| 引用本文: | 张梦杰,王雅静,刘艳蕊,王茜,相聪坤.基于网络药理学及实验验证探究双夏汤治疗失眠的作用机制[J].中国现代应用药学,2025,42(2):1-14. |
| ZHANG Mengjie,WANG Yajing,LIU Yanrui,WANG Xi,XIANG Congkun.Exploring the mechanism of Shuangxia Decoction in treating insomnia based on network pharmacology and experimental validation[J].Chin J Mod Appl Pharm(中国现代应用药学),2025,42(2):1-14. |
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| 基于网络药理学及实验验证探究双夏汤治疗失眠的作用机制 |
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张梦杰1, 王雅静1, 刘艳蕊1, 王茜1, 相聪坤2
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1.河北中医药大学;2.河北省中医院
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| 摘要: |
| 摘要：目的 基于网络药理学及实验验证揭示双夏汤治疗失眠的作用机制，为临床用药提供数据支撑。方法 利用ETCM数据库筛选出双夏汤有效活性成分及靶点，通过CTD数据库筛选出与失眠相关靶点，Venn图获取双夏汤-失眠的交集靶点；STRING数据库构建PPI后导入Cytoscape3.9.1获取核心靶点并进行可视化分析；DAVID数据库进行GO富集分析和KEGG通路富集分析，初步预测双夏汤治疗失眠的作用机制。再以对氯苯丙氨酸（4-chlorophenylacetic acid, PCPA）失眠模型小鼠为研究对象，进行高架十字迷宫行为学实验，采用液相色谱-质谱联用（liquid chromatograph mass spectrometer, LC-MS）检测5-羟色胺（5-hydroxy tryptamine, 5-HT）、5-羟吲哚乙酸（5-hydroxyindole acetic acid, 5-HIAA）和5-羟基色氨酸（5-hydroxytryptophan, 5-HTP）的含量变化，以实时荧光定量PCR（quantitative real-time PCR, qRT-PCR）检测白介素1β（interleukin-1β, IL-1β）、白介素6（interleukin-6, IL-6）、肿瘤坏死因子α（tumor necrosis factor-α, TNF-α）、5-羟色胺1A受体（5-hydroxytryptamine 1A receptor, 5-HT1AR）、5-羟色胺2A受体（5-hydroxytryptamine 2A receptor, 5-HT2AR）、5-羟色胺转运蛋白（5-hydroxytryptamine transporter, 5-HTT）和色氨酸羟化酶2（tryptophan hydroxylase 2, TPH2）的基因表达，尼氏染色观察脑组织形态，免疫组化检测5-HT1AR、5-HT2AR、5-HTT和TPH2的蛋白表达。结果 网络药理学分析得到双夏汤治疗失眠的化学活性成分49种，主要作用成分11种，药物疾病交集靶点107个，核心靶点17个，推测双夏汤的主要活性成分可能是通过抗炎、保护神经元等作用达到镇静安眠效果，其关键靶点主要作用于5-羟色胺能突触，苯丙胺酸代谢，cAMP及神经活性配体-受体相互作用等信号通路。动物实验结果显示，与正常组相比，模型组小鼠在高架十字迷宫开臂中的活动距离、停留时间以及进入次数显著降低（P＜0.05，P＜0.01），脑组织IL-1β、IL-6、TNF-α基因表达明显升高（P＜0.01），尼氏染色显示神经元细胞水肿，空泡变性，尼氏小体染色浅且数目较少，分布不均，前额叶皮质（prefrontal cortex, PFC）5-HT、5-HTP和5-HIAA含量明显降低（P＜0.01），5-HT2AR的基因、蛋白表达明显升高（P＜0.01，P＜0.05），5-HT1AR、5-HTT的基因、蛋白表达均明显下降（P＜0.01）；中缝背核（dorsal raphe nucleus, DRN）中TPH2的基因、蛋白表达明显下降（P＜0.01）。与模型组相比，双夏汤组小鼠在高架十字迷宫开臂中的距离、停留时间以及进入次数明显升高（P＜0.05，P＜0.01）；各给药组小鼠IL-1β、IL-6、TNF-α基因表达明显降低（P＜0.01），脑组织神经元细胞损伤有不同程度好转，结构相对清晰，尼氏小体相对规则，阳性组、双夏汤组小鼠PFC中5-HT、5-HTP和5-HIAA含量以及5-HT1AR、5-HTT的基因、蛋白表达明显升高（P＜0.01，P＜0.05），5-HT2AR的基因、蛋白表达明显下降（P＜0.05，P＜0.01）；DRN中TPH2的基因、蛋白表达明显升高（P＜0.05，P＜0.01）。半夏组、夏枯草组小鼠5-HT2AR蛋白表达明显降低（P＜0.01，P＜0.05），TPH2蛋白表达明显升高（P＜0.01，P＜0.05）。结论 双夏汤对PCPA失眠小鼠的治疗作用可能是通过抗炎、保护神经元并且增加5-HTP含量，促进5-HT的合成、重摄取及代谢实现的。 |
| 关键词: 双夏汤 失眠 PCPA 网络药理学 5-HT信号通路 |
| DOI： |
| 分类号:R256.23 |
| 基金项目:河北省中医药管理局项目 |
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| Exploring the mechanism of Shuangxia Decoction in treating insomnia based on network pharmacology and experimental validation |
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ZHANG Mengjie1, WANG Yajing1, LIU Yanrui1, WANG Xi1, XIANG Congkun2
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1.Hebei University of Chinese Medicine;2.Traditional Chinese Medicine Hospital in Hebei Province
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| Abstract: |
| ABSTRACT: OBJECTIVE Based on network pharmacology and experimental validation to reveal the mechanism of action of Shuangxia Decoction in the treatment of insomnia and to provide data support for clinical use. METHODS The effective active ingredients and targets of Shuangxia Decoction were screened out by ETCM database, the targets associated with insomnia were screened out by CTD database, the meeting points between insomnia and Shuangxia Decoction were obtained by Venn diagram. In order to acquire the core targets and perform visual analysis, the STRING database's PPI is imported into Cytoscape3.9.1. DAVID's database was used to analyze the role of GO and the pathway enrichment of KEGG, and initially hypothesized how Shuangxia Decoction would work to cure insomnia. Using 4-chlorophenylacetic acid (PCPA) insomnia model mice as the primary research subject, conducting behavioral experiments on the elevated cross maze, 5-hydroxytryptamine (5-HT), 5-hydroxyindole acetic acid (5-HIAA) and 5-hydroxytryptophan (5-HTP) content was detected using liquid chromatograph mass spectrometer (LC-MS), interleukin 1β (IL-1β), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), 5-hydroxytryptamine 1A receptor (5-HT1AR), 5-hydroxytryptamine 2A receptor (5-HT2AR), 5-hydroxytryptamine transporter protein (5-HTT) and tryptophan hydroxylase 2 (TPH2) gene expression was detected using quantitative real-time PCR (qRT-PCR), Nissl staining to observe the morphology of brain tissue, as well as protein expression of 5-HT1AR, 5-HT2AR, 5-HTT and TPH2 detected by immunohistochemical. RESULTS The network pharmacological analysis obtained 49 kinds of chemically active ingredients, 11 kinds of main acting ingredients, 107 drug-disease intersection targets and 17 core targets of Shuangxia Decoction for the treatment of insomnia. It is hypothesized that the main active ingredient in Shuangxia Decoction may achieve sedative and tranquilizing effects through anti-inflammatory, protection of nervous cells and that the key targets are mainly in the signaling pathways of Serotonergic synapse, Amphetamine addiction, cAMP signaling pathway, and Neuroactive ligand-receptor interactions. The results of the animal experiments showed that, compared with the normal group, the distance, dwell time and number of entries in the open arm of the elevated cross maze were significantly lower in the model group (P < 0.05, P < 0.01); the gene expression of IL-1β, IL-6, and TNF-α were significantly higher (P < 0.01), Nissl staining showed oedema of neuronal cells, vacuolar degeneration, Nissl's bodies were lightly stained and fewer in number and unevenly distributed; the contents of 5-HT, 5-HTP and 5-HIAA were significantly lower in the prefrontal cortex (PFC) of mice (P < 0.01); the gene and protein expression of 5-HT2AR was significantly higher in the PFC of mice (P < 0.01, P < 0.05), as well as the gene and protein expression of 5-HT1AR and 5-HTT were significantly decreased (P < 0.01); the gene and protein expression of TPH2 in the dorsal raphe nucleus (DRN) were significantly decreased (P < 0.01). Compared with the model group, the distance, residence time and number of entries in the open arm of the elevated cross maze were significantly higher in the Shuangxia Decoction groups (P < 0.05, P < 0.01); the gene expression of IL-1β, IL-6, and TNF-α were significantly decreased (P < 0.01), brain tissue neuronal cell damage has improved to varying degrees, with relatively clear structures and relatively regular Nissl's bodies; the contents of 5-HT, 5-HTP and 5-HIAA in the PFC of mice were significantly higher (P < 0.01, P < 0.05); in the positive and Shuangxia Decoction groups, the gene and protein expression of 5-HT2AR in the PFC of mice were significantly decreased (P < 0.05, P < 0.01) and the gene and protein expression of 5-HT1AR and 5-HTT increased significantly (P < 0.05, P < 0.01); the gene and protein expression of TPH2 increased significantly in DRN (P < 0.05, P < 0.01). Compared with model group, in the Pinelliae Rhizoma and Prunellae Spica groups, the protein expression of 5-HT2AR was significantly lower (P < 0.01, P < 0.05) and the protein expression of TPH2 in DRN was significantly increased (P < 0.01, P < 0.05). CONCLUSION Treatment of PCPA insomnia model mice by Shuangxia Decoction may be achieved by anti-inflammation, protection of neurons and increasing the level of 5-HTP and promoting the synthesis, reuptake and metabolism of 5-HT. |
| Key words: shuangxia decoction insomnia PCPA network pharmacology 5-HT signaling pathway |
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