基于cAMP/PKA/CREB通路的神牡安神胶囊延长深度睡眠作用机制探讨  

Shenmu Anshen Capsules Prolong Deep Sleep via cAMP/PKA/CREB Pathway

作　　者：尤诗晴 徐畅 陈静婷 肖驿泽 刘莹 杨晓菲 吴嘉怡 王金朝 褚福浩[1] 张薇 YOU Shiqing;XU Chang;CHEN Jingting;XIAO Yize;LIU Ying;YANG Xiaofei;WU Jiayi;WANG Jinzhao;CHU Fuhao;ZHANG Wei(School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 102488,China;Vast Sky Biotechnology(Beijing)Technology Co.,Ltd.,Beijing 100020,China)

机构地区：[1]北京中医药大学中药学院,北京102488 [2]瀚天生物(北京)科技有限公司,北京100020

出　　处：《中国现代中药》2025年第4期685-694,共10页Modern Chinese Medicine

基　　金：北京中医药大学青年科学家培育计划项目(BUCM-2019-QNKXJC008);国家中医药管理局高水平中医药重点学科建设项目(zyyzdxk-2023258)。

摘　　要：目的:探讨神牡安神胶囊对对氯苯丙氨酸(PCPA)致失眠小鼠睡眠的改善作用。方法:通过腹腔注射PCPA制备C57雄性小鼠失眠模型,随机分为对照组,模型组,阳性药地西泮组,神牡安神胶囊低、中、高剂量(0.41、1.23、3.69 g·kg^(-1))组,造模后连续给药7 d,观察小鼠精神状态、体质量等指标,通过旷场实验和高架十字迷宫实验观察小鼠自发活动情况,反映其焦虑状态;苏木素-伊红(HE)染色法检测海马区神经元形态变化;酶联免疫吸附(ELISA)法检测脑组织中γ-氨基丁酸(GABA)、多巴胺(DA)、皮质醇(Cort)、褪黑素(MT)、环磷酸腺苷(cAMP)、脑源性神经营养因子(BDNF)含量;蛋白质免疫印迹法检测脑组织中γ-氨基丁酸A型受体亚基β3(GABRA3)、蛋白激酶A(PKA)、cAMP反应元件结合蛋白(CREB)蛋白质表达水平;通过脑电图(EEG)协同肌电图(EMG)监测和分析失眠小鼠给药后8 h内的睡眠结构,分别记录苏醒时长、非快速眼动睡眠时期(NREM)时长和快速眼动睡眠时期(REM)时长。结果:造模成功后失眠小鼠出现暴躁易怒、昼夜节律紊乱的现象。与对照组比较,模型组小鼠在旷场实验中穿格数减少(P<0.05)、中央区域停留时间缩短(P<0.05)、总运动距离减少(P<0.05)、中央区域运动距离减少(P<0.01)、总停留时间显著延长(P<0.01);高架十字迷宫实验中进入开放臂次数占比和开臂时间占比显著降低(P<0.01);脑组织中GABA、MT、cAMP、BDNF表达水平降低(P<0.05,P<0.01),Cort、DA表达水平升高(P<0.05),GABRA3、PKA、CREB蛋白质表达水平降低(P<0.05);苏醒时长延长(P<0.05),NREM和REM时长缩短(P<0.05)。与模型组比较,神牡安神胶囊各剂量组及地西泮组小鼠在旷场实验中穿格数增加(P<0.05,P<0.01),中央区域运动距离、总运动距离增加(P<0.01),中央区域停留时间延长(P<0.05),总停留时间缩短(P<0.05,P<0.01);高架十字迷宫实验中进入开放臂次数占比和开臂时间占比均升高(P<0.05Objective:To study the effects of Shenmu Anshen Capsules in improving the deep sleep of the mouse model of p-chlorophenylalanine(PCPA)-induced insomnia.Methods:The mouse model of insomnia was established by intraperitoneal injection of PCPA in C57 male mice,and mice were randomized into control,model,diazepam,and low-,medium-,and high-dose(0.41,1.23,and 3.69 g·kg–1,respectively)Shenmu Anshen Capsules groups.Mice were administrated with drugs for 7 consecutive days after modeling.The mental state and body weight of mice in each group were observed,and the open field test and elevated plus maze test were conducted to evaluate the spontaneous activities reflecting the anxiety status of mice in each group.Morphologic changes of neurons in the hippocampus were observed by hematoxylin-eosin staining.The levels of gamma-aminobutyric acid(GABA),dopamine(DA),cortisol(Cort),melatonin(MT),cyclic adenosine monophosphate(cAMP),and brain-derived neurotrophic factor(BDNF)in the brain tissue of mice were measured by enzyme-linked immunosorbent assay.The protein levels of GABA A receptor subunit β3(GABRA3),protein kinase A(PKA),and cAMP-response element binding protein(CREB)in the brain tissue of mice were determined by Western blot.Electronencephalography(EEG)and electromyography(EMG)were employed to analyze the sleep structure of mice within 8 h after administration with Shenmu Anshen Capsules.The awake duration,non-rapid eye movement(NREM)sleep duration and rapid eye movement(REM)sleep duration of mice in each group were recorded.Results:The successfully modeled mice showed irritability and circadian rhythm disruption.Compared with the control group,the model group showed reductions in the number of crossings(P<0.05),time in the central zone(P<0.05),total movement distance(P<0.05),and movement distance in the central zone(P<0.01),and prolonged total residence time(P<0.01)in the open field test.In the elevated plus maze test,the model group showed decreases in the percentage of mice entering the open arm and the percentage

关 键 词：神牡安神胶囊 失眠 神经衰弱 镇静催眠 深度睡眠 环磷酸腺苷/蛋白激酶A/环磷酸腺苷反应元件结合蛋白信号通路 

分 类 号：R285.5[医药卫生—中药学]