40 Hz听觉刺激对AD小鼠认知功能的影响及其机制研究

摘要/Abstract

摘要：

目的分析40 Hz声刺激对睡眠剥夺小鼠的学习记忆能力及海马组织内淀粉样前体蛋白、磷酸化tau蛋白、突触相关蛋白、炎症因子、脑源性神经营养因子（brain derived neurotrophic factor，BDNF）/酪氨酸激酶受体B（tyrosine kinase receptor B，TrkB）通路蛋白的影响，探究40 Hz声刺激改善睡眠剥夺诱导的认知功能受损的作用机制。方法将18只C57BL/6j雄性小鼠随机分成对照组、阿尔茨海默病（Alzheimer’s disease，AD）模型组、AD+40 Hz组，每组6只，通过腹腔注射D-半乳糖和灌胃AlCl₃的方法构建AD小鼠模型。AD+40 Hz组小鼠在构建模型后施加14 d，每天1 h的40 Hz听觉刺激，应用新物体识别（new object recognition，NOR）、Y迷宫（Y Maze）评估各组小鼠认知功能，蛋白质印迹检测前额叶皮层、海马组织的淀粉样前体蛋白（amyloid precursor protein，APP）、p-Tau-S404、神经胶质纤维酸性蛋白（glial fibrillary acidic protein，GFAP）、突触后密度蛋白95（postsynaptic density protein 95，PSD95）、突触素重组蛋白（synaptophysin，Syp）、脑源性神经营养因子（brain derived neurotrophic factor，BDNF）和酪氨酸激酶受体B（tyrosine kinase receptor B，TrkB）的蛋白表达水平，实时荧光定量（quantitative real-time PCR，RT-qPCR）检测前额叶皮层、海马组织促炎因子白介素-1β（interleukin-1β，IL-1β）、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α） mRNA的表达水平。结果与对照组相比，AD组小鼠在Y迷宫中的路程占比、持续时间占比显著降低，差异有统计学意义（P<0.05）；前额叶皮层、海马中的APP、p-Tau-S404蛋白水平和IL-1β、TNF-α的mRNA水平升高，差异有统计学意义（P<0.05）；前额叶皮层、海马中的GFAP蛋白水平下降，差异有统计学意义（P<0.05）；PSD95、Syp、BDNF、TrkB的蛋白水平下降，差异有统计学意义（P<0.05）。与AD组相比，AD+40 Hz组小鼠在Y迷宫中的路程占比、持续时间占比显著升高，差异有统计学意义（P<0.05）；前额叶皮层、海马中的APP、p-Tau-S404蛋白水平和IL-1β、TNF-α mRNA水平降低，差异有统计学意义（P<0.05）；GFAP的蛋白水平降低，差异有统计学意义（P<0.05）；PSD95、Syp、BDNF、TrkB的蛋白水平升高，差异有统计学意义（P<0.05）。结论 40 Hz听觉刺激可以改善AD小鼠β-淀粉样蛋白、磷酸化tau蛋白的病理变化，可能通过调节BDNF/TrkB信号通路来改善前额叶皮层和海马的突触可塑性，从而改善随AD导致的认知障碍。

关键词: 阿尔兹海默症, 40 Hz听觉刺激, β-淀粉样蛋白, 磷酸化tau蛋白, BDNF/TrkB通路

Abstract:

Objective To analyze the effects of 40 Hz auditory stimulation on the learning and memory ability，amyloid precursor proteins，phosphorylated tau proteins，synapticproteins，inflammatory factors，and brain-derived neurotrophic factor（BDNF）/tyrosine kinase receptor B（TrkB） pathway proteins in hippocampal tissue in sleep deprived mice，explore the mechanism of 40 Hz auditory stimulation to improve cognitive impairment induced by sleep deprivation. Methods Totally 18 C57BL/6j male mice were randomly divided into control group，the Alzheimer's disease（AD） model group，Alzheimer's disease model +40 Hz auditory stimulation （AD+40 Hz） group，six in each group. AD mouse model was constructed by intraperitoneal injection of D-galactose and gavage of AlCl₃. The AD+40 Hz group mice were stimulated with 40 Hz auditory stimulation for 14 days after model construction. The new object recognition（NOR） and Y maze were used to evaluate the cognitive function of mice in each group. The Western blotting was used to detect the protein expression levels of amyloid precursor protein（APP），p-Tau-S404，glial fibrillary acidic protein（GFAP），postsynaptic density protein 95（PSD95），synaptophysin（Syp），brain derived neurotrophic factor（BDNF） and tyrosine kinase receptor B（TrkB） in the prefrontal cortexand hippocampus. The quantitative real-time PCR（RT-qPCR） was used to detectthe expression levels of proinflammatory factors interleukin-1β（IL-1β） and tumor necrosis factor-α（TNF-α） mRNA in the prefrontal cortex and hippocampus. Results Compared with the control group，the proportion of distance and duration of mice in the Y maze in the AD group was significantly reduced，and the differences were statistically significant（all P<0.05）. The expression levels of APP and p-Tau-S404 protein and the levels of IL-1β and TNF-α mRNA in theprefrontal cortex and hippocampus were significantly increased，and the differences were statistically significant（all P<0.05）.The expression level of GFAP protein in the prefrontal cortex and hippocampus were significantly decreased，and the differences were statistically significant（all P<0.05），the expression levels of PSD95，Syp，BDNF，and TrkB protein were significantly decreased（all P<0.05）.Compared with the AD group，the proportion of distance and duration of mice in the Y maze in AD+40 Hz group were increased significantly，and the differences were statistically significant（all P<0.05）. The expression levels of APP and p-Tau-S404 protein and the levels of IL-1β and TNF-α mRNA in the prefrontal cortex and hippocampus were significantly decreased，and the differences were statistically significant（all P<0.05）.The expression level of GFAP protein was significantly decreased（P<0.05），the expression levels of PSD95，Syp，BDNF，and TrkB protein were significantly increased，and the differences were statistically significant（all P<0.05）. Conclusion 40 Hz auditory stimulation can improve the pathological changes of β-amyloid protein and phosphorylated tau protein in AD mice，and improve synaptic plasticity in the prefrontal cortex and hippocampus by regulating the BDNF/TrkB signaling pathway，thereby improving cognitive impairment caused by AD.

Key words: Alzheimer's disease, 40 Hz auditory stimulation, Amyloid β-protein, Phosphorylated tau protein, BDNF/TrkB pathway

中图分类号:

R-332

戴心宇, 佘晓俊, 马科锋, 付波, 赵凤红, 崔博. 40 Hz听觉刺激对AD小鼠认知功能的影响及其机制研究. [J]职业与健康, 2026, 42(8): 1065-1070.

DAI Xinyu, SHE Xiaojun, MA Kefeng, FU Bo, ZHAO Fenghong, CUI Bo. Study on effect of 40 Hz auditory stimulation on cognitive function in AD mice and its mechanism. [J]OCCUPATION AND HEALTH, 2026, 42(8): 1065-1070.

图/表 6

图1 40 Hz听觉刺激频率频谱和单次听觉刺激频谱

表1 目的基因及内参基因引物序列

基因名称（Gene）	序列	长度
TNF-α	F：5′-CAAGGGACAAGGCTGCCCCG-3′	23
TNF-α	R：5′-GCAGGGGCTCTTGACGGCAG-3′	19
IL-1β	F：5′-TCCAGGATGAGGACATGAGCAC-3′	22
IL-1β	R：5′- GAACG CACACACCAGCAGGTTA-3′	21
β-ACTIN	F：5′-GTCCACCCGCGAGTACAACCTTCT-3′	20
β-ACTIN	R：5′-TCCTTCTGACCCATACCCACCATC-3′	22

图2 Y迷宫和新物体识别行为学实验（n=5）注：Control—对照组；AD—AD造模；AD+40 Hz—AD造模并给予40 Hz声刺激；A—Y迷宫的次数占比；B—Y迷宫的路程占比；C—Y迷宫的持续时间占比；D—新物体识别的认知指数；Control vs AD aP<0.05，bP<0.01；AD vs AD+40 Hz cP<0.05。

图3 前额叶皮层和海马组织APP和p-Tau-S404蛋白含量（n=3）注：Control—对照组；AD—AD造模；AD+40 Hz—AD造模并给予40 Hz声刺激；PFC—前额叶皮层；HIP—海马；APP—淀粉样前体蛋白；p-Tau-S404—磷酸化的tau蛋白；A—前额叶皮层APP、p-Tau—S404蛋白水平表达；B、C—前额叶皮层APP、p-Tau-S404定量分析；D—前额叶APP、p-Tau-S404蛋白水平表达；E、F—海马APP、p-Tau-S404定量分析；Control vs AD aP<0.05，bP<0.01；AD vs AD+40 Hz cP<0.05，dP<0.01。

图4 前额叶皮层、海马星形胶质细胞蛋白含量和炎症因子mRNA表达水平（n=3）注：Control—对照组；AD—AD造模；AD+40 Hz—AD造模并给予40 Hz声刺激；PFC—前额叶皮层；HIP—海马；GFAP—神经胶质纤维酸性蛋白；TNF-α—肿瘤坏死因子-α；IL-1β—白介素-1β；Relative mRNA level—相对mRNA表达水平；A、B—前额叶皮层、海马GFAP蛋白表达水平；C、D—前额叶皮层、海马GFAP蛋白表达水平定量；E、F—前额叶炎症因子mRNA表达水平表达；G、H—海马炎症因子mRNA表达水平表达；Control vs AD aP<0.05；AD vs AD+40 Hz bP<0.05。

图5 前额叶皮层和海马组织突触相关蛋白和BDNF、TrkB蛋白含量（n=3）注：Control—对照组；AD—AD造模；AD+40 Hz—AD造模并给予40 Hz声刺激；PFC—前额叶皮层；HIP—海马；BDNF—脑源性神经营养因子；TrkB—酪氨酸激酶受体B；Syp—突触素重组蛋白；PSD95—突触后密度蛋白95；A、B—前额叶皮层和海马PSD95、Syp、BDNF、TrkB蛋白水平表达；C-J—前额叶皮层和海马PSD95、Syp、BDNF、TrkB定量分析；Control vs AD aP<0.05，bP<0.01，cP<0.001；AD vs AD+40 Hz dP<0.05。

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