基于NRG1/ErbB4信号通路研究安寐丹对睡眠剥夺大鼠海马神经元损伤及突触微环境的保护作用  

Protective effect of Anmeidan on hippocampal neuronal damage and synaptic microenvironment in sleep-deprived rats based on NRG1/ErbB4 signaling pathway

作　　者：谢光璟 徐波 夏婧 徐子绚 李非洲 王平[1,4] XIE Guang-jing;XU Bo;XIA Jing;XU Zi-xuan;LI Fei-zhou;WANG Ping(Engineering Research Center,Ministry of Education,Hubei University of Chinese Medicine,Wuhan 430065,China;School of Basic Medical Sciences,Hubei University of Chinese Medicine,Wuhan 430065,China;College of Traditional Chinese Medicine,Hubei University of Chinese Medicine,Wuhan 430065,China;Hubei Shizhen Labortary,Wuhan 430065,China)

机构地区：[1]湖北中医药大学教育部工程研究中心,湖北武汉430065 [2]湖北中医药大学基础医学院,湖北武汉430065 [3]湖北中医药大学中医学院,湖北武汉430065 [4]湖北时珍实验室,湖北武汉430065

出　　处：《中国中药杂志》2024年第18期4957-4966,共10页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(82305354);湖北省自然科学基金项目(2022CFB829);湖北省中医药管理局2023—2024年度中医药科研项目(ZY2023Q043);湖北省教育厅中青年人才项目(Q20222008)。

摘　　要：探讨安寐丹对睡眠剥夺大鼠海马神经元及突触微环境的影响。60只SD大鼠随机分为空白组、模型组、安寐丹组和褪黑素组,每组15只。采用多平台水环境法制备睡眠剥夺模型,安寐丹组给药剂量为18.18 g·kg^(-1)·d^(-1),褪黑素组给药剂量为100 mg·kg^(-1)·d^(-1),空白组和模型组给予等容纯水,大鼠灌胃给药,时间为4周。动物行为视频系统评估大鼠自发活动变化,尼氏染色观察海马锥体神经元及尼氏体变化,原位末端转移酶标记技术观察神经元凋亡,透射电镜观察神经元突触超微结构,高尔基染色观察海马树突棘变化,免疫荧光检测海马胶质纤维酸性蛋白(GFAP)与γ-氨基丁酸A受体(GABAA)、谷氨酸受体N1(GLUN1)表达及突触后致密蛋白95(PSD95)、突触素(SYP)、脑源性神经营养因子(BDNF)、神经生长因子(NGF)的表达,蛋白免疫印迹法检测神经调节素1(NRG1)、酪氨酸激酶受体4(ErbB4)和谷氨酸受体A1(GLUA1)、GLUN1及谷氨酸受体N2A(GLUN2A)的表达。结果显示,与空白组比较,模型组水平得分、垂直得分及修饰次数、中央区路程比例显著降低,海马锥体细胞数量减少,尼氏体减少,凋亡指数增加,NRG1、ErbB4及GLUA1、GLUN1、GLUN2A蛋白表达下调,GFAP^(+)/GABAA^(+)及GFAP^(+)/GLUN1^(+)阳性表达下降,PSD95、SYP及BDNF、NGF荧光强度下降,同时突触囊泡密度不均,突触界面曲率及突触后致密物质厚度降低,树突棘减少。与模型组比较,安寐丹组水平得分、垂直得分及修饰次数、中央区路程比例升高,海马锥体细胞及尼氏体增加,凋亡指数下降,NRG1、ErbB4、GLUA1、GLUN1、GLUN2A蛋白表达上调,GFAP^(+)/GABAA^(+)及GFAP^(+)/GLUN1^(+)阳性表达增加,PSD95、SYP及BDNF、NGF荧光强度升高,且突触损伤减轻,树突棘增加。研究结果表明,安寐丹缓解睡眠剥夺大鼠海马神经元损伤可能与上调NRG1/ErbB4信号通路,促进谷氨酸受体、神经营养因子、突触蛋白表达,改善突触This study aims to investigate the effect of Anmeidan on hippocampal neurons and synaptic microenvironments in sleep-deprived rats.Sixty SD rats were randomly divided into blank,model,Anmeidan,and melatonin groups,with 15 rats in one group.The study used the multi-platform method to prepare the sleep deprivation model.A dosage of 18.18 g·kg^(-1)·d^(-1)was administered to the Anmeidan group,a dosage of 100 mg·kg^(-1)·d^(-1)to the melatonin group,and pure water to the blank and model groups.All rats were administered by gavage for 4 weeks.The spontaneous activity of rats was assessed by using a behavioral analysis system.Hip-pocampal pyramidal neurons and Nissl bodies were observed by Nissl staining.Neuronal apoptosis was observed by terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling(TUNEL)staining.The ultrastructure of synaptic was observed by transmission electron microscopy,and dendritic spine changes were observed by Golgi staining.The expression of glial fibrillary acidic protein(GFAP),γ-aminobutyric acid A(GABAA),glutamate receptor N1(GLUN1),postsynaptic density-95(PSD95),synaptophysin(SYP),brain-derived neurotrophic factor(BDNF),and nerve growth factor(NGF)were detected by immunofluorescence staining.The expression of neuregulin 1(NRG1),epidermal growth factor receptor(ErbB4),glutamate receptor A1(GLUA1),GLUN1,and glutamate receptor N2A(GLUN2A)were detected by Western blot.While compared with the blank group,the results showed that the model group had a significant decrease in the horizontal score,vertical score,modified times,ratio of the central region,a decline in the number of hippocampal pyramidal cells and Nissl bodies,and an increase in the apoptotic index.The expression of NRG1,ErbB4,GLUA1,GLUN1,and GLUN2A proteins was down-regulated,and the positive expression of GFAP^(+)/GABAA^(+)and GFAP^(+)/GLUN1^(+)was decreased.The fluorescence intensity of PSD95,SYP,BDNF,and NGF decreased,and the synaptic vesicle density was uneven.Synaptic interface curvature and postsynaptic density t

关 键 词：安寐丹 睡眠剥夺 突触微环境 NRG1/ErbB4信号通路 

分 类 号：R285.5[医药卫生—中药学]