Impacts Of Porphyromonas Gingivalis On Adhesion And Inflammatory Mediators Of Gingival Epithelial Cells And Regulatory Effects Of Polyphenols On Cellular Inflammatory Responses In Vitro

ObjectivesChronic periodontitis is a kind of chronic, multifactorial, destructive and inflammatory disease involving periodontal microorganisms as initiating factors and the immune host defense. Among the periodontal microorganisms, Porphyromonas gingivalis is identified as a proof-abundant periodontal suspicious pathogen. It produces a broad array of potential virulence factors such as fimbriae, which can promote P. gingivalis to adhere to and subsequently invade into epithelial cells and result in direct destruction of periodontal tissue. Meanwhile, several signaling pathways of epithelial cells are activated under the stimulation of P. gingivalis as well as its products and cellular immunoinflammatory responses are induced. Mechanism of interaction between P. gingivalis and epithelial cells is one of the hot spots in the field of periodontology.In this study, western blotting analysis was used to detect the degradation effects of P. gingivalis ATCC 33277 and its fimA-deficient mutants on paxillin and focal adhesion kinase (FAK) of gingival epithelial cells, which was to better understand the pathogenic effects of P. gingivalis fimbriae, to further investigate the pathogenesis of P. gingivalis. Enzyme linked immunosorbent assay (ELISA) and real-time reverse transcription-polymerase chain reaction (RT-PCR) were performed to investigate prostaglandin E₂ (PGE₂) secretion and the mRNA expression of cyclooxygenase-2 (COX-2), interleukin (IL)-6, -8 and matrix metalloproteinase (MMP)-1 and -3, which was to explore the cellular inflammatory responses induced by P. gingivalis vesicles. Then hop bract polyphenol (HBP), apple condensed polyphenol (ACT) and epigallocatechin gallate (EGCg) were used to investigate whether they had inhibitory effects on the cellular inflammatory responses induced by P. gingivalis vesicles. The aim of this study was to explore the pathogenesis of P. gingivalis, to enrich the etiology of periodontology and to find an alterative way for the prevention and treatment of periodontitis.Materials and methodsMaterialsP. gingivalis ATCC 33277 wild strains and its fimA -deficient mutants (provided by Central laboratory of dental school in Osaka University)Immortalized human gingival epithelial cells (IHGE cells) (provided by Professor Murakami of Osaka University)Keratinocyte specific medium (HuMedia KG2, Kurabo, Osaka, Japan)DMEM (Sigma Chemicals, St. Louis, MO)Monoclonal antibodies against paxillin and FAK (Transduction Laboratories, Lexington, KY)ECL Plus Western blotting detection reagents (Amersham Biosciences, UK)PGE₂ immunoassay reagents (R&D Systems, Abingdon, UK)TRIzol reagent (Invitrogen, Carlsbad, CA)Iscript kit (Invitrogen, Carlsbad, CA)SYBRGreen reagents (QIAGEN, Hilden, Germany)Ultralow freezer (Sanyo, UltraLow, Japan)Anaerobic incubator (PLAS-LABS, USA)CO₂ incubator (Heraeus, Germany)electrophoresis apparatus trophoresis and Western blot instruments (BIORAD, USA)thermal cycler (LightCycler 2, Roche Molecular Biochemicals, Mannheim, Germany)HBP (Fundamental Research Laboratory, Asahi Breweries Ltd, Japan)ACT (Fundamental Research Laboratory, Asahi Breweries Ltd, Japan)EGCg (Funakoshi Co., Tokyo, Japan)Experimental methods1. P. gingivalis cultureP. gingivalis ATCC 33277 wild strains and its fimA -deficient mutants were kindly provided by the Central Laboratory of Dental School, Osaka University. P. gingivalis was cultured in trypticase soy broth (TSB) containing yeast extract, menadione and hemin under an atmosphere of 5% CO₂, 5% H₂ and 90% N₂ at 37℃.2. Preparation of vesiclesP. gingivalis cells were removed from the culture by centrifugation and filtration. Vesicles in the supernatant were collected by ultra-centrifugation at 100,000×g for 1 hour at 4℃before resuspended in 500μl phosphate-buffered saline (PBS). The protein content of P. gingivalis vesicles was determined using a bicinchoninic acid (BCA) protein assay kit. The concentration of P. gingivalis vesicles was adjusted to 1mg/ml for stock.3. Cell cultureImmortalized human gingival epithelial cells (IHGE cells) were grown in keratinocyte specific medium (HuMedia KG2). Hela cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum(FCS) under an atmosphere of 95% O₂, 5% CO₂, saturated humidity at 37℃.4. Preparation of polyphenolHBP, ACT and EGCg were dissolved in dimethyl sulfoxide (DMSO) and the final concentration of DMSO is 0.05% (v/v).5. Western blot ananysis for paxillin and FAKIHGE cells and Hela cells were grown in 6-well plates in DMEM until sub-confluence and were cultured with P. gingivalis ATCC 33277 or its fimA -deficient mutants at the indicated MOI (Multiplicity of infection) (0, 50, 200 and 500) for various periods (0, 30, 60 and 120min). P. gingivalis-infected cells were lysed with Triton-lysis buffer. The soluble fractions were collected by centrifugation for Western blot analysis. Samples were subjected to sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and then the protein blots were electrically transferred from the gels onto Polyvinylidene Fluoride (PVDF) membranes. After the membranes were incubated with primary antibodies against paxillin or FAK and horseradish peroxidase (HRP) labeled secondary antibody, they were scaned.6. ELISA for detection of PGE₂ secretion induced by P. gingivalis vesiclesIHGE cells were grown in 96-well plates in HuMedia KG2 until sub-confluence. P. gingivalis vesicles were diluted by DMEM beforehand and the final concentration of vesicles was 25 and 50μg/ml. IHGE cells were cultured in DMEM with or without vesicles. After 6h and 24h, PGE₂ secretion was detected by ELISA.7. Real-time RT-PCR for quantitative detection of proinflammatory mRNA expression induced by P. gingivalis vesiclesIHGE cells were grown in 6-well plates in HuMedia KG2 until 40% confluence and then incubated in DMEM for 24h until sub-confluence. P. gingivalis vesicles were added and the final concentration was 50μg/ml. The monolayers of IHGE cells were treated by TRIzol and the total RNA were extracted at 0,1, 3, 6, 9, 12 and 24h. A thermal cycler and a reagent named SYBRGreen were utilized for quantitative detection of mRNA expression of COX-2, IL-6, IL-8, MMP-1 and MMP-3.8. ELISA for detection of the effect of HBP, ACT and EGCg on PGE₂ secretionHBP, ACT and EGCg were diluted by DMEM beforehand and the final concentrations were 1, 10 and 25μg/ml. IHGE cells were stimulated with P. gingivalis vesicles (50μg/ml of final concentration) in the presence or absence of polyphenols at various concentrations for 24h. Then PGE₂ secretion was detected by ELISA.9. Real-time RT-PCR for detection of the effect of HBP, ACT and EGCg on proinflammatory mRNA expression P. gingivalis vesicles were added and the final concentration was 50μg/ml. Meanwhile HBP, ACT and EGCg were added and the final concentrations were 1, 10 and 25μg/ml. IHGE cells were cultured in DMEM with or without P. gingivalis vesicles, HBP, ACT and EGCg for 6h. Then monolayers of IHGE cells were treated by TRIzol and the total RNA were extracted. A thermal cycler and a reagent named SYBRGreen were utilized for quantitative detection of proinflammatory mRNA expression of COX-2, IL-6, IL-8, MMP-1 and MMP-3.10. Statistical analysisAll data are expressed as the mean±standard deviation (SD). An unpaired Student's t-test was used for comparative analyses. Differences in the data were considered significant when the probability value was less than 1% (P<0.01).Results1. Effect of P. gingivalis on paxillin and FAKP. gingivalis ATCC 33277 wild strain and its fimA -deficient mutant induced degradation of paxillin and FAK both in IHGE cells and in Hela cells. ATCC 33277 fimA -deficient mutant has a markedly weaker degradation ability than the wild strain. In IHGE cells, paxillin and FAK were degraded in a time-dependent and MOI-dependent manner.2. Cellular inflammatory responses of gingival epithelial cells induced by P. gingivalis vesicles(1) IHGE cells were stimulated by P. gingivalis vesicles (10 and 50μg/ml) and PGE₂ production was detected by ELISA. At 24h, P. gingivalis vesicles (10 and 50μg/ml) significantly induced PGE₂ production of IHGE cells.(2) IHGE cells were stimulated by P. gingivalis vesicles (50μg/ml) and mRNA expression of COX-2, IL-6, IL-8, MMP-1 and MMP-3 at 0, 1, 3, 6, 9, 12 and 24h was detected by Real-time RT-PCR. P. gingivalis vesicles significantly up-regulated mRNA expressions of COX-2, IL-6, IL-8, MMP-1 and MMP-3.3. Effects of HBP, ACT and EGCg on P. gingivalis vesicles-induced cellular inflammatory responses of gingival epithelial cells(1) Effects of HBP, ACT and EGCg on P. gingivalis vesicles-induced PGE₂ productionHBP significantly inhibited PGE₂ production at all the concentrations (1, 10 and 25μg/ml) in a dose dependent manner. However, EGCg had such inhibitory effect only at 10 and 25μg/ml. In contrast, ACT had no significant inhibitory effect on PGE₂ production.(2) Effects of HBP, ACT and EGCg on P. gingivalis vesicles-induced mRNA expression of COX-2, IL-6, IL-8, MMP-1 and MMP-3HBP significantly inhibited mRNA expression of COX-2, IL-6 and IL-8 at 10 and 25μg/ml and the mRNA expression of MMP-1 and MMP-3 was inhibited by HBP of 25μg/ml; while EGCg only clearly suppressed the expression of COX-2 at 10 and 25μg/ml. Other mRNA expressions were not suppressed by EGCg. In contrast, ACT has no inhibitory effect on these mRNA expressions at all.Conclusions1. P. gingivalis ATCC 33277 can induce degradation of focal adhesion components like paxillin and FAK, which may be promoted by fimbriae-mediated adhesion to and invasion into epithelial cells. IHGE cells are more sensitive to P. gingivalis than Hela cells. IHGE cells may be more suitable for the study of periodontal pathogens than Hela cells.2. Interaction of P. gingivalis or its products and gingival epithelial cells is the key point for its invasion into periodontal tissue. In addition to its direct destruction to periodontal tissue, P. gingivalis vesicles can markedly induce cellular inflammatory responses of gingival epithelial cells by up-regulating the gene expression of proinflammatory mediators and promoting their production. Excessive immunoinflammatory responses are suggested to be responsible for the initiation and progression of periodontitis.3. HBP has significant inhibitory effects on P. gingivalis vesicles- induced cellular inflammatory responses. It would be an alternative way for the prevention and treatment of periodontitis as a potential anti- inflammatory reagent.