摘要
目的:通过网络药理学及分子对接技术探讨补阳还五痛风汤治疗高尿酸血症合并痛风性关节炎的潜在分子生物机制,并通过动物实验初步验证.方法:利用中药系统药理数据库和分析平台(TCMSP)等数据库获取补阳还五痛风汤中的活性成分及靶点;使用DisGeNET和GeneCards数据库获取高血酸尿症及痛风性关节炎相关的疾病靶点,并对药物靶点与疾病靶点取交集获得关键靶点;使用R语言ClusterProfiler包和Python进行基因本体(GO)富集分析和京都基因组百科全书(KEGG)富集分析;利用Cytoscape 3.9.1软件绘制药物-关键靶点-功能-通路的调控网络图并针对关键靶点绘制蛋白质-蛋白质相互作用(PPI)网络,通过拓扑结构分析获得hub基因;最后运用Auto Dock、PyMOL等软件进行分子对接,探究补阳还五痛风汤对高尿酸血症合并痛风性关节炎可能的治疗机制.动物实验验证,通过建立腹腔注射氧嗪酸钾致高尿酸血症结合改良的Coderre法致痛风性关节炎的复合大鼠模型;通过苏木素-伊红(HE)染色、尿酸测试、酶联免疫吸附测定法(ELISA)、蛋白免疫印迹法(Western blot)和实时荧光定量聚合酶链式反应(Real-time PCR)等实验,观察补阳还五痛风汤对高尿酸血症合并痛风性关节炎的分子机制及关键靶点的影响.结果:经过筛选和去掉重复值后,最终获得76个活性成分,15个关键靶点;GO富集分析得到补阳还五痛风汤治疗高尿酸血症合并痛风性关节炎的过程中主要与急性炎症反应、星形胶质细胞活化、白细胞介素(IL)-8产生的调节、核受体活性、生长因子受体结合等显著相关;KEGG通路富集分析得到关键靶点基因与IL-17信号通路、晚期糖基化终末产物/晚期糖基化终末产物受体(AGE/RAGE)信号通路、抗炎性等通路显著相关;PPI网络表明白蛋白(ALB)、过氧化物酶体增殖活化受体-γ(PPAR-γ)、IL-6、IL-1β、C反应蛋白(CRP)等为关键蛋白靶点;分子对接结果显示,ALB与β-胡萝卜素结合力最强.生化结果显示,补阳还五痛风汤组血尿酸有所下降;HE染色结果显示,补阳还五痛风汤低、中、高剂量(7.76、15.53、31.05 g·kg-1·d-1)组均有不同程度缓解,高剂量组缓解最明显;高剂量组滑膜关节纤维组织增生伴炎性细胞浸润及肾组织炎性细胞浸润明显减轻,中、低剂量次之;ALB、PPAR-γ、IL-6、IL-1β、CRP表达均有不同程度下调.结论:补阳还五痛风汤通过调节ALB、PPAR-γ、IL-6、IL-1β、CRP等靶点,通过抑制PPAR-γ/核转录因子-κB(NF-κB)通路,减少AGE/RAGE介导的炎症,在高尿酸血症合并痛风性关节炎的治疗中发挥抗炎镇痛、活血利尿的作用.
Abstract
Objective:This paper aims to investigate the potential molecular biological mechanism of Buyang Huanwu Tongfeng decoction in treating hyperuricemia and gouty arthritis by network pharmacology and molecular docking technology and preliminarily verify the mechanism through animal experiments.Methods:The active ingredients and targets in the Buyang Huanwu Tongfeng decoction were obtained by the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)and ETCM databases.The DisGeNET and GeneCards databases were utilized to acquire disease targets associated with hyperuricemia and gouty arthritis.These disease targets were then intersected with drug targets to identify key targets.The R language ClusterProfiler package and Python were employed for conducting gene ontology(GO)enrichment analysis and Kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis.The regulatory network diagram of the drug-key target-function-pathway was visualized using Cytoscape 3.9.1 software,and the protein-protein interaction(PPI)network for key targets was depicted.Finally,the hub gene was determined through topological analysis.Auto Dock,PyMOL,and other software were used for molecular docking to explore the possible therapeutic mechanism of Buyang Huanwu Tongfeng decoction for hyperuricemia and gouty arthritis.In animal experiments,a composite rat model of hyperuricemia induced by intraperitoneal injection of oteracil potassium combined with gouty arthritis induced by the modified Coderre method was established.Through hematoxylin-eosin(HE)staining,uric acid test,enzyme linked immunosorbent assay(ELISA),Western blot,and real-time polymerase chain reaction(Real-time PCR),the molecular mechanism and key targets of Buyang Huanwu Tongfeng decoction for treating hyperuricemia and gouty arthritis were observed.Results:After screening and removing duplicate values,76 active ingredients and 15 key targets were finally obtained.GO enrichment analysis yielded that the treatment of hyperuricemia and gouty arthritis with Buyang Huanwu Tongfeng decoction was significantly associated with acute inflammatory response,astrocyte activation,regulation of interleukin(IL)-8 production,nuclear receptor activity,and binding of growth factor receptor.KEGG pathway enrichment analysis obtained that the key target genes were significantly associated with the IL-17 signaling pathway,advanced glycosylation end/receptor of advanced glycation endproducts(AGE/RAGE)signaling pathway,anti-inflammatory,and other pathways.PPI network indicated that albumin(ALB),peroxisome proliferator-activated receptor-γ(PPAR-y),IL-6,IL-1β,and C-reactive protein(CRP)were the key protein targets.The molecular docking results showed that ALB had the strongest binding force with beta-carotene(β-carotene).Biochemical results showed that blood uric acid decreased in the Buyang Huanwu Tongfeng decoction groups.HE staining results showed that the low-dose(7.76 g·kg-1·d-1),medium-dose(15.53 g·kg-1·d-1),and high-dose(31.05 g·kg-1·d-1)groups of Buyang Huanwu Tongfeng decoction had different degrees of remission,and the remission of the high-dose group was the most obvious.Fibroblastic tissue hyperplasia in synovial joints accompanied with inflammatory cell infiltration,as well as inflammatory cell infiltration in renal tissue of the high-dose group was significantly reduced,followed by the medium-dose and low-dose groups,and the expression of ALB,PPAR-γ,IL-6,IL-1β,and CRP was down-regulated to different degrees.Conclusion:By regulating the targets such as ALB,PPAR-γ,IL-6,IL-1β,and CRP,inhibiting the PPAR-γ/nuclear transcription factor(NF)-κB pathway,and reducing AGEs/RAGE-mediated inflammation,Buyang Huanwu Tongfeng decoction exerts anti-inflammatory and analgesic effects and activates blood circulation and diuresis in the treatment of hyperuricemia and gouty arthritis.
国家科技期刊平台
NSTL
维普
万方数据