Regulation Of Osteoclast Differentiation And Inflammatory Signaling By TCF8 In Periodontitis

Research Background:Periodontitis is a common chronic inflammatory disease that is increasing globally and significantly affects oral health and quality of life.It damages both natural teeth and dental implants,characterized by gingival inflammation,alveolar bone loss,and the formation of periodontal pockets,which can lead to tooth mobility and loss.The major issue in periodontitis is the resorptive bone destruction in the alveolar bone.Periodontitis is primarily caused by bacterial adhesion on tooth surfaces,forming dental plaque.Bacteria in the plaque release endotoxins called lipopolysaccharides(LPS),which can bind to Toll-like receptor 4(TLR4)on the surface of immune cells,leading to the release of inflammatory mediators.At this stage,gingival epithelial cells are activated,resulting in the release of inflammatory cytokines such as tumor necrosis factor-alpha(TNF-α),interleukin-1 beta(IL-1β),and interleukin-6(IL-6).These inflammatory mediators can activate the nuclear factor kappa B(NF-κB)pathway within cells.Notably,activation of the NF-κB signaling pathway has the potential to induce the differentiation of macrophages into large multinucleated osteoclasts.Receptor activator of nuclear factor kappa-B(RANK)is a transmembrane receptor on osteoclasts that can bind to its ligand,receptor activator of nuclear factor-κB ligand(RANKL),inducing osteoclast differentiation,activation,and bone resorption.The expression of RANKL is regulated by various inflammatory mediators,such as IL-1 and TNF-α,which are the main causes of bone loss in periodontitis.Studies have demonstrated that excessive osteoclast generation is associated with alveolar bone loss in periodontitis,and interventions targeting abnormal osteoclastogenesis may be effective strategies for treating the disease.The NF-κB pathway is a central factor in various human diseases related to bone resorption,including periodontitis.Inactivation of the NF-κB signaling pathway can reduce the number of osteoclasts after periodontal infection,suggesting that the NF-κB pathway may be a promising therapeutic target for periodontitis.RANKL is a crucial inducer of osteoclast differentiation,and literature reports suggest that RANKL can activate the NF-κB signaling pathway in mouse monocyte/macrophage leukemia cells(RAW264.7),which can differentiate into osteoclasts.Importantly,RANKL overexpression can upregulate and activate the two-handed zinc-finger homeodomain transcription factor 8(TCF8),which has shown potential in regulating the NF-κB signaling pathway.Therefore,TCF8 may be involved in osteoclast differentiation and the regulation of the NF-κB signaling pathway in RANKL-induced RAW264.7 cells.TCF8 is a transcription factor belonging to the two-handed zinc-finger family,which can bind to E-box sequences.Previous studies have demonstrated the regulatory role of TCF8 in epithelial-mesenchymal transition(EMT),a crucial process in embryonic development,hematopoietic stem cell differentiation,and tumor development.TCF8 expression has been validated in dental mesenchyme and tooth germs,suggesting its potential role in regulating tooth development.Importantly,TCF8 is highly expressed in oral epithelial cells and gingival tissues after infection with Porphyromonas gingivalis(Pg),a major pathogen in periodontitis.Additionally,TCF8 regulates the inflammatory response induced by IL-1β or TNF-α in gingival fibroblasts and promotes osteoclast differentiation during breast cancer bone metastasis.However,the role of TCF8 in periodontitis remains largely unexplored.Based on the above findings,we hypothesize that TCF8 may be involved in the acute inflammation and osteoclast differentiation processes in periodontitis.This study aims to explore the potential role of TCF8 in osteoclastogenesis and inflammation during periodontitis and investigate the protective effects of TCF8 gene silencing on osteoclast differentiation and inflammation induced by Porphyromonas gingivalis lipopolysaccharide(Pg-LPS)in rats with periodontitis and RANKL-induced RAW264.7 cells.Furthermore,we aim to evaluate the potential of TCF8 as a therapeutic target for periodontitis.Research Content:The research is divided into in vivo experiments and in vitro experiments.In the in vivo experiments,a rat model of periodontitis was established by injecting Porphyromonas gingivalis lipopolysaccharide(Pg-LPS).A recombinant lentivirus carrying short hairpin RNA(sh RNA)targeting TCF8 was used to silence TCF8 in vivo.Immunofluorescence,real-time quantitative polymerase chain reaction(Real-time PCR),and Western blot were used to detect the expression of TCF8 in rat periodontal tissues.Immunofluorescence was used to determine the localization and expression of TCF8,while Real-time PCR and Western blot were used to measure the gene and protein levels of TCF8.In addition,Micro-CT scanning of the rat maxilla was performed to calculate bone volume,total volume,bone volume fraction,Cementoenamel Junction to Alveolar Bone Crest distance(CEJ-ABC),and bone loss.Hematoxylin and eosin(H&E)staining was used to examine the pathological changes in periodontal tissues,and Real-time PCR was used to detect the expression of IL-1β,IL-6,and TNF-α in periodontal tissues.In the in vitro experiments,lentivirus packaging technology was used to construct sh RNA targeting TCF8 and control sequences,which were then infected into RAW264.7 cells.Different concentrations of RANKL(0,25,50,100 ng/m L)were added to induce differentiation,and Real-time PCR and Western blot were used to detect the expression of TCF8 in RANKL-induced RAW264.7 cells.Light microscopy was used to observe osteoclast formation.Further research was conducted to investigate the effect of TCF8 on osteoclast differentiation.RAW264.7 cells infected with lentivirus were induced to differentiate with 50 ng/m L RANKL.Real-time PCR and Western blot were used to measure the knockdown efficiency of TCF8,and tartrate-resistant acid phosphatase(TRAP)staining was performed to quantify the number and activity of osteoclasts after TCF8 knockdown.F-actin immunofluorescence was used to investigate the effect of TCF8 on the formation of the actin ring,and immunofluorescence was used to detect the nuclear translocation of p65 to assess the effect of TCF8 on the NF-κB signaling pathway.Research Results:The in vivo experiments showed that Pg-LPS induced overexpression of TCF8 in rat periodontal tissues,and silencing of TCF8 significantly reduced bone loss,inflammatory cell infiltration,and proinflammatory cytokine IL-1β,IL-6,and TNF-α expression and osteoclastogenesis in Pg-LPS-induced periodontitis rats.The in vitro experiments revealed that TCF8 knockdown inhibited RANKL-induced osteoclast differentiation in RAW264.7 cells,as evidenced by a decrease in the number of TRAP-positive osteoclasts,reduced formation of F-actin rings,downregulation of osteoclast-specific markers,and suppression of inflammatory responses.TCF8 knockdown also inhibited NF-κB signaling in RANKL-induced cells by blocking phosphorylation and nuclear translocation of NF-κB p65.Research Conclusion:Silencing of TCF8 inhibits alveolar bone loss,osteoclast differentiation,and inflammation in periodontitis.TCF8 may be a potential therapeutic target for the treatment of periodontitis,as it exhibits protective effects against osteoclast differentiation and inflammatory responses.This study provides a theoretical basis for the treatment of inflammation-induced bone resorption diseases related to periodontitis and offers new insights into targeted therapy for periodontal tissue inflammation.