The Researches Of NF-κb In Inflammatory HPDLSCs During Osteogenic Differentiation

Periodontitis, with the incidence rate of90%, is the main cause of toothloss in adults. It is caused by bacteria, which adhered to tooth surface, invadinginto periodontal tissue directly and stimulating the chronic inflammationreaction of the body indirectly. The results of periodontitis are destruction of softconnective tissue and alveolar bone, which finally lead to tooth loss. At present,no efficient therapy is available for periodontitis.In normal condition, periodontal tissue possesses the capacity ofregeneration. Alveolar bone and periodontal fiber are remodeling in lifetime tomaintain the integrity of periodontal supporting tissues. Periodontal stem cells(PDLSCs), a group of pluripotent adult stem cell, had been isolated fromperiodontal tissues. PDLSCs, with the ability of self-renew and multi-potentdifferentiation, could differentiate into bone, cartilage, nerve, and blood vesselsin vitro, and format the cementum-periodontal fiber-alveolar bone structure invivo. Therefore, PDLSCs are considered to play an important role in periodontaltissue regeneration. Previous research suggested that local inflammatorymicroenvironment inhibited the regeneration of PDLSCs. But the principleremained generally unknown.NF-κB is the key nuclear transcription factor in immune procedure.Different kinds of acute and chronic inflammation could activate NF-κB pathway to enhance the transcription of immune-related genes. Meanwhile,through modulation of RANK/RANKL ratio, osteogenetic signaling pathwayand bone matrix formation, NF-κcontributed to the signaling regulation of bonedevelopment and bone remodeling. However, most of research focused on thefunction of NF-κB pathway in osteoclasts formation and activation. It is remainsto be understood whether NF-κB pathway contribute to pathogenesis ofperiodontitis, whether it leads to defect of bone formation in periodontitis,whether it inhibit the osteogenic differentiation ability of PDLSCs, and thespecific molecular principle of signaling transduction.In this study, we isolated human PDLSCs from healthy and periodontitisperiodontal tissues to compare the expression of NF-κB pathway. Then weresearched on the principle of how local inflammatory microenvironmentregulate the osteogenic differentiation of PDLSCs through NF-κB pathway. Thepresent study provided a novel understanding of pathology of periodontitis, anda new thread for target treatment based stem cell regeneration.Objective(1) to investigate whether inflammation in the periodontium can influencestem cell characteristics.(2) to investigate influence of periodontitis microenviroment to to PDLSCsand to study the effect of NF-κB pathway in PDLSCs isolated from healthy andperiodontitis-affected during osteogenic differentiation.(3) to investigate whether inflammatory cytokines effect osteogenicdifferentiation of periodontal ligament stem cells through NF-κB pathway andthe roles of NF-κB pathway in hPDLSCs osteogenic differentiation in theinflammation microenviroment. Methods:(1) We used the limiting dilution technique to isolate H-PDLSCs andP-PDLSCs and analyzed these two types of PDLSCs phenotypeidentification by Immunofuorescence Staining and Flow CytometryAnalysis. PDLSCs proliferation potential and multi-differentiation potentialwere determined by colony-forming assays, MTT, flow cytometry analysisof Cell Cycle, alizarin Red and Oil red O staining. The gene and proteinexpressition of osteogenic and adipogenic were determined by real-timeRT-PCR and western blot.(2) We used western blot analysis to determine the expression of NF-κBpathway during osteogenic differentiation of PDLSCs. We combinedfunctional study with bioinformatics approaches to characterize the target ofNF-κB.(3) The expression of NF-κB and osteogenic gene of PDLSCs were determinedafter treated by TNF-(10ng/mL) by real-time RT-PCR and western blot.Results:(1) There is a difference in the multipotent differentiation of H-PDLSCs andP-PDLSCs. The chronic inflammation inhibits differentiation potentials butpromotes proliferation of PDLSCs.(2) NF-κB expression was significantly upregulated in PDLSCs duringosteogenic differentiation. Meanwhile, the expression of NF-κB was moredistinguished in P-PDLSCs compared to H-PDLSCs. BAY11-7082(theinhibitor of NF-κB) can inverse the differentiation potentials of P-PDLSCspartly.(3) TNF-treatment increased NF-κB expression. Consistent with the effectson Runx2and Osterix mRNA, TNF--treatment upregulated NF-κB protein expression and downregulated Runx2and Osterix protein levels. Besides,BAY11-7082can inhibit the effect of TNF-.Conclusions:(1) The chronic inflammatory microenvironment may influence the PDLSCscharacteristics.(2) In a chronic inflammatory microenvironment, NF-κB pathway inhibits theosteogenic differentiation potentials of PDLSCs. BAY11-7082can partlyinverse the potentials.(3) Overproduced inflammatory cytokines, TNF-resulting in suppression ofosteogenic differentiation and upregulating expression of NF-κB ofPDLSCs in inflammatory microenvironment. BAY11-7082can inverse theinhibition effect of TNF-.