中国中药杂志

2021, v.46(04) 855-864

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基于网络药理学和分子对接研究当归四逆汤治疗原发性痛经的机制
Mechanism of Danggui Sini Decoction in treatment of primary dysmenorrhea based on network pharmacology and molecular docking

卻丹华;陈王焕;姜飞鹏;潘菲;杨科;
QUE Dan-hua;CHEN Wang-huan;JIANG Fei-peng;PAN Fei;YANG Ke;Department of Pharmacy,Hangzhou Lin′an Traditional Chinese Medicine Hospital;College of Pharmacy,Zhejiang University of Technology;

摘要(Abstract):

采用网络药理学、分子对接和体内实验研究当归四逆汤治疗原发性痛经(primary dysmenorrhea,PD)的药效基础与潜在的作用机制。通过TCMSP数据库检索当归四逆汤组成药材当归、白芍、通草、细辛、桂枝、大枣、甘草的化学成分,通过DrugBank搜集当归四逆汤化学成分的作用靶点。以"primary dysmenorrhea""dysmenorrhea"为检索词在GeneCards,OMIM和TTD数据库中检索相应的靶点,取当归四逆汤和原发性痛经靶点交集,进而进行反向筛选,获得对应的活性成分和中药,运用Cytoscape 3.6.1软件构建中药-化合物-靶标-疾病网络,利用STRING数据库建立蛋白相互作用网络,借助DAVID数据库进行靶点GO(gene ontology)功能富集分析和KEGG(Kyoto encyclopedia of genes and genomes)通路富集分析,预测其作用机制,并绘制柱状图及气泡图。然后选取PPI(protein protein interaction)网络中相互作用前5的靶标含靶点与最多的化合物进行对接。动物实验采用SD雌性大鼠通过每天腹腔注射己烯雌酚注射液一次的方法建立原发性痛经模型。SD雌性大鼠共60只,随机分为6组:对照组、模型组、当归四逆汤低(1.5 g·kg~(-1))、中(3.0 g·kg~(-1))、高(6.0 g·kg~(-1))剂量组及布洛芬(20 mg·kg~(-1))阳性对照组,每组10只。第4天起,除对照组外,对各组大鼠进行灌胃给药(1次/d),对照组灌胃生理盐水,连续给药7 d。观察各组给药前后的扭体次数、给药后的宫缩抑制率和子宫指数,并采用ELISA法检测各组大鼠组织前列腺素内过氧化物合酶(prostaglandin-endoperoxide synthase 2,PTGS2)和血管内皮生长因子A(vascular endothelial growth factor A,VEGFA)水平以及血清炎症因子白细胞介素-1(interleukin-1,IL-1)、白细胞介素-6(interleukin-6,IL-6)、肿瘤坏死因子-α(tumor necrosis factor alpha,TNF-α)水平。通过网络分析可知,7种中药包含了114个活性成分共149个靶标,与PD共同靶标基因有30个,关键靶点涉及VEGFA,IL6,PTGS2,TNF等;GO功能富集分析得到条目399个(P<0.05),其中生物过程(biological process,BP)条目353,细胞组成(cell composition,CC)条目21个,分子功能(molecular function,MF)条目25个;KEGG通路富集分析筛选得到14条信号通路,其中有3条通路与炎症相关,分别为花生四烯酸代谢、MAPK信号通路和NOD样受体信号通路。当归四逆汤中的化合物可以通过作用于VEGFA,IL6,PTGS2,TNF等靶点调节花生四烯酸、炎症信号通路等发挥治疗PD的作用。
Network pharmacology, molecular docking and in vivo experiments were used to explore the pharmacodynamic basis and potential mechanism of Danggui Sini Decoction in the treatment of primary dysmenorrhea(PD). The chemical constituents of Danggui(Angelicae Sinensis Radix), Guizhi(Cinnamomi Ramulus), Tongcao(Tetrapanacis Medulla), Baishao(Paeoniae Radix Alba), Xixin(Asari Radix et Rhizoma), Gancao(Glycyrrhizae Radix et Rhizoma), and Dazao(Jujubae Fructus) from Danggui Sini Decoction were retrieved through TCMSP(Traditional Chinese Medicine Systems Pharmacology Database), and the action targets of Danggui Sini Decoction were collected through DrugBank. "Primary dysmenorrhea" and "dysmenorrhea" were used as the key words to search the corresponding targets in the GeneCards, OMIM and TTD databases, and then the intersection targets of Danggui Sini Decoction and the primary dysmenorrhea targets were taken for reverse screening to obtain the corresponding active ingredients. Cytoscape 3.6.1 software was used to construct a traditional Chinese medicine-compound-target-disease network; STRING database was used to build a protein-protein interaction(PPI) network; Gene ontology(GO) function enrichment analysis and Kyoto encyclopedia of genes and genomes(KEGG) pathway enrichment analysis were conducted by using DAVID database. The action mechanism of the intersection targets were then predicted, and a histogram chart and bubble chart were drawn for visualization. Then the top five targets in the PPI network were used for docking with the most compounds. In animal experiments, Sprague Dawley(SD) female rats were used to establish a primary dysmenorrhea model by intraperitoneal injection of diethylstilbestrol once a day. A total of 60 SD female rats were randomly divided into 6 groups, namely control group, model group, Danggui Sini Decoction low(1.5 g·kg~(-1)), medium(3.0 g·kg~(-1)), high(6.0 g·kg~(-1)) dose groups, and ibuprofen(20 mg·kg~(-1)) positive control group, with 10 rats in each group. From day 4, except for the control group, rats in the other groups were given intragastric administration of corresponding drugs, and the control group received intragastric administration of normal saline for 7 consecutive days. The number of writhing before and after the administration, the ute-rine contraction inhibition rate and the uterine index after administration were observed, and ELISA assay was used to detect the levels of prostaglandin-endoperoxide synthase 2(PTGS2) and vascular endothelial growth factor A(VEGFA) in the tissues of each group as well as the levels of serum inflammatory factors interleukin 1(IL-1), interleukin 6(IL-6), and tumor necrosis factor-alpha(TNF-α). According to network analysis, 7 Chinese medicines contained 114 active ingredients, 149 targets, and 30 common target genes with PD were obtained. The key targets included VEGFA, IL6, PTGS2, TNF, etc.; GO function enrichment analysis showed a total of 399 terms(P<0.05) were obtained, 353 of which were biological process(BP) terms, 21 were cell composition(CC) terms, and 25 were molecular function(MF) terms. In KEGG pathway enrichment analysis, 14 signaling pathways were obtained, 3 of which were related to inflammation, namely arachidonic acid metabolism, MAPK signaling pathway and NOD-like receptor signaling pathway. The compounds in Danggui Sini Decoction can play a therapeutic role in the treatment of PD by acting on VEGFA, IL-6, PTGS2, TNF and other targets to regulate arachidonic acid and inflammatory signaling pathways.

关键词(KeyWords): 当归四逆汤;原发性痛经;网络药理学;分子对接
Danggui Sini Decoction;primary dysmenorrhea;network pharmacology;molecular docking

Abstract:

Keywords:

基金项目(Foundation): 杭州市卫生科技计划项目(2018B068);; 国家自然科学基金青年基金项目(81803761)

作者(Author): 卻丹华;陈王焕;姜飞鹏;潘菲;杨科;
QUE Dan-hua;CHEN Wang-huan;JIANG Fei-peng;PAN Fei;YANG Ke;Department of Pharmacy,Hangzhou Lin′an Traditional Chinese Medicine Hospital;College of Pharmacy,Zhejiang University of Technology;

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DOI: 10.19540/j.cnki.cjcmm.20201104.401

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