The Effect Of Porphyromonas Gingivalis On The Expression Of Matrix Metalloproteins By Gingival Epithelial Cell

ObjectiveAs an important oral disease,periodotitis has a hign morbidity in China.It is a complex and multi-factors involved disease caused by periodonal microbian. Porphyromonas gingivalis(P.g) is a Gram-negative bacterium and a putative pathogen of periodotitis,while the pathogenesis is not clear yet.Gingival epithelial cell(GEC) is located in outer surface of mucosa and contacted by pathogens firstly.The interaction between P.g and GEC in periodotitis pathogenesis has been focused recently.Matrix metalloproteinases(MMPs) are involved in the degradation of periodonal tissues and play a role in the pathogenesis of periodotitis.The severity of tissue damage in periodotitis is related to some MMPs.In addition,some cytokines are found to play key roles in the tissue destruction,such as TNF-α,IL-6,IL-8 and IL-1β.And these cytokines also have an effect on host inflammation and immunal response.In this test, we measured levels of some cytokines and MMPs in GEC after they were infected with P.g and studied the regulation of TNF-αon expression of some MMPs,then explored the roles of P.g in the pathogenesis of periodotitis.This study presented evidence for GEC involved in the local immunal response and new strategies for diagnostic, prevention and therapy of periodotitis.Methods1,acterial culture:P.g strain ATCC33277 were inoculated cultrues containing yeast extract in 37℃,anaerobic incubator.When the proliferation index reached log stage,they would be used for study.2,ell culturs:Cells were cultured in DMEM with 10%fetal bovine serum in 37℃, CO₂ incubator. 3,ytokin measure:Levels of TNF-α,IL-6,IL-8 and IL-1βin cell supemant were measured by ELISA before P.g inoculation and 1h,3h,6h,12h and 24h after inoculation.And mRNA of these cytokines was extracted and measured by RT-PCR before P.g inoculation and 1h,3h,6h,12h and 24h after inoculation.4,MP measure:Protein levels of MMP-2,MMP-3,MMP-7 and MMP-9 were measured by Western blotting before and after P.g infection.And mRNA levels of these MMPs were measured by real-time PCR.5,easure of upregulation of MMP2 and MMP-9 by TNF-α.GEC was affected by exogenous TNF-αin three different concentrations.Expression of MMP-2 and MMP-9 was measured by real-time PCR.And GEC was also affected by anti-TNF-aneutralizing antibody,expression of MMP-2 and MMP-9 was also determined by real-time PCR.ResultsEffect of p.g on expression of TNF-α,IL-6,IL-8 and IL-1βin GEC.ELISA results:No cytokines was found in GEC cells without p.g infection including TNF-α,IL-6,IL-8 and IL-1β.In 1h,3h,6h,12h and 24h after infection with p.g,all these cytokines were found in cell supernant.Leves of TNF-αwere 11.2±1.7pg/ml,26.7±6.4pg/ml,45.2±11.3pg/ml,82.6±16.4pg/ml and 128.3±21.3pg/ml respectiv.Levels of IL-1βwere 14.6±2.3pg/ml,31.4±7.9pg/ml,50.5±9.8pg/ml, 72.4±14.7pg/ml and 2.6±16.8pg/ml respectively.Levels of IL-6 were 3.4±0.4pg/ml, 8.3±1.1pg/ml,12.6±1.3pg/ml,23.2±3.3pg/ml and 35.3±5.7pg/ml respectively.Levels of IL-8 were 5.7±0.6pg/ml,9.4±1.3pg/ml,22.4±2.8pg/ml,34.7±4.2pg/ml and 52.3±5.3pg/ml.concentration of these cytokines are all time-dependant(P＜0.01).RT-PCR results:TNF-αmRNA,IL-6mRNA,IL-8mRNA and IL-1βmRNAwere not expressed in GEC without p.g infection.And all these cytokine mRNA was expressed in GEC after p.g infection.Peak values of TNF-αmRNA and IL-1βmRNA occur in 6h after infection.And the peak values of IL-6mRNA and IL-8mRNA occur in 12h after infection. Effect of p.g on expression of MMP-2,MMP-3,MMP-7 and MMP-9 in GEC.Western blotting results:GEC has weak expression of MMP-2 and MMP-9 without p.g infection.And expression of MMP-2 and MMP-9 was increased after p.g infection.Peak value of MMP-2 occurs in 6h and that of MMP-9 occurs in 12h after infection.Neigher MMP-3 nor MMP-7 was found in GEC before and after infection.Real-timePCR results:GEC has low levels of MMP-2 and MMP-9 without p.g infection.And levels of MMP-2 and MMP-9 were increased after p.g infection (P＜0.01,respectively).Levels of MMP-2 increased 1.4,4.5,8.7,8.4 and 4.9 times. Peak value of MMP-2 occurs in 6h after infection.Levels of MMP-9 increased 2.3,6.6, 10.8,12.5 and 7.2 times.And peak value of MMP-9 occurs in 12h after infection. Neigher MMP-3 mRNA nor MMP-7 mRNA was found in GEC before and after infection.Regulation of TNF-αon expresion of MMP-2 and MMP-9 in GEC.Real TimePCR results:Expression of MMP-2 mRNA and MMP-9 mRNA was upregulated in TNF-αdose-dependant way(P＜0.01).10ng/ml of TNF-αstimulated expression of MMP-2 and MMP-9 most significantly.And expression of MMP-2 mRNA and MMP-9 mRNA was most obvious in 3h after treatment with exogenous TNF-α.In 3h after treatment with 10ng/ml TNF-α,expression of MMP-2 mRNA and MMP mRNA increased 20.8 times and 36.7 times.After treatment with anti-TNF-αantibody,expresssion of MMP-2 mRNA and MMP-9 mRNA reduced significantly in various time(P＜0.01).Conclusion1,Expression of TNF-α,IL-1β,IL-6 and IL-8 can be increased significantly after p.g infection in GEC.2,Expression of MMP-2 and MMP-9 can be increased significantly after p.g infection in GEC.3,TNF-αcan upregulated expression of MMP-2 and MMP-9 significantly.