Histone Deacetylase Inhibitors Regulate Inflammatory Cytokines Production In Peripheral Blood Mononuclear Cells And Osteogenic Differentiation Of Gingival Stem Cells Stimulated By Lipopolysaccharide

Objective:The aim of this study was to investigate the role of Vorinostat,Trichostatin A and Panobinostat on inflammatory cytokines production in peripheral blood mononuclear cells and osteogenic differentiation of gingival stem cells(GMSCs)stimulated by lipopolysaccharides(LPS).Methods: 1.The effect of histone deacetylase inhibitors on inflammatory cytokines production in LPS-stimulated peripheral blood mononuclear cellsPeripheral blood mononuclear cells(PBMCs)were isolated from human peripheral blood using Ficoll-Paque density gradient medium.After stimulated with lipopolysaccharides for 4 hours,Vorinostat(SAHA),Trichostatin A(TSA)and Panobinostat(LBH589)were added respectively.Then supernatants were collected and inflammatory cytokines were detected by cytometric beads array.2.The effect of TSA,SAHA,LBH589 on osteogenic differentiation of LPS-stimulated gingival stem cells and the molecular mechanisms.GMSCs were separated from patients aging 16-25 years who exhibited a relative healthy gingiva without a history of periodontal disease.After osteogenic differentiation for 5 and 7 days,cells were fixed and stained with an ALP staining kit,and the ALP activity was assayed with ALP activity kit.To detect mineralization,GMSCs were induced for 14 days and stained with Alizarin Red.The expression of HDACs,OCN,RUNX2 and Osx was assessed by real time RT–PCR.After treated with LPS,SAHA,TSA and LBH589,Western-blot was performed to demonstrate the level of p65 and ?-catenin.Results:PBMCs were isolated from healthy human peripheral blood gingiva.After stimulating with LPS,CBA assay showed that IFN-?,IL-6 and TNF-? levels were significantly reduced in the HDAC inhibitors-treated PBMCs.By performing RT-PCR,we found that LPS treatment could upregulate the expression of HDAC1-7,HDAC10 and HDAC11 during osteogenic induction.After osteogenic induction,LPS-treated GMSCs showed reduction osteogenic differentiation potential,while TSA,SAHA and LBH589 could rescue ALP activity and calcium nodule formation.Moreover,RT-PCR showed that mRNA profiles of OCN,RUNX2 and Osx were also elevated in HDAC inhibitors-treated GMSCs.Under LPS stimulation,TSA,SAHA and LBH589 could reduce p65 phosphorylation at Ser536 and increase acetylation of p65 at K310.Fuerthermore,TSA,SAHA and LBH589 treatment resulted in increased level of ?-catenin.Conclusion:TSA,SAHA and LBH589 could suppress inflammatory cytokine production in supernatants of LPS-treated PBMCs and rescue impaired osteogenic differentiation potential of LPS-treated GMSCs.It could be inferred that application of HDAC inhibitors(TSA,SAHA and LBH589)might be a promising therapeutic approach in treatments of periodontitis.