Biological Effects And Mechanisms Of F.nucleatum On Periodontal Ligament Stem Cells In The Early Stage Of Infection

Background and objectivesPeriodontitis is a chronic inflammatory disease that occurs in the periodontal tissue,and it is the sixth most prevalent disease in the world,affecting more than 50%of adults worldwide.The pathogenesis of periodontitis is complex and convoluted,including microbial challenge,host genetic variation,and acquired environmental stress.Among the pathogenic factors,the rise in pathogenic bacteria in subgingival plaque is one of the pathogenic causes that is frequently cited as a precursor for the onset of periodontitis.Fusobacterium nucleatum(F.nucleatum)is one of the most commonly detected pathogens in the oral cavity of periodontitis patients.In recent years,it has drawn more and more attention as a result of its tight connection to various systemic diseases.F.nucleatum is an invasive bacterium that causes a range of host responses.Clinical research has demonstrated that the detection rate of F.nucleatum increases along with the progression of periodontitis.Studies have shown that F.nucleatum can invade many kinds of host cells,such as epithelial and endothelial cells,monocytes,and fibroblasts.F.nucleatum infection can initiate host cell inflammatory cascades and induce the secretion of pro-inflammatory cytokines IL-6 and IL-8.The secretion of toxic proteins is crucial for bacteria to exert pathogenicity.F.nucleatum expresses multiple virulence factors to elicit different host responses.For example,RadD and Fap2 cause apoptosis in lymphocytes,while FadA mediates host attachment and invasion in epithelial cells.FadA is currently considered the most typical virulence factor of F.nucleatum.According to reports,the virulence and pathogenicity of F.nucleatum may be closely related to FadA.The study found that the detection rates of F.nucleatum and FadA were positively correlated with the gingival index.They played a role in the occurrence and development of orthodontic gingival inflammation.The key role of FadA in the pathogenic mechanism of F.nucleatum provides a new idea for exploring the relationship between F.nucleatum and periodontitis.Periodontal ligament stem cells(PDLSCs),which comprise most of the periodontal ligament,are essential for maintaining periodontal homeostasis.As periodontal inflammation progresses,the oral mucosal barrier suffers from an impairment,followed by increased invasiveness of periodontal microflora and disorders of the host immune responses,which attenuate the function of resident PDLSCs.These impaired PDLSCs may disrupt the microenvironment by aggravating the host immune responses,and facilitating the osteoclastic activity.However,the effects of F.nucleatum on the biological functions and gene regulatory changes of PDLSCs have not been fully elucidated.In summary,this study aims to investigate the pathogenic role of F.nucleatum and the response of PDLSCs in the early stage of F.nucleatum infection,clarify the toxic effect of F.nucleatum adhesin FadA on PDLSCs,and identify the interaction of FadA in PDLSCs.This study aims to reveal the molecular mechanism of pathogenicity by working with proteins;reveal the gene regulation process of host cells in response to F.nucleatum infection through the changes in gene expression profiles after co-culture of sequential F.nucleatum and PDLSCs;use drug repositioning analysis based on co-expressed genes to screen for candidate drugs for F.nucleatum infection provide new ideas for clinical treatment of periodontitis.Materials and methods1.The pathogenic effects of F.nucleatum on PDLSCsAfter resuscitation and expanded culture,F.nucleatum was identified by PCR analysis and 16S rRNA sequendcing.Periodontal ligament stem cells were obtained from the periodontal ligament tissue of healthy premolars or third molars.To confirm that cultured cells had the potential for multilineage differentiation,osteogenic induction and adipogenic induction were induced.The surface markers were detected via flow cytometry.F.nucleatum was co-cultured with PDLSCs to establish an in vitro F.nucleatum infection model.The effects of F.nucleatum at different multiplicities of infection(MOI)on the proliferation of PDLSCs were detected by cell counting and EdU labeling experiments.The apoptosis of PDLSCs at different time points after F.nucleatum infection was detected by flow cytometry.FerroOrange was used to detect the level of Fe2+ in PDLSCs after F.nucleatum infection,and JC-1 was used to detect the changes of the mitochondrial membrane potential.QRT-PCR and ELISA were performed to detect the expression levels of IL-1 β,IL-6,and IL-8.2.FadA activates PEBP1/IKK/NF-κB and PEBP1/Raf1/MAPK signaling pathways by interacting with PEBP1FadA with His-tag was expressed and purified by the E.coli expression system.The effects of FadA on the expression levels of IL-1 β,IL-6 and IL-8 in PDLSCs were detected via qRTPCR and ELISA.The protein interacting with FadA in PDLSCs was obtained by His pull-down experiment,identified by mass spectrometry,and the potential interacting protein PEBP1 in PDLSCs was screened out.The combination of FadA and PEBP1 in cells was verified by coimmunoprecipitation experiments.The purified His-tagged PEBP1 was obtained through the E.coli expression system,and the binding affinity of FadA to PEBP1 was detected by surface plasmon resonance analysis.Western blot was used to detect the phosphorylation levels of PEBP1,Rafl and IKK in PDLSCs,and the activation of NF-κB and MAPK signaling pathways.Small interfering RNA(small interfering RNA,siRNA)was used to interfere the expression of PEBP1.PDLSCs were infected with CFDA-SE dye-labeled F.nucleatum,and the invasion ability of F.nucleatum on PDLSCs after PEBP1 knockdown was detected by flow cytometry.The adhesion ability of F.nucleatum to PDLSCs after PEBP1 knockdown was detected by bacterial adhesion assay.3.Time course RNA-seq analysis of F.nucleatum-infected PDLSCsPDLSCs infected with or without F.nucleatum were collected at 0 h,1 h,3 h,6 h,and 12 h after infection for RNA-seq sequencing analysis.DESeq2 was used to analyze the significant difference between samples and screen differential expression genes(DEGs).The heatmap was plotted through pheatmap to visualize the expression of differential genes.GO and KEGG were used to enrich for differential genes.After F.nucleatum infection,the activation of signaling pathways in PDLSCs and the changes of related genes on the pathways were visualized by Pathview.In order to mine the potential dynamic characteristics of genes at different time points over time,Mfuzz is used for time series analysis and the results are visualized.The transcriptome data was compared to the human reference genome data using the bowtie2 comparison tool.Differential microRNAs were calculated using DESeq2,and target genes were predicted using miRWalk2.0.The data of differentially expressed genes were transformed by log2 and then kmeans clustered,and the regulatory relationship between modules and within modules was calculated.Use TRRUST to predict transcriptional regulatory factors,and filter the RegNetwork prediction results based on TRRUST results to obtain the corresponding transcriptional regulatory network.4.Screening of pathway-targeted drugs based on the pathogenic mechanisms of F.nucleatumThe differential expressed genes at all time points were clustered using cogena.Next,we used SwissTargetPrediction to predict the potential target genes of candidate drugs.GO enrichment analysis was used to predict the potential target genes of candidate drugs,and drugs that could inhibit the activation of IL-17,TNF,NF-κB,and MAPK signaling pathways were filtered out.The cytotoxicity of candidate drugs was evaluated by CCK-8 assay,and the concentration for follow-up studies was determined.The effects of candidate drugs on the expression of pro-inflammatory cytokines IL-1β,IL-6,and IL-8 in PDLSCs caused by F.nucleatum-infection or FadA-stimulation were detected by qRT-PCR and ELISA.FerroOrange probe and JC-1 probe were used to detect Fe2+ level and mitochondrial membrane potential,respectively.AutoDock Vina was used to perform molecular docking,and PyMOL was used to visualize the docking results.The predicted targets of piperlongumine were verified by western blot experiments,and the effects of piperlongumine on the NF-κB and MAPK signaling pathways activated by F.nucleatum-infection were evaluated.Results1.The pathogenic effects of F.nucleatum on PDLSCsWe successfully cultured and identified F.nucleatum;successfully isolated,cultured and identified PDLSCs.Cell proliferation experiments showed that the proliferation of PDLSCs was significantly inhibited by nucleatum significantly in a time-and concentration-dependent manner.As the concentration of F,nucleatum increased,the ratio of EdU-positive signals gradually decreased,indicating that F.nucleatum can inhibit DNA replication to affect the proliferation ability of PDLSCs.AnnexinV/FITC analysis showed that F.nucleatum could promote the apoptosis of PDLSCs.FerroOrange fluorescent probe staining showed that the fluorescence intensity of Fe2+ in PDLSCs was significantly increased after F.nucleatum infection.By staining with the JC-1 fluorescent probe,it was found that the fluorescence intensity of the JC-1 aggregates decreased significantly after F.nucleatum stimulation The qRTPCR and ELISA detection showed that F.nucleatum infection significantly stimulated the expression of IL-1β,IL-6,IL-8 in PDLSCs.2.FadA activates PEBP1/IKK/NF-κB and PEBP1/Raf1/MAPK signaling pathways by interacting with PEBP1The recombinant FadA with His-tag was obtained successfully.The detection of the expression levels of inflammatory factors showed that stimulation of FadA alone could induce the inflammatory response of PDLSCs.Through His pull-down experiment and mass spectrometry identification,we found that phosphatidylethanolamine binding protein 1(PEBP1)can interact with FadA,and verified the interaction of FadA and PEBP1 by coimmunoprecipitation and surface plasmon resonance analysis.The results of Western blot showed that FadA significantly increased the phosphorylation levels of Rafl and IKK,and ERK/JNK/p38 and p65 were also phosphorylated after adding FadA.After using siRNA to inhibit the expression of PEBP1,the fluorescence intensity of F.nucleatum in cells knocked down by PEBP1 was detected to be significantly reduced,and the number of adherent F.nucleatum colonies was also significantly lower than that of the control group.3.Time course RNA-seq analysis of F.nucleatum-infected PDLSCs CXCL1,CXCL2,RASD1,and TNFAIP3 were upregulated at all time points after F.nucleatum stimulation.All genes were clustered according to the expression pattern by Mfuzz analysis,the control group was clustered into 30 clusters,the experimental group was clustered into 20 clusters,and various pro-inflanmmatory cytokines were clustered into the cluster 20 of the experimental group(such as IL-1β,IL-6 and IL-8),and their expression level gradually increased with the prolongation of infection time.Collectively,these results indicated that F.nucleatum was capable of inducing inflammatory response in PDLSCs,which was associated with activation of the NF-κB signaling pathway and the production of inflammatory chemokines.4.Screening of pathway-targeted drugs based on the pathogenic mechanisms of F.nucleatumCogena analysis divided all differentially expressed genes after F.nucleatum infection into three categories according to their expression patterns.KEGG enrichment analysis results showed that genes in cluster 1 and cluster 2 were highly enriched in immune-related pathways.Taken together with the results of the functional enrichment analysis of drug target genes,6 drugs that could block the activation of IL-17,TNF,NF-κB,and MAPK signaling pathways were filtered out.The expression of inflammatory factors was detected by qRT-PCR.The results showed that the six candidate drugs significantly reduced the expression of IL-1β,IL-6 and IL8 induced by F.nucleatum infection.Piperlongumine and fisetin showed the best inhibitory effects,and can significantly reduce FadA-induced production of pro-inflammatory cytokines.The results of FerroOrange and JC-1 fluorescent probe staining found that piperlongumine and fisetin reversed the trend of ferroptosis induced by F.nucleatum,reduced intracellular Fe2+levels,and restored damaged mitochondrial function.The results of molecular docking and western blot analysis showed that F.nucleatum-infection could increase the phosphorylation of IKK,p65 and p38,and piperlongumine can significantly block the activation of IKK/p65/NFκB and p38/MAPK signaling pathways induced by F.nucleatum.Conclusions1.This study successfully constructed an in vitro Fusobacterium nucleatum-periodontal ligament stem cells co-culture model,and proved that F.nucleatum can inhibit the proliferation of PDLSCs,promote cell apoptosis,ferroptosis and the production of inflammatory cytokines.2.F.nucleatum adhesin FadA,as a virulence factor,could induce the expression of IL-1β,IL-6 and IL-8 in PDLSCs by stimulation itself.Further studies have shown that FadA can bind to PEBP1 of PDLSCs,activate Raf1/MAPK and IKK/NF-κB signaling pathways,and induce inflammatory responses.3.Time-course RNA-sequencing revealed the gene expression profile of PDLSCs after F.nucleatum infection.The analysis revealed a cascade of genes in PDLSCs with the duration of F.nucleatum infection.At 3 h post-infection with F.nucleatum,both NF-κB1 and NF-κB2 were upregulated,and the expression of inflammation related genes in the NF-κB signaling pathway were upregulated.4.Using drug repositioning analysis,we proved that piperlongumine,fisetin,tanespimycin,betulinic acid,lomustine,and tiabendazole could inhibit the inflammatory response of PDLSCs caused by F.nucleatum infection,and piperlongumine and fisetin can inhibit F.nucleatuminduced ferroptosis.Piperlonguming could block the activation of IKK/p65/NF-κB and p38/MAPK signaling pathways.