基于网络药理学、分子对接与动物实验探究百合地黄汤治疗阿尔茨海默病的作用机制

Exploring the Mechanism of Baihe Dihuang Decoction in the Treatment of Alzheimer's Disease Based on Network Pharmacology, Molecular Docking and Animal Experiment

关键词:  百合地黄汤  东莨菪碱  阿尔茨海默病  行为学试验  网络药理学

Key words:  Baihe Dihuang decoction  scopolamine  Alzheimer's disease  behavior experiment  network pharmacology

摘要:

目的 基于网络药理学、分子对接与动物实验探究百合地黄汤治疗阿尔茨海默病的作用机制。方法 利用TCMSP数据库预测百合地黄汤的活性成分及靶点；在GeneCards、OMIM、DRUGBANK等数据库检索阿尔茨海默病靶点，利用Cytoscape 3.8.0建立交集靶点网络，GO富集及KEGG通路分析在Metascape数据库进行；运用AutoDock vina软件进行分子对接，并使用PyMOL等软件进行可视化分析；将小鼠分为空白组、模型组、咯利普兰组和百合地黄汤低、中、高剂量组，给药14 d后各组小鼠进行神经行为学评分，检测脑组织内相关蛋白表达，使用ELISA和Western blotting检测关键基因cAMP、PKA、p-CREB、BDNF的蛋白表达。最后通过呕吐实验观察百合地黄汤的不良反应。结果 获得百合地黄汤活性成分13种，作用于阿尔茨海默病靶点39个，与PRKACA关系密切。动物实验结果表明，与正常组比较，模型组小鼠对新物体的辨认率明显下降，新位置的物体辨认率有下降趋势，Y迷宫中自主交替的反应率明显下降，Morris水迷宫中逃避潜伏期明显延长、平台所在象限停留时间和经过平台次数显著减少，且脑中cAMP、PKA、p-CREB、BDNF蛋白表达下降，而百合地黄汤组与咯利普兰组能够逆转上述行为学以及蛋白指标的变化。在呕吐实验中，咯利普兰组麻醉苏醒时间显著延长，百合地黄汤组无明显影响。结论 百合地黄汤治疗阿尔茨海默病的作用机制可能与其影响cAMP-PKA以及调控cAMP-PKA-CREB-BDNF信号通路有关，且不良反应较咯利普兰轻。

Abstract:

OBJECTIVE To explore the mechanism of Baihe Dihuang decoction in the treatment of Alzheimer's disease(AD) based on network pharmacology, molecular docking and animal experiment. METHODS TCMSP were used to predict the active components and targets of Baihe Dihuang decoction and disease-related targets were collected from GeneCards, OMIM and DRUGBANK databases, respectively. Target protein interactions were analyzed with STRING database and biological function and pathway were analyzed with Metascape database. Lastly relevant results were analyzed with Cytoscape 3.8.0. AutoDock vina software was used for molecular docking to analyze the binding energy of the active components and key targets of Baihe Dihuang decoction. PyMOL software were used to visualize the optimal docking results. ICR male mice were randomly divided into control group, model group, Rolipram group, low, medium and high dose group of Baihe Dihuang decoction. After 14 days of administration, the neurobehavioral scores of mice in each group were collected, and the expression of related proteins in brain tissue was detected, ELISA and Western blotting were used to detect the expression of the key protein cAMP, PKA, p-CREB and BDNF. At last, the adverse reaction of Baihe Dihuang decoction was observed by vomiting experiment. RESULTS A total of 13 active components and 39 key targets were collected from network pharmacology. The docking results showed that the first 10 core targets all performed well and their effects were closely related to PRKACA. Compared with the control group, the model group mice's recognition rate of new objects and the spontaneous alternation reaction rate were significantly reduced, the escape latency was significantly prolonged, and the target quadrant stay time, the number of crossing platforms were significantly reduced; cAMP, PKA, p-CREB and BDNF in the hippocampus of mice was significantly decreased. Baihe Dihuang decoction could reverse the behavior of AD mice and the expression of cAMP, PKA, p-CREB and BDNF. In the vomiting experiment, the anesthesia recovery time of the Rolipram group was significantly prolonged, while that of the Baihe Dihuang decoction group was not significantly affected. CONCLUSION The mechanism of Baihe Dihuang decoction in the treatment of AD may be related to its influence on cAMP-PKA and regulation of cAMP-PKA-CREB-BDNF signal pathway, and the adverse reactions are milder than those of clopramide.