Effects Of Procyanidin B2 On The Expression Of Inflammatory Mediators In Human Periodontal Ligament Cells Induced By P.Gingivalis LPS

ObjectiveThis study was to investigate the effects of procyanidin B2(PC B2)on the expression of inflammatory mediators in human periodontal ligament cells(hPDLCs)induced by Porphyromonas gingivalis(P.gingivalis)Lipopolysaccharide(LPS),and to explore the anti-inflammatory effect of PC B2 on hPDLCs preliminarily.Materials and MethodsPrimary hPDLCs were cultured using modified tissue explant method in vitro.The morphology of the cells was observed under the inverted microscope and the cells were identified by SP immunohistochemical staining.The effects of PC B2 on the cell viability of hPDLCs were detected by MTT assay.The effects of PC B2 on IL-1??IL-6?IL-8 mRNA and protein expression of hPDLCs induced by P.gingivalis LPS were detected by quantitative real-time PCR and ELISA assay.HPDLCs were loaded with DCFH-DA.And then the effects of PC B2 on the production of reactive oxygen species(ROS)in hPDLCs induced by P.gingivalis LPS were observed under the fluorescence microscope.The effects of PC B2 on the total nitric oxide(NO)secretion of hPDLCs induced by P.gingivalis LPS were detected by Griess assay.The data were analyzed by graphpad prism 6 software.Results 1.HPDLCs were cultured successfully.The cultured cells were long spindle or polygonal with slender cellure protrusions,rich cytoplasm and large oval nucleus.The cells were arranged radially or whirlpool-like.The results of SP immunocytochemistry showed that the cultured cells were positive for anti-vimentin staining and negative for anti-keratin staining.2.PC B2 had no effects on the cell viability of hPDLCs within the concentration range from 6.25?g/mL to 200?g/mL on the first day(P>0.05).100 ?g/mL PC B2 could enhance the cell viability of hPDLCs on the second day and the third day(P<0.05).3.IL-1? ?IL-6 ?IL-8 mRNA levels of hPDLCs were significantly increased by P.gingivalis LPS compared with the control group(P<0.001).PC B2 decreased IL-1??IL-8mRNA expression from 50?g/mL(P<0.05)and downregulated IL-6mRNA expression from 25?g/mL(P<0.05).4.IL-1??IL-6?IL-8 protein expression of hPDLCs was significantly increased by P.gingivalis LPS compared with the control group(P<0.01).PC B2 decreased the expression of all three proteins from 50 ?g/mL at the time point of 24h(P<0.05).PC B2 decreased IL-1? protein expression from 50?g/mL(P<0.01),decreased IL-6 protein expression from 25?g/mL(P<0.01)and decreased IL-8 protein expression from 100?g/mL(P<0.01)at the time point of 48 h respectively.5.P.gingivalis LPS increased the expression of cytoplasmic ROS of hPDLCs compared with the control group.The expression of ROS was decreased in hPDLCs pretreated with 100?g /mL PC B2 for 1 hour.6.P.gingivalis LPS promoted the secretion of NO of hPDLCs compared with the control group(P<0.001).The expression of NO was decreased pretreated 100?g/mL PC B2 for 1 hour(P<0.01).ConclusionProcyanidin B2 could downregulate the expression of cytokines and inhibit the production of reactive oxygen species and nitric oxide in human periodontal ligament cells induced by P.gingivalis LPS.It may play an anti-inflammatory role in hPDLCs.