| Background and objectiveFusobacterium nucleatum(F.nucleatum)is an important periodontal pathogen which can promote the occurrence and development of periodontitis and gingivitis.F.nucleatum has a strong ability to polymerize together with the initial,early and late stage colonizing bacteria to form the oral biofilm.It is an important "cartilage"organism in the formation of dental plaque.The relationship between the oral microbiome and systemic diseases is now gradually attracting more attentions.F.nucleatum is one of the most common species of human infections,such as in tropical skin ulcers,peritonitis and septic arthritis,bacterial abscess and liver abscess,intrauterine infection,bacterial vaginosis,urinary tract infection,pericarditis and endocarditis,lung and pleurisy and so on.Furthermore,it contributes to the occurrence,promotion and even metastasis of colorectal cancer.Periodontal disease can be connected with the occurrence of oral cancer,because chronic inflammation is the common cause of these two diseases.Bone is a fiber-reinforced calcified tissue that can be permanently remodeled.This process is mainly regulated by two types of cells-osteoblasts(leading bone matrix formation)and osteoclasts(leading bone matrix absorption).Similarly,the maintenance of alveolar bone homeostasis in the oral cavity is also closely related to the adjustment of the dynamic balance between these two cells.Periodontal disease and apical abscess are both common diseases in oral cavity caused by bacterial invasion.Dental plaque is the initiatal pathogenic factor of periodontal disease,and the absorption and destruction of alveolar bone is an important feature of periodontal disease.Bacteria that invades into the pulp cavity will drain the infected root canal and irrupt the surrounding tissues of the root canal,thus causing extra-root infection,purulent inflammation and the destruction of periapical tissues.Therefore,to figure out the mechanisms that how bacteria lead to bone destruction is important to understand the pathogenesis of periodontal disease and apical inflammation,and is also useful to the treatment and prevention of these diseases.Bacteria can directly invade the alveolar bone and cause bone destruction.It can directly infect osteoblasts and lead to imbalance on their functions.Therefore,we established a model of a long-term infection of F.nucleatum stimulating primary rat osteoblasts in vitro.A series of cytobilogical assays were performed to research the changes of biological activity and osteogenic ability of osteoblasts after this infection,and to verify the related molecular pathways.RNA-seq was used to further study the changes of transcriptomes in osteoblasts at time-series infection of F.nucleatum.The objective of this study was to provide strong theoretical support for further understanding the mechanism of bone loss caused by pathogenic microorganism infection.Materials and Methods1.Culture and identification of F.nucleatum and primary osteoblasts in rats and the establishment of a model of F.nucleatum co-cultured with rat osteoblasts in vitro.The strain of F.nucleatum(ATCC 25586)was obtained from the Key Laboratory of Oral Tissue Regeneration of Shandong University.The preserved strains were thawed and then amplified,cultured,and then identified by 16S rRNA sequencing to confirm that the cultured strains were free of other bacteria.Primary osteoblasts were taken from the skull of newborn(24h-36h)Wistar rats by enzyme digestion combined with tissue block crawling method.Then osteoblasts were identified by Col-1 immunochemical staining,ALP staining and von Kossa staining.F.nucleatum at the logarithmic growth phase was collected,and then added into the osteoblasts at the multiplicity of infection(MOIs)of 0,10,50,100 and 200,to establish an in-vitro model of bacteria-cell co-cultured infection.2.The pathogenic effects of F.nucleatum on the biological activity of primary rat osteoblasts.(1)The effect of F.nucleatum with MO Is of 0,10,50,100 and 200 on the proliferation of osteoblasts was detected by cell counting and EdU proliferation rate detection experiments.The effects of F.nucleatum with different MOIs on cell apoptosis and cell cycles of osteoblasts were analyzed by flow cytometry.The secretion of inflammatory cytokines in the culture supernatant after stimulation with the above-mentioned MOIs of F.nucleatum on osteoblasts was detected by ELISA assays.(2)After repeatedly infection with F.nucleatum for a long time,the osteogenic ability and mineralized nodules formation of osteoblasts were evaluated by ALP test,intracellular calcium detection and Alizarin red staining assays.At the same time,osteoblasts stimulated with F.nucleatum for 7d,14d,21d and 28d were collected,and the expression changes of osteogenic differentiation related genes and proteins in osteoblasts were detected by qRT-PCR and Western blots.(3)Data were analyzed by one-way ANOVA or two-way ANOVA followed by Tukey's honestly significant difference comparison test.The variation between two groups at different times was compared by multiple t-test.A p-value<0.05 indicated statistical significance.3.Time-series RNA-seq analysis of rat primary osteoblasts stimulated with F.nucleatum(1)Osteoblasts were co-cultured with F.nucleatum at MOI=50,then cell samples were separately collected at Id,3d,7d,14d,21 d and 28d after stimulation and analyzed by RNA sequencing.Bioinformatic analyses were used to identify the DEGs at different time points,and the Venn diagram was used to analyze the DEGs at all six time points.STRING database was used for enrichment analysis and PPI analysis of these common genes.(2)GO annotations of all of the DEGs through all time points were performed by the Metascape database analysis,and the DEGs related to the biological process in these experiments were further analyzed.STEM tools were used to analyze the expression trends of genes in osteoblasts stimulated by F.nucleatum at different time points.After that,we focused on the osteogenic related DEGs which showed significant expression-trends,and verified by qRT-PCR.A p-value<0.05 indicated statistical significance.(3)The SNP types and variable shear types of mRNA in osteoblasts after infection with F.nucleatum at each separate time were analyzed.4.The mechanism research of the effects of F.nucleatum on the pathogenicity of primary rat osteoblasts(1)KEGG enrichment analysis was respectively performed on the DEGs at six time points to observe the changed trend of the pathways in osteoblasts stimulated by F.nucleatum at different time points.(2)Pathview database was used to analysis DEGs of MAPK and NF-?B pathways at six time points.(3)Proteins from osteoblasts that stimulated with F.nucleatum(MOI=50)at Omin,5min,15min,30min,60min and 120min were collected and extracted,respectively,then using Western blots assays were used to detect the pathway-related protein levels after stimulation of F.nucleatum.A p-value<0.05 indicated statistical significance.Results1.Culture and identification of F.nucleatum and primary osteoblasts in rats and the establishment of a model of F.nucleatum co-cultured with rat osteoblasts in vitro.The bacteria was identified as Fusobacterium nucleatum ATCC 25586 by PCR and 16S rRNA.The suspension of F.nucleatum is streaked on a BHI culture plate and placed in an anaerobic incubator for 4 to 5 days.Then dozens of single colonies can be seen on the plate.The colony is translucent and flat with a convex center and irregular edges,and with a smell of rotten eggs.The logarithmic growth period is 16h to 28h after culturing.The cultured primary cells were identified by Col-1 immunohistochemical staining,ALP staining and von Kossa staining.Osteoblasts stimulated with F.nucleatum at MOIs of 0,10,50,100 and 200 can be co-cultured for a long-term.Cytoskeleton staining showed that the cytoplasm and shape of the control group were full and regular,triangle or polygon.However,F.nucleatum could attach to the cell surface or enter into the osteoblasts,then cause shrunken,elongated and irregular changes of the cytoplasm,and the synapses between adjacent cells were increased.2.The effects of F.nucleatum on the biological activity of primary rat osteoblasts.(1)The results of cell counting and EdU detected assays indicated that F.nucleatum significantly inhibited osteoblasts proliferation,which showed time-and concentration-dependent manners.Flow cytometry analysis of cell apoptosis showed that low concentration(MOI=10)bacteria had no obvious effect on cell apoptosis.With the increased concentration of F.nucleatum,the proportion of normal cells gradually decreased,while the late stage apoptotic cells and total apoptosis proportions increased accordingly.The results of cell cycle indicated that F.nucleatum mainly affected the cell cycles and inhibit its proliferation by blocking the G2/M phase.ELISA results showed that the secretion of inflammatory cytokines IL-6 and TNF-? increased significantly in osteoblasts after stimulation with F.nucleatum.qRT-PCR results showed that Caspase-8 increased significantly after bacterial stimulation at 6h and continued to 48h,while decreased at 72h.On the contrary,the expression of Bcl-2 was lower before 48h,but increased significantly at 72h;the expression of Rankl increased from 6h and continued to 72h.(2)Different MOIs of F.nucleatum were used to stimulate osteoblasts for 3d,7d and 14d,which significantly inhibited the intracellular ALP activity.The calcium content results indicated that the calcium contents of the experiment groups were significantly reduced by F.nucleatum infection on the 21 d.The Alizarin Red staining revealed that F.nucleatum significantly inhibited the formation of mineralized nodules and calcium deposits in the cells at the 21d and 28d.QRT-PCR and Western blots results indicated that the expression of genes(such as Alpl,Runx2,Col-1,Opg,Bsp,Osx and Ocn)and proteins(such as ALP,RUNX2,COL-1,OPG,BSP and OSX)participated in osteogenesis in osteoblasts decreased significantly,while the expression of RANKL increased significantly.3.Time-series RNA-seq analysis of rat primary osteoblasts stimulated with F.nucleatum(1)The principal component analysis of all samples at each time point showed that the samples of the control group and the F.nucleatum infected groups were significantly separated.As the bacterial stimulation time increases,the correlation coefficient between samples gradually decreased.Transcriptome analysis revealed that there were 1138,1122,1446,1039,1382 and 1048 differential transcripts that were differentially expressed on the 1d,3d,7d,14d,21d and 28d,respectively.235 common genes were differentially expressed through all the time.The enrichment analyses results revealed that these genes were mainly enriched in inflammatory biological processes and immune-related pathways,as well as cytokine-related pathways.The results of PPI interaction network analysis showed that the genes Il-6,Vcam1,Tlr2,Csflr,Cxcl1,C3,Thbsl and Socs3,were more closely related to others and might play a key regulatory role in the network.(2)STEM analysis was performed on the transcriptome data of the experimental groups stimulated by F.nucleatum,and 8042 genes with significant trends were obtained.133 genes both with significant differentially expression differences and with significant expression trends in osteogenesis.These genes mainly enriched in multiple pathways related to cancer.A further analysis showed that 8 core DEGs related to osteogenesis(Cyplbl,Mnda,Comp,Phex,Mmp3,Nfkb2,Fbln5 and Tnfrsf1b),whose expression levels continued to increase/decrease,were obtained,and the significance of these genes was verified by qRT-PCR.(3)Further in-depth analysis of the transcriptome data showed that the stimulation of F.nucleatum contributed to an increase in the number of SNP mutations in the experimental groups.The most common types of variable shearing were TSS and TTS,followed by SKIP and AE.4.The mechanism research of the effects of F.nucleatum on the pathogenicity of primary rat osteoblasts(1)KEGG enrichment analysis was performed on the DEGs at six time points,and the results indicated that the PI3K-AKT signaling pathway,TNF signaling pathway,JAK-STAT signaling pathway and osteoclast differentiation pathways were activated at all time points.In addition,many pathways related to infection and inflammation were activated at the initial stage of infection by F.nucleatum,and many metabolic pathways,such as phospholipase D signaling pathway,drug metabolism-cytochrome P450,arginine and proline metabolism,were activated at the later stages of infection.(2)Through the pathview analysis of MAPK and NF-?B signaling pathways,the expression of multiple genes was changed at different times during F.nucleatum infection.Western blots results proved that representative proteins of AKT/P38,JNK and NF-?B pathway were activated within 120 minutes after stimulation by F.nucleatum.Conclusions1.This experiment successfully constructed a co-cultured model of F.nucleatum and rat primary osteoblasts in vitro.2.F.nucleatum could significantly inhibit the proliferation of osteoblasts and promote cell apoptosis,which showed concentration-and time-dependent;and it mainly inhibited the replication cycles of osteoblasts by blocking the G2/M phase.F.nucleatum significantly stimulated the secretion of inflammatory cytokines and inhibited the osteogenic differentiation and mineralization.3.Most of 235 common DEGs were inflammation-related genes and were enriched in TNF signaling pathways and cytokine-related pathways.In addition,during the infection of F.nucleatum,many cancer-related pathways and genes were activated.4.In the early infection stage of F.nucleatum,the pathways related to infection and inflammation were mainly activated,while with the stimulation time increasing,multiple pathways related to cell metabolism were activated.The AKT/P38/JNK and NF-?B pathways were activated at the initial stage of infection of osteoblasts by F.nucleatum. |
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