基于数据挖掘、网络药理学、分子模拟和实验验证探究中药治疗糖尿病足溃疡用药规律及作用机制

Exploring medication rule and mechanism of traditional Chinese medicine in treatment of diabetic foot ulcers based on data mining,network pharmacology,molecular simulation and experimental validation

目的通过数据挖掘、网络药理学、分子对接及动力学模拟和实验验证等方法研究中药治疗糖尿病足溃疡(diabetic foot ulcer,DFU)的用药规律及其作用机制。方法检索中国知网、万方数据库、维普数据库和《中国药典》筛选建立治疗DFU的中成药数据库。利用中医传承辅助平台分析其用药频次、性味归经等规律;对上述药物进行关联规则和聚类分析,筛选治疗DFU的核心药物。检索获取核心药物活性成分及其对应的DFU疾病靶点;构建上述靶点间网络关系并筛选核心靶点,同时进行基因本体(gene ontology,GO)和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)富集分析;通过分子对接及动力学模拟预测评价核心靶点与其对应的活性成分间的结合能力及稳定性。利用CCK8评价活性成分对细胞增殖率的影响;定量聚合酶链反应(quantitative polymerase chain reaction,qPCR)检测黄芩苷对肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)及木犀草素对磷脂酰肌醇-3-羟激酶(phosphatidylinositol-3-hydroxykinase,PI3K)、蛋白激酶B1(protein kinase B1,AKT1)转录水平的影响;Western blotting检测木犀草素对PI3K及AKT蛋白磷酸化水平的影响。构建大鼠DFU模型用以评价黄芩苷及木犀草素ig治疗后创面愈合状况。结果共筛选得到108种中成药,包含180味中药。寒性和甘味药使用频次最高,归经以肝经、脾经为主;聚类分析得到治疗DFU的5种新药物组合;进一步筛选得到当归、黄芪、冰片、丹参为核心药物,其主要活性成分有木犀草素、黄芩苷、槲皮素等,对应的核心作用靶点为TNF-α、AKT1等;作用机制涉及PI3K-AKT、TNF、丝裂原活化蛋白激酶(mitogen activated protein kinases,MAPK)等信号通路;分子对接和动力学模拟表明木犀草素、黄芩苷与AKT1、TNF-α靶点具有较高的结合亲和力及稳定性。细胞实验显示,黄芩苷可剂量相关性降低脂多糖激活的TNF-α转录表达;木犀草素处理后PI3K和AKT1的转录水平及蛋白磷酸化水平显著升高。与模型组相比,黄芩苷或木犀草素ig治疗能显著促进DFU大鼠的创面愈合。结论筛选出治疗DFU的核心药物为当归、黄芪、冰片、丹参,主要发挥清热解毒、补气止痛的作用,核心成分为木犀草素、黄芩苷,通过作用于PI3K、AKT1和TNF-α等通路靶点发挥疗效。

Objective To explore medication rule and mechanism of traditional Chinese medicine(TCM)in treatment of diabetic foot ulcer(DFU)through data mining,network pharmacology,molecular docking,molecular dynamics simulation,and experimental validation.Methods Retrieving the China National Knowledge Infrastructure,Wanfang Database,VIP Database,and the Chinese Pharmacopoeia and established the Chinese patent medicine database on the treatment of DFU.The medication frequency,properties and meridian tropism were analyzed by using the Traditional Chinese Medicine Inheritance Assisted Platform.The core TCMs for treating DFU were screened based on association rules and cluster analysis.The active ingredients in the core TCMs and their corresponding targets for DFU were subsequently retrieved and identified.The network relationship among the common targets was constructed and the core targets were screened,the gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)enrichment analyses were conducted.The binding affinity and stability between core targets and their corresponding active ingredients were predicted and evaluated using molecular docking and dynamics simulations.CCK8 method was used to evaluate the effects of active ingredients on cell proliferation.The quantitative polymerase chain reaction(qPCR)was used to detect the effects of baicalin on tumor necrosis factor-α(TNF-α),and luteolin on phosphatidylinositol-3-hydroxykinase(PI3K),protein kinase B1(AKT1)transcription levels;Western blotting was used to exam the effects of luteolin on PI3K and AKT protein phosphorylation level.The DFU rat model was established to evaluated the wound healing effects of baicalin or luteolin with gastric infusion.Results A total of 108 Chinese patent medicines were selected,involving 180 TCMs.Among them,the most frequently medicines were cold-natured and sweet-flavored and the main meridians were liver and spleen.Five novel drug combinations for treating DFU were identified by clustering analysis.Further screening results showed that,Danggui(Angelicae Sinensis Radix),Huangqi(Astragali Radix),Bingpian(Borneolum),Danshen(Salviae Miltiorrhizae Radix et Rhizoma)were the core TCMs,and their main active ingredients were luteolin,baicalin,and quercetin.The corresponding core therapeutic targets included TNF-α,AKT1,and the mechanisms involved signaling pathways such as PI3K-AKT,TNF and mitogen activated protein kinases(MAPK).The molecular docking and dynamics simulations showed that luteolin and baicalin exhibited higher binding affinity and stability with AKT1 and TNF-α,respectively.The cell experiments showed that baicalin reduced the expression of lipopolysaccharide-activated TNF-αtranscription in a dose-dependent manner.The transcription levels and protein phosphorylation levels of PI3K and AKT1 were significantly increased after luteolin treatment.Compared with the model group,intragastric administration of baicalin and luteolin can significantly promote wound healing in DFU model rats.Conclusion The core TCMs screened for DFU treatment were Angelicae Sinensis Radix,Astragali Radix,Borneolum,and Salviae Miltiorrhizae Radix et Rhizoma,which mainly played the role of clearing heat and detoxifying,supplementing qi and relieving pain luteolin and baicalin,as the core active ingredients,which exerted therapeutic effect by acting on PI3K,AKT1 and TNF-αpathway targets.