激素性股骨头坏死血管新生相关基因的鉴定及其靶向中药成分筛选验证

Identification of angiogenesis related genes in steroid induced osteonecrosis of femoral head and screening and verification of active components of targeted traditional Chinese medicine

目的基于生物信息学分析筛选激素性股骨头坏死(steroid induced osteonecrosis of femoral head,SONFH)中涉及血管生成相关的基因,进一步探讨其作用机制并筛选靶向中药活性成分。方法通过Perl语言和R软件处理GEO数据,获得SONFH的关键差异基因,使用David工具进行基因本体(gene ontology,GO)和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)分析。然后使用STRING平台和Cytoscape软件分析蛋白互作关系,用MCODE和CytoHubba插件识别重要基因簇模块和核心基因,并进一步验证。使用CTD数据库搜索靶向核心基因的中药活性成分并用autodock vina软件进行分子对接,以及探索核心基因与SONFH的相互作用。利用激素诱导人股骨头骨微血管内皮细胞(bone microvascular endothelial cells,BMECs),通过CCK-8、细胞划痕实验和Transwell实验观察白藜芦醇对细胞增殖及迁移的影响,通过Western blotting法检测白藜芦醇对细胞中血管内皮生长因子A(vascular endothelial growth factor,VEGFA)及磷脂酰肌醇-3-羟激酶(phosphatidylinositol-3-hydroxykinase,PI3K)/蛋白激酶B(protein kinase B,Akt)通路蛋白表达的影响。结果在SONFH中鉴定出118个与血管新生相关的关键基因,GO富集分析显示主要参与血管生成、细胞的增殖和迁移等,KEGG通路主要涉及PI3K/Akt信号通路、黏附斑信号通路、细胞外基质受体互作通路和Ras相关蛋白1(Ras-related protein 1,Rap1)信号通路等。鉴定出10个核心基因依次为PDGFRB、THY1、PTPRC、ITGB1、VEGFA、ALB、INS、IGF1、CD44和COL1A1。通过验证,核心基因的表达差异明显,其中PTPRC、CD44、ITGB1表达上调。受试者工作特征(receiver operating characteristic,ROC)分析提示PTPRC、CD44、IGF1、PDGFRB、ITGB1和VEGFA具有潜在诊断价值。白藜芦醇、雌二醇、槲皮素和姜黄素与核心基因的相互作用指数较高,其中白藜芦醇最高,可能为修复SONFH主要药物活性成分。体外实验表明,白藜芦醇能逆转激素诱导的BMECs增殖活性降低,增加细胞划痕愈合率,促进细胞迁移,增加细胞中VEGF、PI3K和Akt蛋白相对表达量,尤其白藜芦醇浓度为100μmol/L时效果最为显著(P<0.05),当使用PI3K抑制剂3-MA时,细胞增殖及迁移效果明显被抑制(P<0.05)。结论通过生物信息学分析鉴定了核心基因与通路,并筛选其受靶向调控的主要中药活性成分,通过细胞实验证实白藜芦醇可能通过PI3K/AKT/VEGF信号通路参与激素诱导的骨微血管内皮细胞损伤的修复,为SONFH的分子发病机制和新的治疗靶点提供了数据支持。

Objective To screen the genes related to angiogenesis in steroid induced osteonecrosis of femoral head(SONFH)based on the bioinformatics analysis,and further explore its mechanism of action and screen the active components of targeted traditional Chinese medicine.Methods The key differential genes of SONFH were obtained by processing GEO data with Perl language and R software,and the gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)were analyzed with David tool.Then,STRING platform and Cytoscape software were used to analyze protein interactions,and MCODE and CytoHubba plug-ins were used to identify important gene cluster modules and core genes,which were further verified.The CTD database was used to search the active components of traditional Chinese medicine targeting the core gene,and the autodock vina software was used to perform molecular docking,as well as explore the interaction between the core gene and SONFH.Human bone microvascular endothelial cells(BMECs)were induced by hormone,and the effects of resveratrol on cell proliferation and migration were observed by CCK-8,cell scratch assay and Transwell assay.The effect of resveratrol on protein expression of vascular endothelial growth factor A(VEGFA)and phosphatidylinositol-3-hydroxykinase(PI3K)/protein kinase B(Akt)pathway in cells was detected by Western blotting.Results A total of 118 key genes related to angiogenesis were identified in SONFH.GO enrichment analysis showed that it was mainly involved in angiogenesis,cell proliferation and migration.KEGG pathway was mainly involved in PI3K/Akt signal pathway,adhesion plaque signal pathway,extracellular matrix receptor interaction pathway and Ras-related protein 1(Rap1)signal pathway.Ten core genes were identified as PDGFRB,THY1,PTPRC,ITGB1,VEGFA,ALB,INS,IGF1,CD44 and COL1A1.Through verification,the up-regulated expression of PTPRC,CD44 and ITGB1 was significantly different.Receiver operating characteristic(ROC)analysis suggested that PTPRC,CD44,IGF1,PDGFRB,ITGB1 and VEGFA had potential diagnostic value.The interaction index of resveratrol,estradiol,quercetin and curcumin with core genes was higher,and resveratrol was the highest,which could be the main active ingredient in repairing SONFH.In vitro experiments showed that resveratrol could reverse the decrease of proliferation activity of BMECs induced by hormones,increase the scratch healing rate,promote cell migration,and increase the relative expression levels of VEGF,PI3K and Akt in cells,especially the effect was most significant when the concentration of resveratrol was 100μmol/L(P<0.05).When PI3K inhibitor 3-MA was used,cell proliferation and migration were significantly inhibited(P<0.05).Conclusions Through bioinformatics analysis,the core genes and pathways were identified,and the main active components of targeted traditional Chinese medicine were screened.Through cell experiments,it was confirmed that resveratrol might play a role in repairing hormone-induced bone BMECs injury through PI3K/AKT/VEGF signal pathway.Our research provides data support for the molecular pathogenesis of SONFH and new therapeutic targets.