基于网络药理学和实验验证探讨加味孔圣枕中丹治疗痴呆的分子机制

Mechanism of Modified Kongsheng Zhenzhong Pills in the Treatment of Dementia Based on Network Pharmacology and Experimental Validation

目的:基于网络药理学和分子对接技术研究加味孔圣枕中丹(JWKSP)治疗痴呆的作用机制。方法:利用中药系统药理学数据库与分析平台(TCMSP)、中医药百科全书数据库(ETCM)筛选JWKSP活性成分及靶点;利用DrugBank、disGeNET数据库以及治疗靶点数据库(TTD)筛选痴呆靶点。活性成分作用靶点和疾病靶点交集获得JWKSP治疗痴呆的潜在作用靶点,Metascape数据库进行潜在作用靶点的基因本体(GO)富集分析和京都基因与基因组百科全书(KEGG)通路归类分析。Cytoscape 3.6.0软件构建JWKSP-中药-活性成分-潜在作用靶点-通路网络。AutoDock 4.2.6软件进行分子对接验证实验。体内复制大鼠血管性痴呆(VD)模型,JWKSP灌胃给药干预30 d,Morris水迷宫实验评价其记忆力,免疫组织化学法检测大脑皮质脑源性神经营养因子(BDNF)表达水平。结果:筛选获得JWKSP活性成分88个,对应痴呆潜在作用靶点80个,主要涉及血液循环、血管直径调节、化学突触传递调控等生物过程;铵离子结合、儿茶酚胺结合、神经递质结合等分子功能以及膜微区等细胞组分。通路分析显示,JWKSP可能通过神经活性配体-受体相互作用、间隙连接、血管内皮生长因子(VEGF)信号通路、AMP活化蛋白激酶(AMPK)信号通路等发挥抗痴呆作用。分子对接结果显示,JWKSP活性成分槲皮素、熊果酸、丹参酮ⅡA等分别与其对应的潜在作用靶点白蛋白(ALB)、白细胞介素-6(IL-6)、BDNF等具有较好的结合力和稳定性。体内实验结果表明,JWKSP可延长VD大鼠象限内游泳时间,提高大脑皮质BDNF表达水平。结论:JWKSP通过神经活性配体-受体相互作用、AMPK、VEGF等信号通路发挥调节胆碱能系统、抑制凋亡、改善神经炎症、血管新生等作用,这可能是JWKSP治疗痴呆的作用机制。

Objective:To reveal the mechanism of modified Kongsheng Zhenzhong Pills(JWKSP)in the treatment of dementia based on network pharmacology and molecular docking.Methods:The components and targets of JWKSP were screened from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)and The Encyclopedia of Traditional Chinese Medicine(ETCM).Drugbank,DisGeNET,and Therapeutic Target Database(TTD)were used to screen the targets of dementia.The common targets shared by JWKSP and dementia were taken as the potential targets of JWKSP in treating dementia.In addition,Metascape was used for the Gene Ontology(GO)annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment of the predicted targets.The JWKSP-herb-active component-target-pathway network was built in Cytoscape3.6.0.AutoDock 4.2.6 was used for molecular docking of active components with potential targets.The rat model of vascular dementia(VD)was replicated in vivo,and JWKSP was administered orally for 30 days.Morris water maze was used to examine the memory of VD rats.Immunohistochemistry was employed to measure the expression of brain-derived neurotrophic factor(BDNF)in the cerebral cortex.Results:A total of 88 active components corresponding to 80 potential targets of JWKSP in the treatment of dementia were screened out.The GO terms of the targets mainly involved biological processes(such as blood circulation,modulation of chemical synaptic transmission,and regulation of blood vessel diameter),molecular functions(such as ammonium ion binding,catecholamine binding,and neurotransmitter binding),and cell components(such as membrane microdomain).The results of KEGG enrichment showed JWKSP might exert anti-dementia effects via neuroactive ligand-receptor interactions,gap junctions,vascular epithelial growth factor(VEGF),and AMP-activated protein kinase(AMPK)signaling pathways.Molecular docking results showed that the JWKSP active components including quercetin,ursolic acid,and tanshinoneⅡA had strong binding affinity and stability with their corresponding targets albumin(ALB),interleukin 6(IL-6),and BDNF,respectively.The results of in vivo experiments showed that JWKSP prolonged the swimming time in the quadrant and up-regulated the expression level of BDNF in the cerebral cortex of VD rats.Conclusion:JWKSP regulates the cholinergic system,inhibits apoptosis,and reduces neuroinflammation and angiogenesis via neuroactive ligand-receptor interactions and AMPK and VEGF signaling pathways to treat dementia.