Effect Of Quercetin On VEGFA Expression In Synovial Membrane Of Collagen-induced Arthritis Rat And Screening Of Vascular Neovascularization Targets

ObjectiveThis study is designed to investigate the anti-angiogenic effect of quercetin on collagen-induced arthritis(CIA)in rats and its mechanism,and screen for key targets as well as predict potential mechanisms.MethodsBovine type Ⅱ collagen(CⅡ)and incomplete Freund’s adjuvant(IFA)was used to establish a rat CIA model.The rats were randomly divided into the Control group,CIA group,and QUE group,and 6 rats in each group.The QUE group was given quercetin(150 mg/kg)by gavage,and the Control and CIA groups were each given an equal volume of 0.05% DMSO by gavage from the 14 th to the 35 th day of initial immunization.The arthritis score,body weight,and paw volume were measured,and the histopathological changes of the inflamed joints were evaluated by HE staining.Immunohistochemistry(IHC)was used to detect the expression of CD34,HIF-1α,and VEGFA in synovial tissues,along with semi-quantitative analysis of synovial microvessel density.Western blotting was used to detect changes in the expression of CD34 and VEGFA in synovial tissues.Enzyme-linked immunosorbent assay(ELISA)was performed to detect changes in the expression of cytokines such as TNF-α,IL-1β,and IL-17 in the serum of CIA rats.The Network Pharmacology approach was used to investigate the potential mechanisms of quercetin action in RA.ADME property values of quercetin were downloaded from the TCMSP database.Potential targets of quercetin and related targets of RA were obtained from public databases,and quercetin and RA overlapping genes were obtained and drug-target networks were created by R software and R package.The protein-protein interactions(PPI)network was analyzed by String database,and the PPI network was visualized using Cytoscape software and its plug-in Cyto NCA,and topological analysis was performed to screen the core genes based on multicentricity.Finally,Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis of overlapping genes was performed using R software.Potential targets and signaling pathways were predicted.ResultsAfter 14 days of initial immunization,arthritis scores and foot and paw volumes were increased(all P<0.01),and body weight was decreased(P<0.05)in the CIA group of rats compared with the normal control group.Compared with the CIA group,the arthritis score and foot and paw volume in the QUE group decreased(all P<0.05),and there was no significant difference in body weight.Inflammatory cell infiltration,synovial hyperplasia,vascular opacification,and cartilage erosion were reduced in the synovial membrane of rats in the QUE group compared with the CIA group(all P<0.05).Compared with the Control group,microvessel density,expression of CD34,VEGFA,HIF-1α in synovial tissue and expression of TNF-α,IL-1β,IL-17 in rat serum were increased in CIA rats(all P<0.01);compared with the CIA group,the microvessel density,expression of CD34,VEGFA,HIF-1α and expression of TNF-α,IL-1β and IL-17 in rat serum were decreased in the synovial tissue of the QUE group(all P<0.05).A comprehensive web-based pharmacological analysis showed oral bioavailability(OB)values of ≥30% and drug-like(DL)values of ≥0.18 properties for quercetin,exceeding the ADME general screening criteria for active compounds.A total of 184 potential target genes were obtained from the database prediction.By PPI network analysis and multicentricity screening,10 significant target genes were obtained,namely SRC,IL-6,MAPK1,HSP90AA1,TP53,JUN,EGFR,TNF,VEGFA,and RELA.By enrichment analysis,quercetin was found to be associated with RA angiogenesis mainly through eight signaling pathways,including VEGF,HIF-1,PI3 KAkt,Apelin,MAPK,Erb B,FAK,and Ras signaling pathways.Conclusion1.Quercetin attenuates joint inflammation,synovial hyperplasia,vascular opacification formation,and bone destruction in CIA rats,and improves the protective effects on articular cartilage and bone.2.Quercetin could exert anti-angiogenic effects by inhibiting the expression of VEGFA in the synovial membrane of CIA rats.3.Network pharmacology studies show that HIF-α,VEGF,MAPK1,PI3K/AKT,FAK/SRC signaling pathways may play a key role in the anti-RA angiogenesis process of quercetin.