Study On The Mechanism Of Compound Sophora Flavescens Injection In The Treatment Of Nasopharyngeal Carcinoma Based On Network Pharmacology And Proteomic

BackgroundNasopharyngeal carcinoma(NPC)is a common malignant tumor of head and neck cancer and radiotherapy is its main treatment.For early-stage NPC,radiotherapy alone is the main treatment.For locally advanced NPC,more treatment methods are needed,and concurrent chemoradiotherapy(CCRT)is the basis of its treatment.Compound Kushen injection(CKI)is often used in the adjuvant treatment of NPC,which has the effects of clearing heat and dampness,cooling blood and detoxifying,dispersing nodules,and relieving pain.However,the molecular mechanism of CKI in the treatment of NPC remains unclear.ObjectiveIn this study,the methods of network meta-analysis,network pharmacology,proteomics and molecular biology were used to investigate the clinical evaluation and mechanism of CKI in the intervention of NPC,hoping to provide reference for its rational clinical application and interpretation of molecular mechanism.Methods1 Network Meta-analysisThe randomized controlled trials of Chinese herbal injection combined with CCRT in the treatment of NPC were searched in CNKI,Wanfang,VIP,SinoMed,PubMed,Embase and Cochrane Library databases from the date of database establishment to October 05,2020.NoteExpress software was used to screen all the literatures.Two researchers independently read the title and abstract of the literature and obtained the literature read in full-text.Then,the data were extracted,summarized,and risk assessment were conducted for the studies that met the inclusion and exclusion criteria.WingBUGS 1.4.3 software was used to analyze the data and rank the area under the curve.Stata 13.0 software was used to draw the network relationship diagram,detect publication bias,and perform two-dimensional clustering and visualization of the results.R 4.0.3 software was used to perform three-dimensional cluster analysis of the primary outcome measures.2 Network pharmacologyThe components of CKI were searched from TCMSP,PubMed and other databases,and the SMILES structure was searched by PubChem database and ChemDraw software.The data of compounds were uploaded to SuperPred,PharmMapper and SwissTargetPrediction databases for target prediction.NPC related targets were collected through TTD,OMIM,GeneCards and PharmGKB databases.The intersection of compound-targets and NPC-targets was taken,then protein-protein interaction(PPI)network was taken and the core targets were screened.To screen out the core pathways and targets,ClusterProfiler package in R 4.0.3 software was used to perform Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG),and Hallmark enrichment analysis.AutoDock and AutoDock Vina software were used for molecular docking simulation of potential targets and their corresponding compounds.3 Molecular biology analysisTandem mass tag proteomics was used to explore the protein localization and quantitative information of NPC C666-1 cells after the intervention of CKI.Enrichment analysis was used to analyze the core targets and pathways of CKI in NPC cells.The effect of CKI on the proliferation of NPC cells was observed by SRB colorimetric method,clone formation assay and cell cycle detection.Wound healing assay and Transwell assay were used to observe the effect of CKI on the migration and invasion of NPC cells.Annexin V-FITC/PI was used to detect the effect of CKI on the apoptosis of NPC cells.Then RT-qPCR and Western blot were used to detect the effects of CKI on the mRNA and protein expression of NPC cells.Results1 The clinical efficacy of CKI combined with CCRT for NPCA total of 37 randomized controlled trials involving 10 interventions and 2581 NPC patients were included.Among them,CKI was included in 12 randomized controlled trials,including 483 patients with CKI combined with CCRT.Compared with CCRT,CKI+CCRT could significantly improve the clinical effective rate(OR=0.54;95%CI,0.29-0.96),and significantly reduced the incidence of leukopenia(OR=3.71;95%CI,1.76-8.66),nausea and vomiting(OR=2.51;95%CI,1.13-5.80)and>3 radiation-induced oral mucositis(OR=2.73;95%CI,1.28-10.15).The results of cluster analysis showed that Kangai injection combined with CCRT and CKI combined with CCRT had a better ranking than other interventions in the comprehensive evaluation of clinical effective rate,nausea and vomiting and leukopenia.2 The potential mechanism of CKI on NPC by network pharmacologyA total of 16 potential active ingredients,285 potential targets of CKI and 681 targets related to NPC were obtained.There were 55 intersection targets between compounds and diseases.According to PPI network,13 key targets were obtained,including AKT1,MAPK3,MYC,EGFR,MAPK1,CASP3,RELA,IL6,TNF,CCND1,PTEN,ESR1,and HIF1A.The GO enrichment analysis showed that the effect of CKI on NPC was related to biological processes such as peptidyl-serine modification.KEGG enrichment analysis was related to Pathways in cancer,Prostate cancer,Hepatitis B,PI3K/Akt signaling pathway,Cell cycle,and other pathways.The Hallmark analysis included Apoptosis,PI3K/Akt MTOR signaling,TNFa signaling via NF-κB,G2M checkpoint and other items.Molecular docking also revealed high affinity between the active components and the target proteins,including sophoridine to TNF,adenine to MAPK3,and matrine to PTEN,CCND1,IL6 and MYC.3 The mechanism of CKI on NPC by proteomics analysisA total of 655 differentially expressed proteins were obtained,including 309 up-regulated proteins and 346 down-regulated proteins and MYC,E2F4 and E2F5 were down-regulated.There were 371 DEPs located in the nuclear and RNA recognition motif ranked first in domain prediction.NHP2L1,SRSF1,MYC,and POLR2E ranked high in degree values of PPI analysis.GO enrichment analysis showed that CKI mainly affected biological processes such as RNA splicing,and KEGG pathway and Hallmark enrichment analysis were related to RNA splicing,MYC and E2F targets.4 The effect of CKI on NPC cellsThe results showed that CKI could inhibit the proliferation,migration,and invasion of NPC 5-8F cells and C666-1 cells,and induce the apoptosis of C666-1 cells.The results of cell cycle assay showed that CKI could arrest the G0/G1 phase of cell cycle.The results of RTqPCR and Western blot showed that the expression of P15 in 5-8F cells and C666-1 cells was increased,and the expression of E2F4,E2F5,c-Myc,CCND1 and P107 was decreased after the intervention of CKI.ConclusionCKI combined with CCRT can significantly improve the clinical effective rate and reduce the incidence of leukopenia,nausea and vomiting,and≥grade 3 radioactive oral mucositis in NPC patients.The results of network pharmacology and proteomics showed that the mechanism of CKI in the treatment of NPC was related to RNA splicing and cell cycle pathway.The results of in vitro experiments showed that CKI could inhibit the proliferation,migration,and invasion of NPC cells,induce the apoptosis of NPC C666-1 cells,regulate the targets of cell cycle,and arrest the G0/G1 phase of cell cycle.