Effects Of Anti-TLR4 And Pyrrolidine Dithioearbamate On Lipopolysaccharide-stimulated Human Periodontal Ligament Cells

ObjectiveThe purpopse was to investigate the effects of anti-TLR4 and PDTC,a NF-κB inhibitor,on lipopolysaccharide-mediated nuclear translocation of NF-κB and cytokine IL-1βand IL-8 expression in human periodontal ligament cells,and study the mechanism of TLR4-NF-κB signaling pathway in periodontal injure.MethodsHuman periodontal ligament cells were prepared by using explant culture technique.Collecting of the 3-6 passage cells in logarithmic growth phase were used for the following experiment.There were four groups in this study.Control group: cells were treated with Dulbecco's modified Eagle's medium(DMEM) containing 1% FBS.LPS group:cells were treated with DMEM containing 1%FBS and 10μg/ml LPS.TLR4 group:cells were incubated with anti-TLR4 for 30min before DMEM containing 1%FBS and 10μg/ml LPS was added.PDTC goup:cells were incubated with PDTC for 30min before DMEM containing 1%FBS and 10μg/ml LPS was added.Cells of all groups were cultured at 37℃in 5%CO2 for 24 hours.Then the amounts of interleukin 1βand interleukin 8 secreted into the culture medium were determined by an enzyme-linked immunosorbent assay(ELISA) respectively.TLR4 and nuclear translocation of NF-κB in the cells was evaluated by indirect immunofluorescence.Results1.The amounts of IL-1βand IL-8 secreted into the culture medium decreased when cells were incubated with anti-TLR4 for 30 minutes,which had statistic difference compared to cells without anti-TLR4(P＜0.05).2.PDTC suppressed cell proliferation,significantly different from the control (P＜0.05).The effects of 1000μmol/ml and 200μmol/ml PDTC were more significant. 50μmol/ml PDTC and above incubated cells for 24 hours promoted apoptosis.3.The secretion of IL-1βand IL-8 was not inhibited by 10μmol/ml PDTC,and, more pronounced inhibition of 100μmol/ml and 1000μmol/ml PDTC showed there was significant difference from the control(P＜0.05). 4.Cytoplasmic staining by TLR4 antibody was obvious and it had no significant difference before and after LPS stimulation.No TLR4 antibody staining could be observed with anti-TLR4 treatment.The NF-κB antibody staining showed strong green fluorescence signals in the nuclei with weak cyroplasmic staining after LPS stimulated.The anti-TLR4 and PDTC treatment strongly reduced the intensity of nuclear staining.Conclusions1.TLR4 exsits on human periodontal ligament cells surface and can recognize lipopolysaccharide.2.PDTC can suppress human periodontal ligament cells proliferation and induce apoptosis.3.Nuclear translocation of NF-κB and IL-1βand IL-8 expression in LPS-stimulated human periodontal ligament cells can be inhibited by anti-TLR4 and PDTC,which indicates that TLR4- NF-κB signaling pathway play a significant role in periodontal injure.Blocking the pathway can be a new treatment with periodontal disease.