| Background and Objective:Chronic periodontitis is a prevalent oral inflammatory infected disease in the adult population.Periodontitis is initiated by a large number of gram-negative bacteria,which gather around the teeth and result in the gingival tissue inflammation and the formation of periodontal pockets.A large number of bacteria can break through the epithelial barrier and penetrate into the deep connective tissue,and then lead to alveolar bone loss and eventually induce the teeth loosening and shedding.Fusobacterium nucleatum(F.nucleatum),as a crucial periodontal pathogen,plays a key role in promoting the development of periodontitis.It has been reported that F.nucleatum possesses a variety of lectins and presents strong adhesion ability,which can adhere to the surface of epithelial cells,fibroblasts,endothelial cells,polymorphonuclear leukocytes and other host cells.However,the pathogenic effect of F.nucleatum on human oral tissues and cells has not been fully evaluated.Gingival fibroblasts(GFs)are the major constituents of gingival connective tissue and therefore involved heavily in the maintenance,repair and regeneration of the integrity of periodontal connective tissue.Also,some gingival mesenchymal stem cells(GMSCs)are isolated from the gingival tissues and have been confirmed to play crucial roles in the repairing of periodontal destruction tissues.However,whether F.nucleatum can invade into GFs and GMSCs,and the effects of F.nucleatum on the cell biological properties alteration and the underneath molecular mechanism based on the bacteria invasion has not been fully revealed.In addition,few reports are focused on the effective drugs for the treatment of F.nucleatum infected diseases and their mechanism has rarely been studied.This study aimed to explore the detailed biological activities alteration of GFs and GMSCs after time-series F.nucleatum infection,including cell proliferation,migration,differentiation,inflammatory cytokines and reactive oxygen species(ROS)production,and the potential molecular mechanisms of each biological properties changes have also been investigated.Furthermore,to elucidate the mechanism of F.nucleatum on cell biological properties alteration,high-throughput RNA-sequencing(RNA-seq)technology was used to detect all transcriptome genes of GFs and GMSCs after time-series F.nucleatum infection.Based on the differentially expressed genes(DEGs)in time-series F.nucleatum infected GFs,we computationally identified several U.S.Food and Drug Administration(FDA)-approved drugs that could protect F.nucleatum infected GFs,and biological experiments were performed to confirm the effects of these drugs.Our study aimed to find out drugs that can protect tissues with bacteria invasion,which would provide new therapies for clinical treatment of inflammatory infectious diseases.In addition,based on the DEGs in time-series F.nucleatum infected GMSCs,biological information methods were applied to investigate the molecular mechanism of F.nucleatum affecting GMSCs osteogenic differentiation.Materials and methods:1.Culture and identification of F.nucleatum and gingiva-derived cells,and the construction of the co-cultured modelF.nucleatum subsp.nucleatum ATCC 25586 was provided by Shandong Provincial Key Laboratory of Oral Tissue Regeneration(Jinan,China).Stock cultures were routinely propagated in brain heart infusion(BHI,Haibo,Qingdao,China)broth supplemented with 5 μg/mL hemin and 1 μg/mL menadione in an anaerobic atmosphere.The purified single colony was inoculated in liquid medium for extended culture,and polymerase chain reaction(PCR)assay and 16S rRNA gene sequencing were used to authenticate and verify the purity of F.nucleatum.Gingival tissue samples were collected from volunteers undergoing third molar extraction in clinic.The excised gingival tissues were transported from the clinic to the laboratory and cut into two pieces in the sterile stage.Half of each gingival tissue sample was used for hematoxylin and eosin(H&E)staining assay to detect the tissues inflammation state.The other half was used for cell isolation.Large amounts of GFs were obtained By enzyme digestion and GMSCs were isolated by limited dilution method.Crystal violet staining was used to identify the clonal ability of GMSCs and the surface markers CD29,CD44,CD45 and CD73 were detected by flow cytometry.F.nucleatum cultured in the logarithmic growth phase was harvested and the co-cultured model between F.nucleatum and GFs/GMSCs(F.nucleatum:GFs=10,50,100,200,400;-F.nucleatum:GMSCs=10,50,100)were constructed.The cell morphology of co-cultured GFs and GMSCs were identified by trypan blue and crystal violet staining assay2.Effects and mechanisms of F.nucleatum on GFs biological propertiesGFs were cultured and infected with F.nucleatum for 2 h,6 h,12 h,24 h and 48 h at the multiplicity of infection(MOI)equal to 100(F.nucleatum:cell=100:1),and then the total RNA of all samples was extracted and subjected to RNA-seq analysis.DESeq2 and PoissonDis algorithms were used to detect the differentially expressed genes(DEGs)between the control groups and the F.nucleatum-treated groups.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)database were used to annotate DEGs.The expression signatures of the DEGs involved in the GO biological processes,including cell proliferation,apoptotic process,response to reactive oxygen species,and defense response were presented by venn diagrams and heatmaps.Moreover,GFs were co-cultured with different densities of F.nucleatum for various time points.Cell count assay and EDU-label assay were used to evaluate the effect of F.nucleatum on cell proliferation.Flow cytometry was used to detect the apoptosis rates and ROS generation in F.nucleatum-infected GFs.qRT-PCR and ELISA were used to detect the gene and protein expression of IL-6,IL-8,IL-1β and TNF-α.Western blot and cell immunocytochemistry assays were used to evaluate the signaling pathway activation of NF-κB,MAPK and AKT.Also,the pathview database was used to detect the gene variation at NF-κB,MAPK,AKT signaling pathways after time-series F.nucleatum-infection,and some of the genes expression,such as SOD2,A20,was validated by qRT-PCR.3.Screening of drugs against F.nucleatum and their potential mechanism in protecting GFsBased on all DEGs generated in time-series F.nucleatum infected GFs,we firstly analyzed the gene function by GO and KEGG,and then computationally identified several U.S.FDA-approved drugs that could protect F.nucleatum infected GFs via a cogena-based drug repositioning approach.Western blot and flow cytometry were used to validate the effects of the predicated drugs on protecting GFs by detecting the signaling pathways activation of NF-κB,MAPK,AKT and ROS generation in GFs pretreated by the drugs and followed by F.nucleatum-infection.4.Effects and mechanisms of F.nucleatum on GMSCs biological propertiesGMSCs were cultured and infected with F.nucleatum for 24 h,48 h,72 h(3 d),7 d,14 d,21d and 28 d at MOI of 10,50 and 100(F.nucleatum:cell=10:1,50:1,100:1).Flow cytometry was used to detect the surfaces markers CD29,CD44,CD73 expression in F.nucleatum-infected GMSCs.Cell count assay and EDU-label assay were used to evaluate the effect of F.nucleatum on GMSCs proliferation.Transwell assay were used to detect GMSCs migration ability after F.nucleatum infection.qRT-PCR and ELISA were used to detect the gene and protein expression of chemokines and cytokines in GMSCs infected with various concentration of F.nucleatum.Also,alkaline phosphatase(ALP)activity,mineralized nodule formation,and osteogenesis-related genes and proteins expression in F.nucleatum-infected GMSCs were detected to evaluate GMSCs osteogenic differentiation abilities.To explore the mechanism of F.nucleatum affecting GMSCs osteogenic differentiation,RNA-seq analysis was used to seek out DEGs in GMSCs co-cultured with F.nucleatum(MOI=100)for 3d,7d,14d and 21d.GO and Reactome database were used to analyze the gene function of specific DEGs at each time point and the co-expression DEGs at all the time points.In addition,cell differentiation related genes were screened out according to the GO biological process,and the gene function was enriched by GO,disease ontology(DO),KEGG and DisGeNET database.5.Study of F.nucleatum on GMSCs tumor associated genes(TAGs)expression in the process of osteogenic differentiationBased on the transcriptome data of GMSCs co-cultured with F.nucleatum at 3 d,7 d,14 d and 21 d,microarray significant profiles(maSigPro)analysis was applied to obtain the differentially co-expressed genes(co-DEGs).GO and KEGG were used to analyze the gene function.Overlapped analysis between co-DEGs and all TAGs was used to seek out F.nucleatum-induced TAGs expression in GMSCs,and some of the gene expressions,such as CD163,CCND2,were validated by qRT-PCR.A short time-series expression miner(STEM)analysis was used to analyze the time-series gene expression clusters and the dynamic genes in GMSCs with or without F.nucleatum-infection,and gene function of similar clusters in the control group and the experimental groups was analyzed.To obtain the dynamic DEGs,the overlapped analysis was performed to compare the co-DEGs and the dynamic genes generated in F.nucleatum-infected GMSCs.Based on the dynamic DEGs,GeneMANIA was used to construct the functional enrichment network,and the related transcription factor(TFs)and microRNA(miRNA)were predicated.Finally,cytoscape software was used to construct the TFs-mRNAs-microRNA integrated network.Results:1.Culture and identification of F.nucleatum,GFs and GMSCs,and the construction of the co-cultured modelBlood agar plate cultures of F.nucleatum presented specific colony morphology,a flat,dim edge,central bulge translucent colony.Agarose gel electrophoresis assay indicated the bacterium could be amplified by F.nucleatum specific primer and the products length is 146bp.The logarithmic growth phase of the single colony of F.nucleatum was maintained between 15 h and 30 h.Primary cultures of GFs and GMSCs from human gingival tissues exhibited a spindle-shaped fibroblast-like morphology.A trypan blue staining assay indicated that low concentration of F.nucleatum(MOI=10,50,100)could adhere to GFs surface,while high concentration of F.nucleatum(MOI=200,400)induced the cell morphology changed from long spindle to round.GMSCs possessed unique clone formation ability and were uniformLy positive(>99%)for mesenchymal stem cells markers CD29,CD44 and CD73,and negative for hematopoietic stem cell marker CD45(<1%).A crystal violet staining assay indicated that F.nucleatum(MOI=10,50,100)induced no significant change in GMSCs morphology.2.F.nucleatum facilitated GFs apoptosis,ROS generation,and inflammatory cytokine production by activating AKT/MAPK and NF-κB signaling pathwaysWhen GFs co-cultured with F.nucleatum(MOI=10,50,100,200,400)for different time points,the gene expression level and biological properties in GFs were dramatically altered.RNA-seq analysis confirmed that F.nucleatum time-dependently increased the DEGs number in GFs.A total of 62 DEGs was generated in GFs co-cultured with F.nucleatum at all the time points,and these DEGs were significantly enriched in TNF-α,IL-17,NF-κB signaling pathways according to GO and KEGG database.Cell counting assay indicated that F.nucleatum inhibited GF proliferation in a time-and dose-dependent manner and EDU-label assay indicated high concentration of F.nucleatum significantly decreased GF proliferation rate.GO enrichment analysis indicated the gene levels of CCL2,SOD2 and NFKBIA significantly increased at all F.nucleatum infected time points and played key roles in regulating GF proliferation.In addition,F.nucleatum significantly promoted GF apoptosis and intracellular ROS generation.SOD2 was significantly involved in the process of cell apoptosis and ROS generation.qRT-PCR results indicated that F.nucleatum activated the toll-like receptor(TLR)-2 and TLR4,and promoted the proinflammatory cytokines IL-6,IL-8,IL-1β and TNF-α expression.ELISA results indicated that F.nucleatum significantly promoted IL-6 and IL-8 release,while not influence IL-1β and TNF-α protein expression.Western blot assay results indicated F.nucleatum activated NF-κB,MAPK,AKT signaling pathways by increasing the protein level of p-p65,p-IKBa,p-AKT,p-p38,p-ERK and p-JNK.Cell immunofluorescence assay showed that F.nucleatum could also activate NF-κB signaling pathway by increasing the translocation of p65 and p-p65 proteins from cytoplasm to nucleus.Pathview analysis indicated that F.nucleatum could regulate most genes expression in NF-κB,MAPK,AKT and apoptosis signaling pathways,and large number of genes at the downstream of the signaling pathways,such as Bcl-2,COX-2,A20,IκBα and MIP2,were activated after F.nucleatum infection from 2 h to 48 h,which play key roles in inhibiting cell proliferation,promoting cell apoptosis,ROS generation and inflammatory cytokines release.3.Screening of drugs against F.nucleatum and its potential mechanism in protecting GFsBased on the time-series RNA-seq data,we found out 971 DEGs and all these DEGs could be divided into three coexpression clusters.Genes in cluster 1 were upregulated after 12 h of F.nucleatum infection,and genes in cluster 2 were upregulated immediately at 2 h of F.nucleatum infection,while genes in cluster 3 were down-regulated after 12 h of F.nucleatum infection.GO and KEGG functional enrichment analysis revealed that the immune-related signaling pathways were more overrepresented at the early stage,while metabolic pathways were mainly enriched at the late stage.Furthermore,we computationally identified several U.S.FDA-approved drugs that could protect F.nucleatum infected GFs via a cogena-based drug repositioning approach,and finally,six drugs including etravirine,zalcitabine,wortmannin,calcium D-pantothenate,ellipticine and tanespimycin were identified.Biologically,we confirmed that these 6 drugs could significantly decrease F.nucleatum-induced ROS generation and block the protein kinase B(PKB/AKT)/mitogen-activated protein kinase(MAPK)signaling pathways,which play key roles in protecting GFs.4.F.nucleatum triggered GMSCs chemokine/cytokine release,and inhibited cell proliferation and osteogenic differentiationGMSCs exhibited a spindle-shaped fibroblast-like morphology and maintained at the fourth generation.GMSCs co-cultured with F.nucleatum still possessed uniformLy positive(>99%)for mesenchymal stem cells markers CD29,CD44,and CD73.Cell counting assay and EDU-label assay indicated that F.nucleatum dose-dependently inhibited GMSC proliferation.Transwell assay indicated that F.nucleatum(MOI=50,100)significantly promoted GMSCs migration.qRT-PCR results indicated that F.nucleatum significantly activated chemokines CCL2,CCL5,CCL20,CXCL1,CXCL2,CXCL3 expression.In addition,F.nucleatum significantly elevated PTGS2,SOD2,CSF2,IL-6,IL-8 and IL-1β gene expression.ELISA results indicated that F.nucleatum significantly promoted IL-6 and IL-8 release,while did not influence IL-1β protein release in GMSCs.F.nucleatum could inhibit GMSC osteogenic differentiation partly by decreasing ALP activity and mineralized nodule formation,and osteogenesis-related gene and protein expression.qRT-PCR assay results indicated that F.nucleatum significantly decreased osteogenic-related genes ONC,ALP,Runx2,OCN,BSP and OPN expression.Western blot assay results indicated F.nucleatum decreased osteogenic-related protein ALP,COL1,BMP2,Runx2,Osterix and BSP expression.RNA-seq analysis confirmed that GMSCs co-cultured with F.nucleatum for 3,7,14 and 21 d generated 258,323,591 and 245 DEGs,respectively,and a total of 8 DEGs was generated in GMSCs co-cultured with F.nucleatum at all the time points.In addition,149,140,292 and 84 DEGs were specifically identified at 3,7,14 and 21 d of F.nucleatum infection,respectively.The reactome functional enrichment analysis showed that 149 DEGs specifically induced by F.nucleatum infection at 3 d were predominantly enriched in the interferon-related signaling pathway;140 DEGs specifically produced after 7 d of F.nucleatum infection were mostly enriched in extracellular matrix organization,collagen formation,collagen biosynthesis,and modifying enzymes;and 292 unique DEGs generated after 14 d of F.nucleatum infection were mainly enriched in IL-10,IL-4,and IL-13 signaling,chemokine receptors bind chemokines,and O-linked glycosylation signaling pathway;84 DEGs specifically produced at 21 d of F.nucleatum infection were largely enriched in G protein-coupled receptors(GPCRs)-related signaling pathways.A total of 64 cell differentiation related DEGs was selected out according to the GO biological processes,such as Bcl2,IL-6,TGFβ2.All of the DEGs were enriched in cytokine activity,cytokines and growth factor binding-related signaling pathway or growth factor complex,cell surface,and extracellular matrix signaling pathways.In addition,we discovered that 64 cell differentiation-related DEGs were associated with human cancers,such as bone cancer,osteosarcoma,and thyroid cancer according to the DO enrichment analysis,which suggests that persistent exposure to F.nucleatum greatly increases the risk of GMSCs carcinogenesis during the process of osteogenic differentiation.5.F.nucleatum triggered GMSCs tumor associated genes(TAGs)expression in the process of cell osteogenic differentiationTime-course transcriptome analysis indicated F.nucleatum inhibited GMSCs osteogenic differentiation and activated TAGs expression.maSigPro analysis results indicated that a total of 790 co-DEGs was generated in GMSCs co-cultured with F.nucleatum for 3 d,7d,14 d and 21 d.Among the 790 DEGs,50 TAGs including L3MBTL4,CD163,CCND2,CADM1,BCL7A,and IGF1 were identified.In addition,five core dynamic DEGs(PLCG2,CHI3L2,L3MBTL4,SH2D2A and NLRP3)were identified during the process of F.nucleatum infection.Results from a GeneMANIA database analysis showed that PLCG2,CHI3L2,SH2D2A and NLRP3 and 20 otherproteins formed a complex network and most of these genes were enriched in cancer-related pathways.Based on the five core dynamic DEGs,a total of 43 transcription factors(TFs)and 50 related microRNAs(miRNAs)were obtained,and the integrated TFs-mRNAs-microRNA network were constructed.Conclusions:1.Successfully cultured F.nucleatum ATCC 25586,GFs and GMSCs,and constructed the co-cultured model of GFs/GMSCs and F.nucleatum.2.F.nucleatum facilitated GF apoptosis,ROS generation,and inflammatory cytokine production by activating AKT/MAPK and NF-κB signaling pathways.3.The cogena-based drug repositioning method could be used as a new way to screen out drugs and assist in the research of clinical antimicrobial therapy.Etravirine,zalcitabine,wortmannin,calcium D-pantothenate,ellipticine and tanespimycin could significantly decrease F.nucleatum-induced ROS generation and block PKB/AKT)/MAPK signaling pathways,which play key roles in protecting GFs.4.F.nucleatum triggered GMSCs chemokine/cytokine release and inhibited cell proliferation and osteogenic differentiation.Persistent exposure to F.nucleatum greatly increased the risk of GMSC carcinogenesis during the process of osteogenic differentiation,which suggested that inflammatory microenvironment should be reconsidered while applying stem cell based tissue regeneration engineering.5.F.nucleatum inhibited osteogenic differentiation and promoted oncogene expression of GMSCs.F.nucleatum significantly changed core dynamic DEGs expression,including PLCG2,CHI3L2,L3MBTL4,SH2D2A and NLRP3.These genes were regulated by 43 TFs(such as CREB3,GATA2 and SOX4)and 50 miRNAs(such as miR-372-3p,miR-603,and miR-495-3p). |
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