基于网络药理学联合动物实验探究温阳生肌膏治疗糖尿病创面的作用机制

Mechanism of Wenyang Shengji Ointment in treating diabetic wounds based on network pharmacology and animal experiments

目的从网络药理学角度探讨温阳生肌膏治疗糖尿病创面的作用机制,并通过动物实验进行验证。方法采用中药系统药理学数据库与分析平台(TCMSP)及相关文献筛选温阳生肌膏(WYSJO)活性成分及对应靶点;通过Gene Cards、Online Mendelian Inheritance in Man(OMIM)、Drug Bank、Pharm Gkb和Therapeutic Target(TTD)等数据库检索糖尿病创面的作用靶点;使用Cytoscape 3.9.0绘制出WYSJO活性成分-糖尿病创面靶标网络;应用检索相互作用基因/蛋白质的搜索工具(STRING)平台搭建蛋白互作网络(PPI);应用京都基因与基因组百科全书(KEGG)与基因本体(GO)富集分析WYSJO与糖尿病创面的信号通路;应用Auto Dock 1.5.6对核心成分与靶点进行分子对接;将18只大鼠随机分为对照组、模型组和WYSJO组(n=6),以模型组和WYSJO组制备糖尿病大鼠难治性创面模型。分别在处理第0、5、9、14天时观察创面愈合,苏木素-伊红(HE)染色观察创面组织形态,实时荧光定量聚合酶链式反应(q PCR)检测核心基因表达情况。结果网络药理学筛选出WYSJO药物有效成分76种,药物靶点206个,糖尿病创面疾病靶点3797个,以及WYSJO治疗糖尿病创面作用靶点167个;通过构建WYSJO活性成分-糖尿病创面作用靶标网络,发现槲皮素、大豆苷元、山奈酚、鼠李素、鼠李柠檬素、异长春花苷内酰胺和邻苯二甲酸二异丁酯(DIBP)7个核心靶点。GO富集分析表明温阳生肌膏治疗糖尿病创面可能涉及细胞对脂多糖、细菌来源分子、金属离子、外来刺激、化学应激、营养水平、缺氧及氧化应激等生物过程。KEGG富集分析表明温阳生肌膏治疗糖尿病创面可能涉及高级糖基化终末产物-受体(AGE-RAGE)信号通路、p53信号通路、白介(IL)-17信号通路、肿瘤坏死因子(TNF)信号通路、缺氧诱导因子-1(HIF-1)信号通路、细胞凋亡、脂质和动脉粥样硬化等相关通路发挥治疗糖尿病创面的作用。动物实验显示WYSJO可显著加快糖尿病创面的愈合速度(P<0.05),减轻炎症反应,促进肉芽组织生长,同时也可下调网络药理学预测的E1A结合蛋白p300(EP300)、Jun激酶(JUN)、骨髓细胞瘤癌基因(MYC)、缺氧诱导因子1A(HIF1A)、丝裂原活化蛋白激酶14(MAPK14)、特异性蛋白1(SP1)、肿瘤蛋白p53(TP53)以及雌激素受体1(ESR1)等8个核心基因表达(P<0.05)。结论WYSJO治疗糖尿病创面的作用机制可能与AGE-RAGE、p53、HIF-1等通路密切相关,通过此研究可为WYSJO的药理学研究提供新思路,并为其进一步转化应用提供基础。

Objective To explore the mechanism of Wenyang Shengji Ointment(温阳生肌膏,WYSJO)in the treatment of diabetic wounds from the perspective of network pharmacology,and to veri-fy it by animal experiments.Methods The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)and related literature were used to screen active compounds in WYSJO and their corresponding targets.GeneCards,Online Mendelian Inheritance in Man(OMIM),DrugBank,PharmGkb,and Therapeutic Target Database(TTD)databases were employed to identify the targets associated with diabetic wounds.Cytoscape 3.9.0 was used to map the ac-tive ingredients in WYSJO,which was the diabetic wound target network.Search Tool for the Retrieval of Interaction Gene/Proteins(STRING)platform was utilized to construct protein-protein interaction(PPI)network.Kyoto Encyclopedia of Genes and Genomes(KEGG)and Gene Ontology(GO)enrichment analyses were performed to identify signaling pathways be-tween WYSJO and diabetic wounds.AutoDock 1.5.6 was used for molecular docking of core components in WYSJO to their targets.Eighteen rats were randomly divided into control,model,and WYSJO groups(n=6).The model and WYSJO groups were used to prepare the model of refractory wounds in diabetes rats.The wound healing was observed on day 0,5,9,and 14 after treatment,and the wound tissue morphology was observed by hematoxylin-eosin(HE)staining.The expression levels of core genes were detected by quantitative real-time polymerase chain reaction(qPCR).Results A total of 76 active compounds in WYSJO,206 WYSJO drug targets,3797 diabetic wound targets,and 167 diabetic wound associated WYSJO targets were screened out through network pharmacology.With the use of WYSJO-diabetic wound target network,core targets of seven active compounds encompassing quercetin,daidzein,kaempferol,rhamnetin,rham-nocitrin,strictosamide,and diisobutyl phthalate(DIBP)in WYSJO were found.GO enrich-ment analysis showed that the treatment of diabetes wounds with WYSJO may involve lipopolysaccharide,bacteria-derived molecules,metal ions,foreign stimuli,chemical stress,nutrient level,hypoxia,and oxidative stress in the biological processes.KEGG enrichment analysis showed that the treatment of diabetes wounds with WYSJO may involve advanced glycation end products(AGE-RAGE),p53,interleukin(IL)-17,tumor necrosis factor(TNF),hypoxia inducible factor-1(HIF-1),apoptosis,lipid,atherosclerosis,etc.The results of animal experiments showed that WYSJO could significantly accelerate the healing process of diabetic wounds(P<0.05),alleviate inflammatory response,promote the growth of granulation tis-sues,and down-regulate the expression levels of eight core genes[histone crotonyltrans-ferase p300(EP300),protoc gene-oncogene c-Jun(JUN),myelocytomatosis(MYC),hypoxia inducible factor 1A(HIF1A),mitogen-activated protein kinase 14(MAPK14),specificity pro-tein 1(SP1),tumor protein p53(TP53),and estrogen receptor 1(ESR1)]predicted by the net-work pharmacology(P<0.05).Conclusion The mechanism of WYSJO in treating diabetes wounds may be closely related to AGE-RAGE,p53,HIF-1,and other pathways.This study can provide new ideas for the phar-macological research of WYSJO,and provide a basis for its further transformation and appli-cation.