摘要
目的:基于网络药理学研究补肾助孕方治疗复发性流产(RSA)的主要成分、作用靶点及作用通路,应用分子对接和体外细胞实验验证,从而揭示其潜在作用机制。方法:首先,中药系统药理学数据库与分析平台(TCMSP),中医药研究综合数据库(TCMID)检索和收集既往文献补肾助孕方的活性成分,应用DrugBank数据库,SwissTargetPrediction数据库等平台查找预测药物作用靶点,检索人类基因注释数据库(GeneCards),人类孟德尔遗传数据库(OMIM)中等数据库获得复发性流产靶点,将药物与疾病预测靶点取交集。应用Cytoscape 3.7.1软件绘制"补肾助孕方-化合物-靶点-复发性流产"网络,蛋白质相互作用平台(STRING)平台构建蛋白质-蛋白质相互作用(PPI)网络,分析获取核心网络。其次,采用R语言软件对共同靶点分别行基因本体(GO)功能富集分析和京都基因与基因组百科全书(KEGG)通路富集分析。最后,使用Autodock Vina 1.1.2软件用来行分子对接及体外细胞实验验证补肾助孕方对人脐静脉内皮细胞(HUVEC)磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/AKT)通路的激活作用。结果:发现补肾助孕方的49个潜在活性成分可能靶向133个复发性流产相关作用靶点。GO功能富集分析获得470个细胞生物学过程,主要涉及血管形态发生、发育、细胞增殖、氧化活性等生物学过程。KEGG通路富集分析获得103条信号通路(P<0.05),主要与PI3K/AKT信号通路、糖基化终末产物/糖基化终末产物受体(AGE/RAGE)信号通路、肿瘤坏死因子(TNF)信号通路相关。分子对接提示补肾助孕方关键活性成分山柰酚与核心蛋白蛋白激酶B1(AKT1),血管内皮生长因子A(VEGFA)的Vina得分最低且相近。体外细胞实验证实,补肾助孕方可通过活化PI3K/AKT信号通路,上调VEGFA的表达及下游AKT1蛋白的表达水平,促进血管的生成。结论:该研究初步揭示补肾助孕方作用于血管形态发生发育、细胞增殖,明显激活PI3K/AKT通路进而上调VEGFA的表达,最终促进母胎界面血管生成利于妊娠早期胎盘的形成及早孕的维持,起到治疗RSA的作用,为后续新药研发供思路。
Objective:To explore the active components,targets,and signaling pathways responsible for Bushen Zhuyun prescription in treating the recurrent spontaneous abortion(RSA)based on network pharmacology and uncover its potential mechanism by molecular docking and in vitro cell experiments.Method:The active components of Bushen Zhuyun prescription were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),Traditional Chinese Medicine Integrated Database(TCMID)and the published articles,followed by the prediction of drug action targets based on such platforms as DrugBank and SwissTargetPrediction.GeneCards and Online Mendelian Inheritance in Man(OMIM)were searched to obtain the RSA targets,which were then intersected with the targets of Bushen Zhuyun Decoction.Following the plotting of Bushen Zhuyun prescription-compound-target-RSA network by Cytoscape 3.7.1,the protein-protein interaction(PPI)network was then constructed with STRING for screening the core network.The resulting common targets were then subjected to Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis using R software.Autodock Vina 1.1.2 was used for molecular docking.The activation of phosphatidylinositol 3-kinase/protein kinase B(PI3 K/AKT)signaling pathway by Bushen Zhuyun prescription was verified in human umbilical vein endothelial cells(HUVEC)in vitro.Result:It was found that 49 potential active components of Bushen Zhuyun prescription might act on 133 RSA targets.GO enrichment analysis yielded 470 biological processes,with angiogenesis,vascular development,cellular proliferation,and oxidative activity mainly involved.KEGG enrichment analysis revealed 103 signaling pathways(P<0.05),and the PI3 K/AKT signaling pathway,advanced glycation end product(AGE)/receptor for advanced glycation end product(RAGE)signaling pathway,and tumor necrosis factor(TNF)signaling pathway were the main ones.As indicated by molecular docking,the Vina scores of the main active component kaempferol with AKT1 and vascular endothelial growth factor A(VEGFA)were the lowest and similar.It was confirmed in vitro cell experiments that Bushen Zhuyun prescription activated the PI3 K/AKT signaling pathway and up-regulated the expression of VEGFA and downstream AKT protein to promote angiogenesis.Conclusion:Bushen Zhuyun prescription promotes angiogenesis at the maternal-fetal interface by regulating angiogenesis and cellular proliferation,activating the PI3 K/AKT pathway,and up-regulated the VEGFA expression,which is beneficial to the formation of placenta in early pregnancy and the maintenance of early pregnancy.This study has provided ideas for new drug development.
作者
侯梓桐
苏晓慧
隋娟
孟云霄
周天一
秦瑜玲
陈瑞雪
HOU Zi-tong;SU Xiao-hui;SUI Juan;MENG Yun-xiao;ZHOU Tian-yi;QIN Yu-ling;CHEN Rui-xue(Guang'anmen Hospital,China Academy of Chinese Medical Sciences,Beijing 100053,China;Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China;Beijing University of Chinese Medicine,Beijing 100029,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2021年第14期60-69,共10页
Chinese Journal of Experimental Traditional Medical Formulae
基金
中央本级重大增减支项目(2060302)
中国中医科学院基本科研业务费自主选题项目(ZZ070843)。
关键词
补肾助孕方
复发性流产(RSA)
网络药理学
作用机制
细胞实验
Bushen Zhuyun prescription
recurrent spontaneous abortion(RSA)
network pharmacology
mechanism of action
cell experiment
作者简介
第一作者:侯梓桐,在读硕士,从事复发性流产、不孕症的中医药临床与研究,E-mail:1171061659@qq.com;通信作者:陈瑞雪,主任医师,博士生导师,从事复发性流产、不孕症的中医药临床与研究,E-mail:chenruixue@gamyy.cn。
