基于UPLC-Q-TOF-MS结合网络药理学探讨黑逍遥散改善失眠大鼠学习记忆能力的药效物质基础及作用机制  

Exploring Pharmacodynamic Material Basis and Mechanism of Hei Xiaoyaosan in Improving Learning and Memory Ability of Insomnia Rats Based on UPLC-Q-TOF-MS and Network Pharmacology

作　　者：刘佳敏 黄海 梁朋朋 王雅乐 李桂云 吴红彦 LIU Jiamin;HUANG Hai;LIANG Pengpeng;WANG Yale;LI Guiyun;WU Hongyan(Shenzhen Hospital,Shanghai University of Traditional Chinese Medicine,Shenzhen 518004,China)

机构地区：[1]上海中医药大学深圳医院,广东深圳518004

出　　处：《中国实验方剂学杂志》2024年第21期19-30,共12页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：全国老中医药专家学术经验继承项目(国中医药人教函[2022]76号);广东省名中医传承工作室建设项目(粤中医办函[2020]1号);广东省自然科学基金项目(2020A1515010217);广东省中医药管理局科研项目(20222198);罗湖区软科学研究计划项目(LX202202129)。

摘　　要：目的:基于超高效液相色谱-四极杆-飞行时间质谱法(UPLC-Q-TOF-MS)、网络药理学及药效学,考察黑逍遥散改善失眠大鼠学习记忆能力的药效物质基础和作用机制。方法:采用UPLC-Q-TOF-MS对黑逍遥散煎液的化学成分进行表征。应用网络药理学构建“活性成分-交集靶点-通路”网络,对关键成分与核心靶点进行分子对接。选取8周龄雄性SD大鼠60只,随机分为空白组、模型组、黑逍遥散低、中、高剂量组(3.82、7.65、15.30 g·kg^(-1)),酒石酸唑吡坦组(0.5 mg·kg^(-1)),每组10只,除空白组外连续4 d腹腔注射对氯苯丙氨酸(PCPA)诱导大鼠失眠模型。各给药组按相应剂量灌胃给药,1次/d,连续14 d。以Morris水迷宫试验测试大鼠学习记忆能力,透射电镜观察海马突触超微结构,酶联免疫吸附测定法(ELISA)检测海马组织5-羟色胺(5-HT)及白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)含量,蛋白免疫印迹法(Western blot)检测海马组织肿瘤蛋白p53(TP53)、大鼠肉瘤病毒(RAS)、表皮生长因子受体(EGFR)、环磷酸腺苷反应元件结合蛋白(CREB)结合蛋白(CREBBP)、糖原合成酶激酶-3β(GSK-3β)、蛋白激酶B1(Akt1)、一氧化氮合酶1(NOS1)、磷酸化(p)-Akt1、p-GSK-3β蛋白表达情况,实时荧光定量聚合酶链式反应(Real-time PCR)检测海马组织TP53、RAS、EGFR、CREBBP、GSK-3β、Akt1、NOS1 mRNA表达情况。结果:在黑逍遥散中鉴定出176个成分,主要为黄酮类、三萜皂苷类、苯丙素类等化合物。网络药理学分析表明,TP53、V-Ha-Ras肉瘤病毒癌基因同源物(HRAS)、成神经细胞瘤肉瘤病毒癌基因同源物(NRAS)、EGFR、CREBBP、GSK-3β、Akt1、NOS1为黑逍遥散治疗失眠的核心靶点,主要富集于环磷酸腺苷(cAMP)、RAS、Ras相关蛋白1(Rap1)、糖基化终产物(AGE)/糖基化终产物受体(RAGE)、EGFR等多条信号通路。8-姜烯酚、洋川芎内酯F、6-姜辣素、欧当归内酯A、川芎内酯E等5个成分为黑逍遥�Objective:Based on ultra-high performance liquid chromatography-quadrupole-time-offlight mass spectrometry(UPLC-Q-TOF-MS),network pharmacology and pharmacodynamics,to investigate the pharmacodynamic material basis and mechanism of Hei Xiaoyaosan in improving learning and memory ability of insomnia rats.Method:UPLC-Q-TOF-MS was used to characterize the chemical constituents of Hei Xiaoyaosan.Network pharmacology was applied to construct the network of active ingredients-intersecting targets-pathways,and molecular docking was performed on key ingredients and core targets.Sixty 8-week-old male SD rats were selected and randomly divided into blank group,model group,Hei Xiaoyaosan low,medium,and high dose groups(3.82,7.65,15.30 g·kg^(-1)),and zolpidem tartrate group(0.5 mg·kg^(-1)),with 10 rats in each group.Except for the blank group,the insomnia model was induced by intraperitoneal injection of p-chlorophenylalanine(PCPA)for 4 consecutive days.Rats in each dosing group were administered the corresponding dose by gavage,once a day for 14 consecutive days.Morris water maze test was utilized to assess the learning and memory ability of rats,transmission electron microscopy was employed to examine the ultrastructure of hippocampal synapses,enzyme-linked immunosorbent assay(ELISA)was conducted to analyze the levels of 5-hydroxytryptamine(5-HT),interleukin-6(IL-6),and tumor necrosis factor-α(TNF-α)in hippocampal tissues,and Western blot was performed to detect the expression levels of tumor suppressor protein p53(TP53),rat sarcoma virus(RAS),epidermal growth factor receptor(EGFR),cyclic adenosine monophosphate(cAMP)-response element binding protein(CREB)binding protein(CREBBP),glycogen synthase kinase-3β(GSK-3β),protein kinase B1(Akt1),nitric oxide synthase 1(NOS1),phosphorylated(p)-Akt1,and p-GSK-3βin hippocampal tissues.Additionally,real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was used to assess the mRNA expression levels of TP53,RAS,EGFR,CREBBP,GSK-3β,Akt1 and NOS1.Result:A total

关 键 词：黑逍遥散 失眠 学习记忆能力 超高效液相色谱-四极杆-飞行时间质谱法(UPLC-Q-TOF-MS) 网络药理学 物质基础 海马组织 

分 类 号：R22[医药卫生—中医基础理论] R28[医药卫生—中医学] R256.23[理学—分析化学] O657[理学—化学]