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

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个成分为黑逍遥散煎液治疗失眠的关键活性成分。动物实验结果表明，黑逍遥散中、高剂量组能显著升高失眠大鼠的体质量、缩短睡眠潜伏期、延长睡眠时间（P<0.01）；显著缩短逃避潜伏期、增加穿越平台的次数（P<0.01），并能改善海马突触超微结构的病理变化。同时显著上调海马组织中5-HT含量、Akt1 mRNA表达、Akt1和p-Akt1蛋白表达的水平（P<0.01）；显著下调海马组织中炎症因子含量（P<0.01）；显著下调海马组织中TP53、RAS、NOS1、EGFR、CREBBP、GSK-3β、p-GSK-3β蛋白，以及TP53、RAS、NOS1、EGFR、CREBBP、GSK-3β mRNA的表达水平（P<0.01）。结论 该研究确定了黑逍遥散中5种关键活性成分可能通过调控cAMP、RAS、EGFR等信号通路改善失眠大鼠的学习记忆能力，为其机制研究与临床应用提供了重要参考。

Abstract:

Objective Based on ultra-high performance liquid chromatography-quadrupole-time-of-flight 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-3β in 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 of 176 components were identified in Hei Xiaoyaosan， mainly flavonoids， triterpene saponins， phenylpropanoids and other compounds. Network pharmacological analysis revealed that TP53， V-Ha-Ras Harvey Rat sarcoma viral oncogene homolog（HRAS）， neuroblastoma sarcoma viral oncogene homolog（NRAS）， EGFR， CREBBP， GSK-3β， Akt1 and NOS1 were the key targets of Hei Xiaoyaosan in treating insomnia. The core targets were predominantly associated with cAMP， RAS， Ras-associated protein 1（Rap1）， advanced glycation end products（AGE）/receptor for AGE（RAGE）， and EGFR signaling pathways， and the key active ingredients of Hei Xiaoyaosan in treating insomnia were 8-shogaol， ligustilide F， 6-gingerol， levistilide A and senkyunolide E. Animal experiment results demonstrated that Hei Xiaoyaosan medium and high dose groups significantly increased body weight， shortened sleep latency and prolonged sleep duration in insomnia rats（P<0.01）， significantly decreased escape latency and increased platform crossing frequency（P<0.01）， and improved the pathological changes of hippocampal synaptic ultrastructure. Meanwhile， the two groups could significantly elevate 5-HT level， Akt1 mRNA expression， Akt1 and p-Akt1 protein expression（P<0.01）， reduce inflammatory factor levels（P<0.01）， and down-regulate protein expression levels of TP53， RAS， NOS1， EGFR， CREBBP， GSK-3β and p-GSK-3β（P<0.01）， as well as mRNA expression levels of TP53， RAS， NOS1， EGFR， CREBBP and GSK-3β in hippocampal tissues（P<0.01）.Conclusion This study determined that the five key active ingredients（8-shogaol， ligustilide F， 6-gingerol， levistilide A and senkyunolide E） in Hei Xiaoyaosan may improve the learning and memory ability of insomnia rats by regulating signaling pathways such as cAMP， RAS， and EGFR， providing an important reference for its mechanism research and clinical application.