基于网络药理学探讨益肾通络方治疗糖尿病肾脏疾病的药效作用机制及关键调控通路的验证

Pharmacodynamic mechanism of Yishen Tongluo Formula in treatment of diabetic kidney disease based on network pharmacology and verification of key regulation pathway

目的探讨益肾通络方治疗糖尿病肾脏疾病(DKD)的活性成分、作用靶点及可能的药效作用机制。方法利用中药系统药理学数据库和分析平台(TCMSP)、中医药百科全书数据库(ETCM)和BATMAN-TCM数据库筛选得到益肾通络方的有效成分及相关作用靶点,通过文献检索对上述化学成分和作用靶点信息进行补充,进一步与GeneCards数据库、OMIM数据库、TTD数据库、DrugBank数据库中与DKD有关的靶点信息进行关联。基于String 11.0软件构建的蛋白相互作用(PPI)网络,得到与益肾通络方中主要活性成分相对应的关键靶点,运用cytoscape 3.8.0构建“成分-疾病-靶点”中药复方调控网络图,初步预测益肾通络方治疗DKD的药效作用机制。同时利用R语言BioConductor与pathview软件进行基因本体论(GO)富集分析和京都基因与基因组百科全书(KEGG)富集分析,揭示相关靶基因和通路的功能。以高糖诱导的小鼠肾小球系膜细胞(SV40-MES-13)为主要研究对象,采用荧光素酶报告基因检测、实时荧光定量PCR、蛋白质印迹检测研究益肾通络方对关键靶基因表达及相关通路调控作用的影响,对其药效作用机制进行验证。结果筛选得到益肾通络方中108个具有潜在DKD治疗作用的活性成分及相对应的417个作用靶点。“成分-疾病-靶点”网络图提示山柰酚、黄芪甲苷等是益肾通络方中发挥DKD治疗作用的主要活性成分,蛋白激酶(Akt)、白细胞介素-6(IL-6)等是上述化学成分作用的关键靶点。GO功能注释及KEGG通路富集结果表明,筛选所得关键靶点主要涉及药物免疫反应、氧化应激反应、炎症反应、信号转导等一系列生物学反应过程,主要参与晚期糖基化终末产物及其受体(AGE-RAGE)、PI3K/Akt等信号通路的调控。实验结果表明,益肾通络方有效改善高糖诱导SV40-MES-13细胞中炎症相关指标TNF-α、IL-6以及纤维化相关指标TGF-β1、α-SMA、Col-Ⅰ等的激活(P<0.05)。结论益肾通络方对DKD的治疗具有多成分、多靶点、多途径的特点,其可能通过调控AGE-RAGE、PI3K/Akt信号通路,抑制炎症反应及肾脏组织纤维化发挥治疗DKD的作用。

Objective To explore the active components,targets and possible mechanism of Yishen Tongluo(Kidney-boosting Collateral-unblocking)Formula(YSTLF)in the treatment of diabetic kidney disease(DKD)based on network pharmacology.Methods The effective components and related targets of YXTLF were screened from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),The Encyclopedia of Traditional Chinese Medicine(ETCM)and A Bioinformatics Analysis Tool for Molecular mechanism of Tradition Chinese Medicine(BATMAN-TCM),and further supplemented with literature research.Then these components and targets were associated with the target information related to DKD in GeneCards,OMIM,TTD and DrugBank databases.Based on the protein-protein interaction(PPI)network constructed by String11.0 software,the key targets corresponding to the main active components in YSTLF were obtained.The"component-disease-target"regulatory network diagram with TCM compounds was constructed by cytoscape 3.8.0,and the pharmacodynamic mechanism of YSTLF in the treatment of DKD was preliminarily predicted.At the same time,gene ontology(GO)enrichment analysis and Kyoto Encyclopedia of Gene and Genome(KEGG)enrichment analysis were carried out using R language BioConductor and pathview software to reveal the functions of related target genes and pathways.With the mesangial cells(SV40-MES-13)of mice induced by high glucose as the main research object,the effects of YSTLF on the expression of key target genes and the regulation of related pathways were verified by luciferase reporter gene detection,RT-qPCR and western blot.Results 108 active components of YSTLF with potential therapeutic effect of DKD and 417 corresponding targets were screened.The"component-disease-target”network diagram suggested that kaempferol and astragaloside were the main active components in YSTLF in the treatment of DKD,and Akt and IL-6 were the key targets of the above-mentioned chemical components.The result of GO functional annotation and KEGG pathway enrichment showed that the selected key targets were mainly involved in a series of biological reactions,such as drug immune response,oxidative stress response,inflammatory response,and signal transduction,etc.,and were mainly involved in the regulation of AGE-RAGE and PI3 K/AKT signal pathways.The result showed that YSTLF could effectively improve the activation of inflammation related indexes TNF-αand IL-6 and fibrosis related indexes TGF-β1,α-SMA and Col-Ⅰin SV40-MES-13 cells in mice induced by high glucose.Conclusion Yishen Tongluo Formula seems to be multi-component,multi-target and multi-pathway in the treatment of DKD.It may play a role in the treatment of DKD by regulating AGE-RAGE and PI3 K/AKT signal pathways,thus inhibiting inflammatory reaction and renal tissue fibrosis.