西中医药大学针灸推拿学院 [3]陕西省针药结合重点实验室 [4]陕西中医药大学附属医院

出　　处：《中国康复医学杂志》2025年第2期171-179,共9页Chinese Journal of Rehabilitation Medicine

基　　金：国家自然科学基金项目(82074555);陕西省教育厅重点实验室项目(21JS022)。

摘　　要：目的:观察电针干预对肌萎缩侧索硬化小鼠(amyotrophic lateral sclerosis,ALS)小胶质细胞和大脑皮质兴奋性的影响,探讨电针干预改善ALS动物模型运动功能的中枢机制。方法:(1)按随机数字法将16只h SOD1G93A小鼠分为模型组和电针组,hSOD1G93A阴性小鼠为空白组,每组8只。电针组电针干预百会、双侧天柱、天枢。10min/次,5次/周,1周为1个疗程,连续3个疗程。采用后肢紧抱实验、旷场实验评估各组小鼠运动功能;ELISA法检测血清炎症因子IL-1β及IL-6的含量;免疫荧光染色法和流式细胞术观察大脑皮质Iba-1阳性细胞率及细胞表型。(2)21只h SOD1G93A小鼠,7只同窝野生鼠。向小鼠大脑皮质M1区注射化学遗传兴奋病毒[rAAV-CaMKIIa-hM3D(Gq)-EGFP-WPRE-hGH polyA],待病毒表达21天后,随机选取3只SOD1G93A小鼠和1只h SOD1G93A阴性小鼠于荧光显微镜下观察病毒表达情况,将剩余SOD1G93A小鼠随机分为3组:模型组、电针组和化学遗传兴奋病毒+电针组[腹腔注射氯氮平-N-氧化物(CNO)+电针],每组6只。采用免疫荧光法观察大脑皮质c-Fos阳性细胞率,旷场实验评估各组小鼠运动功能。结果:与空白组比较,模型组小鼠旷场运动总距离缩短、神经学评分升高(P<0.01),血清IL-1β、IL-6含量、大脑皮质Iba-1、c-Fos阳性细胞率均升高(P<0.01),M1型、M2型小胶质细胞占比升高(P<0.01,P<0.05)。与模型组比较,电针组小鼠旷场运动总距离延长、神经学评分降低(P<0.01,P<0.05),血清IL-1β、IL-6含量、大脑皮质Iba-1、c-Fos阳性细胞率均降低(P<0.01,P<0.05),M2型小胶质细胞占比升高(P<0.05);病毒+电针组c-Fos阳性细胞率降低、旷场运动总距离延长。结论:电针干预可以改善早期ALS模型小鼠运动功能,其机制可能与抑制小胶质细胞的活性,降低炎症因子表达,降低M1皮质神经元兴奋性有关。Objective:To observe the effect of electroacupuncture(EA)on microglia and cortical excitability in hSOD1G93A mice and to explore the potential central mechanisms of EA intervention to improve motor function in amyotrophic lateral sclerosis(ALS)mice.Method:The present study includes 2 parts.①Sixteen hSOD1G93A mice were randomly divided into model group and EA group(n=8 per group).Eight hSOD1G93A-negative mice were used as the blank group.The EA group was applied to the Baihui,bilateral Tianzhu and bilateral Tianshu acupoints(10 minutes/session,5 sessions/week,for 3 weeks).After 3 weeks of EA,hind limb functional neurologic score and open field test were used to evaluate the changes in motor function of mice.The concentrations of IL-1βand IL-6 were detected by ELISA.Immunofluorescence staining and flow cytometry were employed to observe the rate of Iba-1 positive cells and cell phenotypes in the cerebral cortex.②Twenty-one hSOD1G93A mice and seven wild-type mice were included.The Chemical genetic excitatory virus[rAAV-CaMKIIa-hM3D(Gq)-EGFP-WPRE-hGH polyA]was injected into the primary motor cortex(M1)area of the mice.After 21 days,four mice(3 SOD1G93A and 1 wild-type)were randomly selected for fluorescent microscopy to confirm viral expression.The remaining hSOD1G93A mice were randomly divided into 3 groups:model,EA group,and chemogenetic excitatory virus(intraperitoneal injection of clozapine-N-oxide[CNO])combined with EA group,with 6 mice per group.The rate of c-Fos positive cells in the cerebral cortex was observed by immunofluorescence.The open field experiment was used to evaluate the changes in motor function of mice.Result:Compared with the blank group,the model group showed significantly reduced total distance in the open-field test and increased neurologic scores(P<0.01),elevated serum IL-1βand IL-6 levels,increased cortical Iba-1 and c-Fos expression(P<0.01),and higher proportions of M1 and M2 microglia(P<0.01,P<0.05).Compared with the model group,the total distance of open field movement was prolong

关 键 词：肌萎缩侧索硬化症 电针干预 小胶质细胞 神经炎症 大脑皮质 

分 类 号：R245[医药卫生—针灸推拿学] R493[医药卫生—中医临床基础]