Research Of Peroxisome Proliferator Activated Receptor-? Regulating Inflammation And Bone Metabolism In Periodontitis

PPARy was a nuclear receptor transcription factor found in the past 20 years.It can regulate the metabolism of glucose and lipid,cell proliferation,differentiation,apoptosis,and participate in the process of inflammation,tumor,atherosclerosis,diabetes,obesity and other diseases.In the resting state,PPARy forms a heterodimer with the retinoic acid X receptor in the promoter region of the target gene.This heterodimer together with the co-inhibitor of the nuclear receptor maintains the target gene in a transcriptional inhibitory state.Under the activation of ligands,PPARy undergoes a conformational change.On the one hand it dissociates with the nuclear co-suppressor,on the other hand it specifically binds with the peroxisome proliferator-responsive element which is the DNA sequence of the promoter region of the target gene,then initiate the transcription process of the target gene.PPARy can regulate the transcription process of the downstream gene,and then regulate the occurrence and development of the disease indirectly.Periodontitis is a chronic progressive inflammation characterized by the surrounding tissue including the gingival,cementum,periodontal ligament and alveolar bone destruction.Subgingival plaque(include gram-negative bacteria and their metabolites such as lipopolysaccharide)is a driving factor,the local environment around the teeth and systemic factors is a contributing factor which caused periodontitis.The clinical manifestations of periodontitis are mainly inflammation of soft tissue such as gum and damage of bone tissue such as alveolar bone.PPARy involved in inflammation and bone metabolism regulation,it is necessary to explore the role of PPARy in periodontitis.Periodontal ligament connects the teeth and periodontal tissue.The loss of periodontal attachment caused by inflammation stimulus is the pathological basis of periodontitis.PPAR? mainly expressed in adipose tissue,but also a small amount of it expressed in immune cells,endothelial cells and other tissue cells.In this study,we used LPS to stimulate periodontal ligament cells to simulate periodontitis,and to study the effect of PPARy on the inflammatory response of periodontal ligament cells and its mechanism.At the same time,periodontal ligament cells also have a multi-directional differentiation function,can form both the soft tissue cells such as fibroblasts and hard tissue cells such as osteoblasts,osteoblasts and so on.In this study,the effect and mechanism of PPARy on the mineralization of periodontal ligament cells under noninflammation and inflammation condition were discussed.Objective1.To investigate the changes of NF-?B signaling pathway and the expression of IL-1? and TNF-? in primary cultured hPDLCs,and to explore the effect of PPAR? on hPDLCs in periodontitis and its mechanism.2.Observing the changes of osteogenesis-related genes,mineralization and ?-catenin signaling pathway in different groups,to discuss the influence and mechanism of PPAR? on osteogenesis in hPDLCs in inflammatory and noninflammatory situation.Materials and Methods1.Healthy hPDLCs were cultured by tissue block method and identified.The cells were divided into control group,LPS stimulation group,dimethylsulfoxide(DMSO)group and rosiglitazone group.The expression of PPAR? and NF-?B p65 were detected by immunoblotting,and the expression and location of NF-?B p65 and p-p65 were detected by immunofluorescence.Real-time qPCR and ELISA were used to detect the RNA and protein expression of IL-1? and TNF-?.2.Culturing hPDLCs,stimalaing cells in inflammatory and noninflammatory environment with PPAR? agonist(rosiglitazone)and anagonist(GW9662).Cells divided into control group,RGZ group,RGZ+GW9662 group,LPS group,LPS+RGZ group and LPS+RGZ+GW9662 group for a period of 3,7,14 and 21 days treated with mineralization induction medium seperately.Detecting ALP activity with ALP activity detection kit and finding out the gene expression of RUNX2,OCN,OPG,RANKL,ColI,BMP2 and ALP using qPCR;Observing the mineralization nodules with alizarin red staining method and examining the percentage changes of ?-catenin in total protein and nucleic protein through western blot analysis.Result1.Stimulated by LPS,the expression of PPARy protein decreased and NF-?B p65 protein increased in the nucleus of the hPDLCs.And then RNA and protein expression of IL-1? and TNF-? were increased.When added with rosiglitazone,the expression of PPARy protein increased and NF-?B p65 decreased in the nucleus.What's more,the RNA and protein expression of IL-1?,TNF-? were decreased.The difference were statistically significant(P<0.01).2.Either in inflammatory status or noninflammatory status,PPAR? agonist improves RNA and protein of ALP activity(P<0.05).promotes mineralization nodules and reduces ?-catenin content in total protein and nucleic protein(P<0.05),PPAR?anagonist can reduce RNA and protein of ALP activity(P<0.05)?decrease mineralization nodules improved by PPARy agonist,but can't reverse the change of ?-catenin in hPDLCs treated with mineralization induction medium 3,7,14 days;PPAR?agonist inhibits generation of mineralization nodules in 21 days.Compared to noninflammatory situation,PPAR? agonist can accelerate gene expression of OPG and BMP2 in inflammatory situation(P<0.05).Conclusion1.PPAR? can block NF-?B signaling pathway,inhibit the RNA expression and protein secretion of cytokine such as IL-1?,TNF-?when stimulated by LPS in hPDLCs,and further regulate the inflammatory response in periodontitis.2.PPAR? contributes to the osteogenesis of hPDLCs in both inflammatory and noninflammatory situation without relying on ?-catenin signaling pathway,and this provides new ideas for clinical treatment of periodontitis.