安寐丹“生熟枣仁”同用及拆方调控Orexin信号通路改善失眠大鼠模型突触可塑性的差异及机制探究  

Study of the Differences and Mechanisms in Synaptic Plasticity by the Orexin Signalling Pathway in the Rat Models of Insomnia by Using“Raw and Cooked Jujube Kernel”Together and Removing the Formula of AMD

作　　者：徐波[1,2] 孙康 王平[1,2] 谢光璟 叶子靖[1,2] 秦庆花 夏婧 XU Bo;SUN Kang;WANG Ping;XIE Guangjing;YE Zijing;QIN Qinghua;XIA Jing(Engineering Research Center of TCM Protection Technology and New Product Development for the Elderly Brain Health,Ministry of Education,Hubei University of Chinese Medicine,Wuhan 430065,China;Hubei Shizhen Laboratory,Wuhan 430065,China)

机构地区：[1]湖北中医药大学老年脑健康中医药防护技术与新产品研发教育部工程研究中心,武汉430065 [2]湖北时珍实验室,武汉430065

出　　处：《世界科学技术-中医药现代化》2024年第12期3218-3233,共16页Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology

基　　金：国家自然科学基金委员会青年项目(82104712):培元固本安神法调控Orexin信号通路改善睡眠剥夺模型大鼠学习记忆的分子机制研究,负责人:徐波;国家自然科学基金委员会青年项目(82205232):从“昼精夜瞑”角度探讨枣仁生熟同用激活MAPK/MNK/eIF4E通路调控老年模型睡眠-觉醒节律的作用机制,负责人:夏婧。

摘　　要：目的基于食欲素(Orexin)信号通路调控的突触可塑性初步探索清代经典名方安寐丹“生熟枣仁”同用及拆方对失眠大鼠模型学习记忆的影响差异及可能机制。方法腹腔注射对氯苯丙氨酸(PCPA)构建失眠大鼠模型,给予安寐丹“生熟枣仁”同用及拆方水煎液灌胃。利用Etho Vision XT动物运动轨迹跟踪系统观察其24 h自主活动,Morris水迷宫评价学习记忆能力;苏木精-伊红(HE)和尼氏(Nissl)染色观察大鼠海马CA1区神经元改变,高尔基(Golgi)染色和免疫组化(IHC)观察大鼠海马CA1区树突棘变化和突触可塑性蛋白突触素(SYN)、人微管相关蛋白2(MAP-2)、胶质细胞原纤维酸性蛋白43(GFAP-43)表达;蛋白免疫印迹法(Western blot)、实时荧光定量聚合酶链式反应(RT-qPCR)检测海马食欲素A(OXA)/磷脂酰肌醇特异性磷酯酶C_(β1)(PLC_(β-1))/蛋白激酶C_(α)(PKC_(α))/胞外信号调节激酶(ERK_(1/2))信号通路蛋白、基因表达。结果与空白组相比,模型组上平台潜伏期延长、穿越站台次数降低、站台周围范围Ⅰ活动路程降低、狂躁频率和时间增加、静止频率和时间降低(P<0.01),海马CA1区肌纤维受损、细胞核染色不均匀,尼氏小体减少、排列紊乱,树突棘长度、个数均减少(P<0.05),SYN、MAP-2、GFAP-43染色淡、表达下降(P<0.01),海马OXA蛋白、基因表达增高(P<0.01),PLC_(β-1)、PKC_(α)、ERK_(1/2)蛋白和基因表达降低(P<0.01)。与模型组比较,苏沃雷生和安寐丹、安寐丹去生、熟枣仁可增加其静止频率、穿越站台次数、第一象限和站台周围范围活动路程、站台周围范围Ⅰ活动时间(P<0.05,P<0.01);海马肌纤维排列较整齐,尼氏小体增多,树突棘长度、个数增加(P<0.05),SYN、MAP-2、GFAP-43表达升高(P<0.05,P<0.01),OXA表达降低(P<0.01,P<0.05),PLC_(β-1)、PKC_(α)、ERK_(1/2)表达提升(P<0.01,P<0.05)。与安寐丹组比较,安寐丹去生、熟枣仁组均可以增加其穿越站台次数和路�Objective To evaluate the effects and the possible mechanisms based on the synaptic plasticity regulated by the orexin signalling pathway of the classic Qing Dynasty prescription An Meidan(AMD),whose“Raw and cooked jujube kernel”used and without the prescription on learning and memory in a rat model of insomnia.Methods The insomnia model was constructed by intraperitoneal injection of p-chlorophenylalanine(PCPA)into rats,and AMD was administered by gavage and a decoction of the formula was disassembled.Observing 24 h autonomous activity using the Etho Vision XT animal motion trajectory tracking system,Morris water maze was used to evaluate the learning and memory ability;hematoxylin-eosin(HE)and Nissl staining were used to observe the neuronal changes in rat hippocampal CA1 area;Golgi staining and immunohistochemistry(IHC)were used to observe the neuronal dendritic changes in rat hippocampal CA1 area,and the changes in synaptic plasticity proteins,synaptophysin(SYN),microtubule-associated protein 2(MAP-2),glial fibrillary acidic protein43(GFAP-43);Western blotting and Reverse transcription-q PCR(RT-q PCR)were used to detect hippocampal orexin A(OXA)/phosphatidylinositol-specific phosphodiesterase C_(β1)(PLC_(β-1))/protein kinase C_(α)(PKC_(α))/extracellular signalling-regulated kinase(ERK_(1/2))signalling pathway protein and gene expression.Results Compared with the blank group,the model group showed prolonged upper platform latency,decreased number of crossing stations,decreased distance travelled by periplatform range I activity,the frequency and duration of mania increase,while the frequency and duration of stillness decrease(P<0.01),damaged muscle fibres in the CA1 region of the hippocampus,uneven staining of the nuclei,decreased and disorganized Nyctal microsomes,reduced dendritic spine length and number(P<0.05),and faded staining and decreased expression ofSYN,MAP-2,and GFAP-43(P<0.01),hippocampal OXA protein and gene expression increased(P<0.01),and PLC_(β-1),PKC_(α),ERK_(1/2)protein and gene exp

关 键 词：安寐丹 失眠 学习记忆 突触可塑性 OXA/PLC_(β-1)/PKC_(α)/ERK_(1/2)信号通路 

分 类 号：R285.5[医药卫生—中药学]