电针联合跑轮训练对MCAO模型大鼠海马BDNF蛋白表达及学习记忆能力的影响

Effects of Electroacupuncture Combined with Running Wheel Training on the Expression Level of BDNF Protein in Hippocampus and Learning and Memory Ability of MCAO Model Rats

目的:探究电针联合跑轮训练对大脑中动脉闭塞(MCAO)模型大鼠海马脑源性神经营养因子(BDNF)蛋白表达及学习记忆能力的影响。方法:选取30只无特定病原体(SPF)级成年雄性SD大鼠,随机分为3组:对照组(假手术处理,10只)、MCAO模型组(接受MCAO手术,10只)、电针联合跑轮组(接受MCAO手术并进行电针联合跑轮训练治疗,10只)。比较各组大鼠脑梗死体积和神经损伤评分。通过免疫荧光分析大鼠皮质中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)和白细胞介素-10(IL-10)的阳性细胞数。通过蛋白印迹分析BDNF/δ-阿片受体(DOR)/原肌球蛋白相关激酶B(TrkB)的表达。通过逆转录聚合酶链式反应(RT-PCR)分析大鼠海马组织抗N-甲基-D-天冬氨酸受体(NMDAR)、抗α-氨基-3-羟基-5-甲基-4-异噁唑丙酸受体(AMPAR)、γ-氨基丁酸A型受体(GABAAR)和钙/钙调素依赖性蛋白激酶Ⅱ(CaMKⅡ)mRNA转录水平。通过Morris水迷宫测试大鼠逃生潜伏期。通过新物体识别比较大鼠的学习记忆能力。结果:MCAO模型组大鼠脑梗死体积和神经缺陷评分、TNF-α、IL-1β阳性细胞数较对照组上调(P<0.05),电针联合跑轮组大鼠脑梗死体积和神经缺陷评分、TNF-α、IL-1β阳性细胞数较MCAO模型组降低(P<0.05)。MCAO模型组总探索时间、辨别指数、NMDAR、AMPAR、GABAAR和CaMKⅡmRNA表达、IL-10阳性细胞数、BDNF、DOR和TrkB蛋白表达较对照组下降(P<0.05),电针联合跑轮组总探索时间、辨别指数、NMDAR、AMPAR、GABAAR和CaMKⅡmRNA表达、IL-10阳性细胞数、BDNF、DOR和TrkB蛋白表达较MCAO模型组升高(P<0.05)。第2天和第3天MCAO模型组和电针联合跑轮组的逃生潜伏期较对照组延长(P<0.05),第4天和第5天MCAO模型组大鼠逃生潜伏期较对照组延长(P<0.05),电针联合跑轮组逃生潜伏期较MCAO模型组缩短(P<0.05)。结论:电针联合跑轮训练通过DOR/BDNF/TrkB信号通路抑制炎症反应,改善MCAO大鼠学习记忆能力。

Objective:To explore the effect of electroacupuncture combined with running wheel training on the expression level of brain derived neurotrophic factor(BDNF)protein and learning and memory ability in the hippocampus of middle cerebral artery occlusion(MCAO)model rats.Methods:Thirty specific pathogen free(SPF)adult male SD rats were randomly divided into 3 groups:control group(sham operation,n=10),MCAO model group(MCAO operation,n=10),electroacupuncture combined with running wheel group(MCAO operation and electroacupuncture combined with running wheel training,n=10).The cerebral infarction volume and nerve injury score of rats in each group were compared.Tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,and IL-10 positive cells in rat cortex were analyzed by immunofluorescence.The expression of BDNF/Delta-opioid receptors(DOR)/tropomyosin-associated kinase B(TrkB)was analyzed by Western Blot.The transcription levels of N-methyl-D-aspartate receptor(NMDAR),α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor(AMPAR),gamma-aminobutyric acid A receptor(GABAAR),and Ca 2+/calmodulin-dependent protein kinaseⅡ(CaMKⅡ)mRNA in rat hippocampus were analyzed by reverse transcription-polymerase chain reaction(RT-PCR).The escape latency of rats was tested by Morris water maze.The learning and memory ability of rats was compared by new object recognition.Results:Compared with control group,the cerebral infarction volume and neurological deficit score,the number of TNF-αand IL-1βpositive cells in MCAO model group were increased(P<0.05),while the cerebral infarction volume and neurological deficit score,the number of TNF-αand IL-1βpositive cells in electroacupuncture combined with running wheel group were decreased(P<0.05).Total exploration time,discrimination index,NMDAR,AMPAR,GABAAR,and CaMKⅡmRNA expression,IL-10 positive cell number,BDNF,DOR,and TrkB protein expression were decreased in MCAO model group compared with those in control group(P<0.05).Compared with MCAO model group,total exploration time,discrimination index,NMDAR,AMPAR,GABAAR,and CaMKⅡmRNA expression,IL-10 positive cell number,BDNF,DOR,and TrkB protein expression were increased in electroacupuncture combined running wheel group(P<0.05).At the second and third days,the escape latency of rats in MCAO model group and electroacupuncture combined running wheel group was longer than that in control group(P<0.05),and at the fourth and fifth days,the escape latency of rats in MCAO model group was longer than that in control group(P<0.05),and the escape latency of rats in electroacupuncture combined running wheel group was shorter than that in MCAO model group(P<0.05).Conclusion:Electroacupuncture combined with running wheel training can inhibit inflammatory response through DOR/BDNF/TrkB signaling pathway and improve learning and memory ability of MCAO rats.