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多囊卵巢综合征(polycystic ovary syndrome,PCOS)是育龄期女性最常见的一种生殖内分泌疾病,其特征以生化或临床高雄激素血症、无排卵和卵巢多囊样改变等为主要表现[1-2]。流行病学显示PCOS在全世界范围内的总体发病率约为6%~20%[3],而由PCOS引发的排卵障碍所致的不孕症占40%[4]。PCOS不仅影响女性的正常生殖功能,还会导致女性发生代谢系统方面的障碍,如高胰岛素血症、胰岛素抵抗,增加女性继发糖尿病、心血管疾病及子宫内膜癌的风险,对女性健康造成严重不良影响[2]。肥胖是PCOS发生的重要危险因素之一[5]。研究数据表明,28.3%的超重或肥胖的女性患有PCOS[6],多达42%的PCOS患者超重或肥胖[7]。肥胖对PCOS的发展和进展产生显著影响,研究发现,脂肪细胞主要通过分泌脂肪因子,如IL-1、IL-6、瘦素、脂联素等,作用于相应的靶器官、靶组织、靶细胞,如卵巢、肾上腺等,刺激机体产生较多的雄激素,而雄激素又可通过抑制肾上腺素受体等导致体内脂肪分解减少,脂肪大量堆积在体内,导致体内高雄激素水平与肥胖之间形成恶性循环,严重影响PCOS患者的健康状况[3]。与普通女性相比,PCOS患者具有更高的肥胖倾向,且更容易出现腹部脂肪堆积[8];而这种由于腹部脂肪堆积造成的中心性肥胖反过来又可加重PCOS患者的临床或生化表现,导致胰岛素抵抗、高雄激素血症、生殖功能异常等[9]。临床试验结果显示,若患者体质量减轻初始体质量的5%,其体内激素水平、血糖水平得以改善,同时,月经周期和排卵情况趋于正常化,这表明体质量减轻可增加患者排卵和妊娠的可能性[10]。
青蒿素(artemisinin,ART)是一种天然倍半萜内酯化合物,最初由2015年诺贝尔生理学或医学奖获得者屠呦呦从青蒿植物中提取出来并广泛用于抗疟疾治疗[11];青蒿素还用于抗癌、抗炎药物等[12]。近年研究发现,青蒿素及其衍生物还具有预防肥胖的功效:在啮齿动物模型中,青蒿素及其衍生物通过调节p38MAPK/ATF2轴和Akt/mTOR途径等在脂肪生成过程中诱导脂肪细胞褐变,从而预防肥胖并改善肥胖相关的代谢紊乱[13]。Lee等[14]和Jang[15]体外实验数据表明,青蒿素及其衍生物可通过PPARγ途径抑制脂肪生成和脂肪因子的表达。本研究通过网络药理学方法和分子对接方法分析预测青蒿素可用于治疗PCOS的潜在靶点,旨在为深入研究其治疗的作用机制提供参考。
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通过数据库检索共得到青蒿素潜在作用靶点229个,PCOS疾病靶点1292个。利用在线作图软件将青蒿素作用靶点与PCOS疾病靶点进行韦恩图分析,得到二者的交集靶点90个,如图1所示。
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将交集靶点导入String数据库,绘制PPI网络关系图,如图2所示,其中包括网络节点90个,边235条。将共同靶点导入Cytoscape 软件进行核心靶蛋白筛选。如图3所示。综合节点度值及本研究相关度排名靠前的分别为AKT1、ESR1、MMP9、PPARγ、MMP2(见表1)。
表 1 青蒿素作用于PCOS的核心靶点
基因名称 节点度值 排名 ALB 66 1 AKT1 60 2 CASP3 53 3 SRC 51 4 EGFR 50 5 HSP90AA1 49 6 MMP9 48 7 ESR1 48 8 HRAS 47 9 PPARγ 43 10 ERBB2 41 11 MMP2 37 12 -
将得到的90个交集靶点导入DAVID数据库进行GO富集分析,富集结果分别根据基因富集程度进行排序,其中BP前10个条目主要涉及细胞增殖调控、蛋白质磷酸化和RNA聚合酶Ⅱ启动子转录的正调控等生物学过程,MF前10个条目主要与蛋白酪氨酸激酶活性、蛋白激酶活性、蛋白结合和酶结合等分子功能有关,CC前10个条目主要在细胞膜、胞质和胞核等部位富集,如图4所示。KEGG富集分析共筛选到162条信号通路,根据基因富集程度排序,前20个条目主要涉及PI3K/Akt、MAPK、Ras、内分泌抵抗等信号通路,如图5所示。
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将相关靶点及通路文件导入Cytoscape 3.9.1软件,得到药物、疾病、靶点和通路之间的关系图(见图6)。
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分子对接结果显示,青蒿素与核心靶蛋白AKT1、MMP9、ESR1、PPARγ、MMP2之间均存在结合位点。青蒿素与核心靶蛋白的最低结合能分数见表2,结合能越低表示结合活性越高,化合物越容易与该靶点结合。其中青蒿素与核心靶蛋白之间的氢键连接可视化情况如图7所示。
表 2 青蒿素与核心靶点分子对接结果
化合物 核心靶点 最低结合能(kJ/mol) 结合位点 青蒿素 MMP9 −8.2 ARG-143 AKT1 −7.9 HIS-152 ESR1 −7.9 THR-460 PPARγ −7.7 PRO-426、GLN-430、
LEU-431、PHE-432MMP2 −6.4 HIS-190
Exploration on the potential therapeutic mechanism of artemisinin in polycystic ovary syndrome based on network pharmacology and molecular docking technology
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摘要:
目的 应用网络药理学和分子对接技术探索青蒿素对多囊卵巢综合征(polycystic ovary syndrome,PCOS)的潜在作用机制。 方法 通过Pubchem、Swiss Target Prediction、PharmMapper数据库预测青蒿素作用靶点,利用GeneCard、DisGeNET数据库获取与PCOS有关靶点;应用韦恩图分析青蒿素与PCOS的交集靶点;利用String软件对交集靶点进行PPI蛋白网络互作分析,并利用Cytoscape软件进行核心靶点筛选;应用DAVID数据库进行基因本体(gene ontology,GO)功能、京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)富集分析,并借助在线软件对分析结果进行可视化;通过Chemdraw、PyMol、Auto Dock Tools软件及RCSB PDB数据库对青蒿素及核心靶蛋白进行分子对接。 结果 得到青蒿素靶点229个,PCOS靶点1292个,韦恩图分析交集靶点90个,潜在核心靶点5个,分别为丝氨酸/苏氨酸蛋白激酶(serine/threonine-protein kinase,AKT1)、雌激素受体(estrogen receptor 1,ESR1)、基质金属蛋白酶(matrix metalloprotein 9,MMP9)、过氧化物酶体增殖激活受体(peroxisome proliferator-activated receptor gamma,PPARγ)、基质金属蛋白酶(matrix metalloprotein 2,MMP2),主要涉及磷脂酰肌醇-3-激酶(phosphoinositide 3-kinase,PI3K)-蛋白激酶B(protein kinase B,Akt)、丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)、Ras、内分泌抵抗等信号通路。分子对接结果显示青蒿素与对应核心靶蛋白之间存在分子结合位点。 结论 初步预测分析青蒿素可能通过多靶点、多机制对PCOS发挥治疗作用。 Abstract:Objective To explore the potential mechanism of artemisinin in the treatment of polycystic ovary syndrome (PCOS) by network pharmacology and molecular docking technology. Methods The corresponding targets of natural product artemisinin were obtained from PubChem, Swiss Target Prediction and PharmMapper databases, targets related to PCOS were obtained through GeneCards and DisGeNET databases; the intersection target genes of Artemisinin and PCOS were screened by Draw Venn diagram. Then the protein-protein interaction network (PPI) was constructed according to the intersection target genes through the STRING Database, and the core targets were screened by Cytoscape. Besides, gene ontology (GO) function and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis was performed by DAVID Database, and finally the data were analyzed visually by the online platform. Molecular docking of artemisinin and core targets were performed by Chemdraw, Pymol, Auto Dock Tools and RCSB PDB database. Results A total of 229 targets of artemisinin and 1292 targets of PCOS were screened out, 90 overlapping targets were obtained by Draw Venn diagram, and 5 potential core targets, AKT1, ESR1, MMP9, PPARG, MMP2, were mainly act on PI3K Akt, MAPK, RAS, endocrine resistance and other signal pathways. Molecular docking results showed that there were molecular binding sites between artemisinin and core targets. Conclusion It is preliminarily analyzed that artemisinin may play a therapeutic role in PCOS through multiple targets and mechanisms. -
Key words:
- artemisinin /
- polycystic ovary syndrome /
- network pharmacology /
- molecular docking
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表 1 青蒿素作用于PCOS的核心靶点
基因名称 节点度值 排名 ALB 66 1 AKT1 60 2 CASP3 53 3 SRC 51 4 EGFR 50 5 HSP90AA1 49 6 MMP9 48 7 ESR1 48 8 HRAS 47 9 PPARγ 43 10 ERBB2 41 11 MMP2 37 12 表 2 青蒿素与核心靶点分子对接结果
化合物 核心靶点 最低结合能(kJ/mol) 结合位点 青蒿素 MMP9 −8.2 ARG-143 AKT1 −7.9 HIS-152 ESR1 −7.9 THR-460 PPARγ −7.7 PRO-426、GLN-430、
LEU-431、PHE-432MMP2 −6.4 HIS-190 -
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