Preliminary Study On The Effect Of Porphyromonas Gingivalis On Nitric Oxide Signal Path In Cultured Human Vascular Endothelial Cells

Increasing evidence from epidemiological, clinical, animal model and in vitro studies attributes a role for periodontal infections in the pathogenesis of Atherosclerosis and related event. However the role of periodontal disease in the damaging in endothelial function is unclear, particularly the influence of NO signal path from endothelial cells is unclear.In this study, we used Pg 33277 at multiplicity of infection (MOI) 1:10, 1:100 to intervene human umbilical vein endothelial cells (HUVEC) for 4, 8, 12, 24h respectively, detected the invading, the production of NO and the expression of eNOS protein and iNOS protein, then investigated the pathway of Pg in damaging in endothelial function and the role in the onset and development of Atherosclerosis.PartⅠ: Culture of Porphyromonas gingivalis and human vascular endothelial cells in vitroObjective To culture Pg 33277 and HUVEC cells in vitro and identify them by several menthods.Materials and Methods Pg 33277 were grown in trypticase soy broth at 37℃in an anaerobic jar (80%N2, 10%H2, 10% CO2 ), then culture purity was assessed by gram staining and plating on columbia blood agar plate,the bacteria was observed by SEM and TEM. Fimbria genotype of Pg was detected with PCR. HUVEC were cultured in DMEM at 37℃with 5%CO2 and observed by inverted microscope.Results Using gram staining, red bacillus were observed under immersion objective, gram(-); This bacteria had grown up to 2mm diameter and protruded, shiny, glossy, black and round shaped bacterium after cultured in columbia blood agar plate; under SEM, Pg 33277 appeared pole-shaped or sphere shaped; Pg 33277 had a few membrane structure, such as fimbria and membrane-bubble touched with outer- membrane by TEM; fimbria protein genotype of Pg 33277 were identified as I type by PCR. HUVEC presented polygonal and arranged like pavement stones under the inverted microscope.Conclusion Pg 33277 and HUVEC cells were grew stably in vitro, and could be used for the next steps of our experiment.PartⅡ: Invasion of human vascular endothelial cells by Porphyromonas gingivalisObjective To observe the ability of Pg to actively invade vascular endothelial cells.Materials and Methods we used Pg 33277 at MOI 1:10, 1:100 to infect HUVEC cells for 4, 8, 12, 24hours respectively, then the cells were detached with 0.25% trypsin, centrifuged, the cells pellet was fixed with 2.5% gluteraldehyde, postfixed in 1%OsO4, and the ultrathin sections were contrasted with lead citrate and uranyl acetate before examination by TEM.Results Pg 33277 was demonstrated to adhere and invade into HUVEC cells by fimbriae under TEM, and exist in cytoplasm.Conclusion In this study, we demonstrate that Pg can adhere to and invade endothelial cells and exist in cytoplasm. Invasion of host cells is believed to be an important strategy utilized by a number of pathogens, which affords them protection from the host immune system and may effect endothelial cells function.PartⅢ: Effect of Porphyromonas gingivalis on nitric oxide in cultured human vascular endothelial cellsObjective To investigate the effect of Pg on NO in cultured HUVEC cells.Materials and Methods we used Pg 33277 at MOI 1:10, 1:100 to infect HUVEC cells for 4, 8, 12, 24hours respectively, then collected cell supernatant, preserved at -70℃. NO production was assayed by measuring the accumulation of the stable oxidative metabolite, nitrite (NO2–) by nitrate reductase assay in the culture supernatants, HUVEC cells left uninfected were as negative control group. The significance of variability between the results from each group and the corresponding control was determined by analysis of variance.Results Concentrations of NO2–, an indicator of NO production, were measured after adding various concentrations of Pg 33277 to HUVEC. Within 24h, Pg 33277 at MOI of 1:10, 1:100 stimulated the release of nitric oxide in cultured HUVEC cells (P <0.05).Conclusion We demonstrate that Pg can effect NO production from endothelial cells, Pg stimulated the release of NO. Increasing in NO production may provoke directly tissue manage, cytotoxic effect and induce endothelial dysfunction PartⅣ: Effects of Porphyromonas gingivalis on the expression of eNOS and iNOS protein from human vascular endothelial cellsObjective To investigate the effects of Pg on the expression of eNOS protein and iNOS protein from HUVEC cells.Materials and Methods we used Pg 33277 at MOI 1:10, 1:100 to infect HUVEC cells for 4, 8, 12, 24hours respectively, cell lysate were preparated by RIPA buffer, protein concentrations were determined by Lorry assay. Proteins were separated by SDS-PAGE on a minigel apparatus, and then transferred onto PVDF membranes. Membranes were incubated with polyclonal antibody against eNOS (1:1000), polyclonal antibody against iNOS (1:1000) and monoclonal antibodyβ-actin (1:2000) at 4℃overnight respectively, then membranes were incubated with the secondary antibodies for 1hour at room temperature. Bands were visualized with ECL detection system, Quantification of images was performed by Quantity JS-300 analysis software. The significance of variability between the results from each group and the corresponding control was determined by analysis of variance.Results HUVEC expressed a protein of approximately 135 kDa, recognized by specific antibody to eNOS and expressed a protein of approximately 130 kDa, recognized by specific antibody to iNOS. Compared with the negative group, we found a decrease in eNOS protein expression and an increase in iNOS protein expression by Western-blot when HUVEC stimulated with Pg 33277 at MOI 1:10, 1:100 for the indicated times (P <0.05).Conclusion In this study, we demonstrate that Pg can effect the expression of eNOS protein and iNOS protein from HUVEC. Pg can inhibit eNOS expression and induce iNOS expression.We conclude from this study that: Pg can adhere to and invade HUVEC, then exist in cytoplasm. Pg can inhibit eNOS expression, induce iNOS expression and stimulate the release of NO in cultured HUVEC cells. The ability of Pg to invade HUVEC cells and promote the production of NO may be important in the endothelial dysfunction and the pathogenesi of As.