基于网络药理学和实验验证探讨大补肝汤治疗广泛性焦虑症的作用机制

Mechanism of Dabugan Decoction in treatment of generalized anxiety disorder based on network pharmacology and experimental verification

基于网络药理学、分子对接和动物实验探讨大补肝汤治疗广泛性焦虑症(generalized anxiety disorder, GAD)的作用机制。通过网络药理学和分子对接获得大补肝汤治疗GAD的可能靶点和相关信号通路。建立GAD大鼠模型,随机分组后相应药物灌胃,连续干预28 d后检测各组大鼠焦虑状态,并检测大鼠海马病理改变,采用酶联免疫吸附法(ELISA)、蛋白免疫印迹法(Western blot)检测相关蛋白表达水平。初步筛选共得到大补肝汤中化合物65种,作用靶点403个,GAD疾病靶点7 398个,“药物-疾病”共有靶点279个,蛋白-蛋白互作(protein-protein interaction, PPI)网络关键节点为丝氨酸和苏氨酸激酶1(Akt1)、肿瘤坏死因子(tumor necrosis factor, TNF)、白细胞介素(interleukin, IL)-6、细胞肿瘤抗原p53(tumor protein 53,TP53)、IL-1β等;基因本体论(Gene Ontology, GO)功能分析与京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes, KEGG)通路富集分析显示,PI3K-Akt信号通路是最主要途径。分子对接显示,药物核心成分与对应关键靶点均具有较好的结合活性。动物实验显示,大补肝汤可有效改善大鼠焦虑行为,增加大鼠在高架十字迷宫中开臂端运动距离及总距离、进入明箱次数及停留时间、进入旷场中央时间百分比及次数。苏木精-伊红(HE)染色、尼氏染色显示,大鼠海马神经细胞数量增加,排列紧密,细胞体损坏改善,细胞中尼氏体增加,海马组织中TNF-α、IL-6、IL-1β表达下降,TP53、磷酸化丝氨酸和苏氨酸激酶1(p-Akt1)、磷酸化磷脂酰肌醇3-激酶(p-PI3K)表达上升,其机制可能与激活PI3K-Akt信号通路、抑制炎症反应有关。结果表明,大补肝汤对GAD起到很好的治疗调控作用,体现了中药复方整体作用和多靶点、多通路的特点。同时,初步探讨了大补肝汤治疗GAD可能通过激活PI3K-Akt信号通路,抑制炎症反应、抗细胞凋亡,从而改善GAD状态,为大补肝汤的临床应用提供实验数据支持。

This study aims to explore the mechanism of Dabugan Decoction in the treatment of generalized anxiety disorder(GAD)based on network pharmacology,molecular docking,and animal experiments.Network pharmacology and molecular docking technology were used to obtain the possible targets and related signaling pathways of Dabugan Decoction in the treatment of GAD.The GAD rat model was established,and the corresponding drugs were given by gavage after randomization.After 28 days of continuous intervention,the anxiety state of rats was detected,and the pathological changes of the hippocampus were detected in each group.ELISA and Western blot were used to detect the protein expression levels of related molecules.A total of 65 drug compounds in Dabugan Decoction were obtained,involving 403 targets of action,7398 disease targets of GAD,and 279 common targets of"drug-disease".The key nodes in the protein-protein interaction(PPI)network were Akt1,TNF,IL-6,TP53,IL-1β,etc.Function analysis of Gene Ontology(GO)and enrichment analysis of Kyoto Encyclopedia of Genes and Genomes(KEGG)showed that the PI3K-Akt signaling pathway was the most important pathway.The results of molecular docking showed that the core components of the drug had good binding activity with the corresponding key targets.Animal experiments showed that Dabugan Decoction could effectively improve the anxiety behavior of rats and increase the open arm end movement distance and total distance of rats in the elevated cross labyrinth,the number and stay time of entering the open box,and the time(%)and the number of entering the center of the open field.At the same time,HE staining and Nicil staining showed that the number of hippocampal nerve cells in rats increased,and they were closely arranged.The damage to the cell body was improved,and there was an increase in Nissl substances in the cells.The expression of TNF-α,IL-6,and IL-1βin rat hippocampus decreased,and the expression of TP53,p-Akt1,and p-PI3K increased.The mechanism may be related to the activation of the PI3K-Akt signaling pathway and the inhibition of inflammatory response.Dabugan Decoction can play a good therapeutic and regulatory role in GAD,reflecting the overall effect of traditional Chinese medicine(TCM)compound and the characteristics of multiple targets and multiple pathways.At the same time,it is preliminarily discussed that the state of GAD may be improved by Dabugan Decoction via-activating PI3K-Akt signaling pathway and inhibiting inflammatory response and anti-apoptosis,thus providing experimental data support for the clinical application of Dabugan Decoction.