Effects And Mechanism Of Periodontitis Induced By Fusobacterium Nucleatum In The Development Of Atherosclerosis

Atherosclerosis(AS)is a chronic and inflammatory disease that primarily affects large and medium-sized blood vessels and has a high incidence rate globally.It can lead to various cardiovascular and cerebrovascular diseases.Periodontal disease is a chronic and destructive condition that affects the periodontal supporting tissues,leading to adverse effects not only on oral tissues but also on remote organs.Pathogenic periodontal microorganisms have been identified as an independent risk factor for atherosclerosis.Some evidence suggested that periodontal microorganisms exist in atherosclerotic plaques.Fusobacterium nucleatum(F.nucleatum),a gram-negative anaerobic bacterium,contributes to the occurrence of periodontitis and has been shown to promote the progression of AS.Therefore,investigating the role and potential mechanism of F.nucleatum in AS is clinically significant for the treatment of patients with periodontitis complicated with atherosclerosis.The pathogenesis of atherosclerosis is complex and involves multiple cell types,with macrophages being the primary cell group present in atherosclerotic plaques.Macrophages ingest lipids and convert into foam cells,exacerbating lipid load and inflammation in plaques,thus marking the formation of atherosclerotic lesions.Therefore,it is crucial to ascertain whether and how F.nucleatum promotes lipid deposition in macrophage-derived foam cells to explore the mechanisms of F.nucleatum promotion the occurrence of atherosclerosis.It has been proposed that virulence proteins of bacteria may accelerate the formation and progression of plaques in the inner walls of coronary arteries.FadA and Fap2,the currently identified virulence proteins of F.nucleatum,have been shown to promote periodontitis and cancer,but whether they act as key virulence proteins of F.nucleatum promoting lipid deposition in macrophages is not known.Infection of the host by pathogenic bacteria and the corresponding disease symptoms occur as a result of the interaction of virulence factors with the host cells.Macrophages can express pattern recognition receptors to sense microorganisms,which in turn activate a signal transduction cascade response for a rapid innate immune response.For example,lipopolysaccharide(LPS)can stimulate cytokine production via tolllike receptors(TLRs).However,it has not been suggested in which way the virulence proteins of F.nucleatum are recognized and acted by macrophages during lipid deposition by macrophages.In summary,this study aims to evaluate the possibility of atherosclerosis in F.nucleatuminduced experimental periodontitis mice;to clarify whether F.nucleatum is involved in the process of monocyte-derived macrophage foaming;to screen the key virulence protein in F.nucleatum that interacts with macrophages,to explore the receptors on macrophages and elucidate the central role of the interaction of this two protein in inflammatory signaling and cholesterol transport.This study will help to deepen the understanding of the role of periodontitis in promoting the development of atherosclerosis,and discover new pathogenesis of atherosclerosis,so as to provide new targets for the treatment of atherosclerosis.Materials and Methods:1.Detection of F.nucleatum in human atherosclerotic plaque samples and construction of a mouse atherosclerosis model with F.nucleatum-induced experimental periodontitisIn this study,we first downloaded RNA-seq data from atherosclerosis datasets GSE104140 and GSE120521,via the NCBI GEO database,to investigate the presence of F.nucleatum in human atherosclerosis samples.Next,we established an experimental periodontitis model in ApoE-/-mice by topically applying F.nucleatum to the periodontium,while feeding the mice a high-fat diet.To evaluate the effect of F.nucleatum on the resorption of alveolar bone,microCT was used.The state of inflammation in gingival and alveolar bone tissues was assessed by H&E staining,while FISH staining was employed to detect the infiltration of F.nucleatum into gingival and aortic tissues.Aortic plaque formation was examined using MRI and bulk Oil Red O staining.Additionally,plaque area of the aortic sinus was analyzed by H&E and Oil Red O staining,while immunofluorescence staining was used to examine the distribution of macrophages in aortic plaque tissues.RT-qPCR experiments were performed on aortic tissues to detect the expression of inflammatory factors and lipid metabolism-related factors.2.Study on the regulatory effect and mechanism of F.nuclatum on the formation of lipid droplets in THP-1 derived macrophagesTo investigate the effect of F.nucleatum infection on THP-1 derived macrophage,cells were infexted with the bacteria(MOI=50)for 24 h and analyzed using various techniques.Flow cytometry was used to assess apoptosis,while enzyme marker was utilized to measure the contents of total cholesterol,free cholesterol,and cholesterol esters in the infectd cells.Oil Red O staining was performed to visualized intracellular lipid deposition,and RT-qPCR was used to evaluate the mRNA expression levels of inflammatory factors and lipid metabolism factors.In addition,transcriptome gene sequencing was carried out on infected cells at different time points(2,6,12,24,and 48 h).Time-series analysis was conducted using maSigPro and mFuzz,while different gene analysis was performed by limma,GO,and KEGG databases.Furthermore,the protein interaction network was abakyzed using the string database and the Cytascape software.3.Determination and mechanistic studies of the interaction between F.nucleatum virulence proteins and THP-1-derived macrophage host proteinsTo identify the key virulence proteins of F.nucleatum that interact with THP-1-derived macrophages,we utilized a biotin pull-down assay and a high-performance liquid chromatography-mass spectrometry coupling assay.The PET28a-Gbp plasmid was then constructed,and the proteins were expressed and purified in E.coli.The concentration of Dgalactose-binding protein(Gbp)protein,without cytotoxicity,was screened using a cell activity assay for subsequent experiments.Cells were stimulated with Gbp protein for 24 hours,and lipid deposition in the cells was observed using BODIPY staining.mRNA expression levels of inflammatory factors and lipid-metabolizing factors were detected using RT-qPCR.We also screened the receptor proteins that can interact with Gbp protein in macrophages using a Histagged pull-down assay,a high-performance liquid chromatography-mass spectrometry coupling assay,and a Co-immunoprecipitation(Co-IP)assay.Furthermore,we knocked down the expression of this receptor protein in cells by siRNA and observed intracellular lipid deposition by Oil Red O staining.mRNA expression levels of inflammatory factors and lipidmetabolizing factors were detected using RT-qPCR,and the activation of related pathways was detected using the protein immunoblotting technique.4.Intervention study of lipid deposition induced by F.nucleatum infectionTo identify potential intervention agents that target the PI3K-AKT and MAPK pathways,we utilized the cogena tool to analyze overall differentially expressed genes.We then crossreferenced these genes with the DGI database to identify drug targets.Bioactivity assays were performed to determine appropriate drug concentrations for subsequent experiments.Western blot analysis was employed to detect the activation of relevant pathways after the administration of intervention agents.Additionally,intracellular lipid deposition was observed using BODIPY staining.RT-qPCR was used to assess the mRNA expression levels of inflammatory factors and lipid-metabolizing factors.Results1.F.nucleatum promoted the formation of atherosclerotic plaquesWe detected the presence of F.nucleatum in all samples from both GSE104140 and GSE120521 datasets.Results from CIBERSORTx analysis showed that macrophages had the highest percentage of all types of immune cells,and the abundance of F.nucleatum was positively correlated with the percentage of macrophages.Mi croCT and H&E staining revealed that F.nucleatum promoted inflammation and bone loss in the periodontal tissues of mice,while FISH staining showed that F.nucleatum colonized in gingival and vascular plaques.Furthermore,results from MRI and aortic bulk Oil Red O staining showed that F.nucleatum promoted aortic plaque formation in mice,and the total serum cholesterol level was higher in the F.nucleatum group based on serum biochemical indexes.Immunofluorescence staining showed that macrophages were the most abundant cells in atherosclerotic plaques in mice.RTqPCR results revealed an increase in the expression of inflammatory factors and scavenger receptor mRNA levels,while cholesterol efflux-related gene expressions were decreased in the aortic tissues of mice in the F.nucleatum group.2.F.nucleatum promoted lipid deposition in THP-1 derived macrophage via PI3KAKT/MAPK/NF-κB pathwaysThe results showed that F.nucleatum induced macrophage apoptosis at varying degrees when the MOI was greater than 50.Bacteria stimulated with MOI of 50 showed increased intracellular lipid deposition,as indicated by Oil Red O staining,with increased levels of total cholesterol,free cholesterol,and cholesteryl esters.RT-qPCR results demonstrated that F.nucleatum upregulated the expression of inflammatory factors such as int.erleukin(IL)6,IL1β,and matrix metalloproteinase 9(MMP9),promoted the expression of the scavenger receptor CD36 on the surface of macrophages,and inhibited the expression of cholesterol efflux-related factors such as liver X receptor(LXR),ATP-binding cassette transporter(ABC)A1,ABCG1,and peroxisome proliferative activated receptor gamma(PPARG).RNA-seq results revealed that F.nucleatum transiently activated a large number of genes affecting immune inflammatory responses and cholesterol transport processes,mainly through the activation of the PI3KAKT/MAPK/NF-κB signaling pathway.3.Gbp interacted with CypA to promote lipid deposition in THP-1 derived macrophage by activating NF-κB,MAPK and PI3K-AKT pathwaysThe key virulence protein of F.nucleatum that interacts with macrophages was identified using a biotin pull-down assay and high performance liquid chromatography-mass spectrometry.The Gbp protein was successfully expressed and purified in E.coli,and its concentration without cytotoxicity was screened using a CCK8 assay.The Gbp protein was then used to stimulate macrophages,leading to an increase in intracellular lipid deposition.RT-qPCR and western blot results showed that F.nucleatum promoted the expression of inflammatory factors IL1β,IL6,MMP9,and the scavenger receptor CD36,while inhibiting the expression of cholesterol efflux-related factors such as ABCG1,PPARG,and LXR,and activating the NF-κB,MAPK,and PI3K-AKT pathways.The receptor protein that interacts with Gbp in macrophages was identified as CypA using a His-tagged pull-down assay,and it was shown through Co-IP that Gbp could bind to CypA.Knockdown of intracellular CypA expression using small interference revealed a decrease in intracellular lipid deposition.Additionally,ELISA results showed an increase in extracellular CypA content,and CD 147 was identified as the receptor for extracellular CypA.RT-qPCR and western blot results demonstrated that Gbp promoted CD 147 expression at the gene and protein levels.Knockdown of CD 147 using small interference reduced Gbp stimulation-induced lipid deposition and decreased the activity of related pathways.4.Interventions targeting PI3K-AKT and MAPK pathways resisted F.nucleatum-induced lipid depositionThe drug repositioning method based on cogena was used to screen drugs that could reverse the co-expression of differential genes.A total of 3 drugs targeting upregulated genes(Tretinoin,Isotretonoin,Trichostatim)and 3 drugs targeting upregulated genes(Betulin,Tanespimycin,Liothyronine)were obtained.The results of KEGG enrichment analysis of the action targets of these drugs showed that the drugs inhibited the function of NF-κB,MAPK,and PI3K-AKT signal pathways.CCK8 experiments were conducted to screen drug concentrations that do not affect biological activity.Western blot results showed that the drugs can inhibit the activation of MAPK and PI3K-AKT signaling pathways.BODIPY and RT-qPCR results confirmed that these drugs effectively suppressed F.nucleatum-induced lipid deposition.Conclusions1.F.nucleatum had been implicated in the development of atherosclerosis in humans,causing periodontitis in experimental mice,and promoting the formation of atherosclerosis in mice,mainly through its effects on macrophages.2.F.nucleatum infection activated the PI3K-AKT/MAPK/NF-κB signaling pathways,leading to increased immune inflammatory response,increased cholesterol uptake,decreased efflux,and the formation of macrophage foam cells.3.The main virulence protein of F.nucleatum,Gbp,promoted macrophage foaminess by binding to CypA in THP-1-derived macrophages and interacting with the CD 147 receptor to initiate the PI3K-AKT/MAPK/NF-κB signaling pathways.4.Six interventions,including Tretinoin,Isotretonoin,Trichostatin,Betulin,Tanespimycin,and Liothyronine resisted lipid deposition in THP-1-derived macrophages by inhibiting the activation of PI3K-AKT and MAPK pathways.