The Study On The Effects Of Lipopolysaccharide From Different Strains Of Porphyromonas Gingivalis On The Function Of Human Umbilical Vein Endothelial Cells

Objective:to explore the effects of P. gingivalis-LPS in the development of atherosclerosis and the possible role of P. gingivalis-LPS underlying the relationship between periodontitis and atherosclerosis, we observed the effects of Porphyromonas gingivalis lipopolysaccharide (Pgingivalis-LPS) form different fimA genotypes of Porphyromonas gingivalis on the expression of cell adhesion molecules, and secretion of pro-inflammatory cytokines and chemokines in HUVECs.Methods:An aeropack method was employed for P.gingivalis ATCC 33277 (typeⅠfimA gene), WCSP 115 (typeⅡfimA gene) and W83 (typeⅣfimA gene). Pgingivalis-LPS form different fimA genotypes of Porphyromonas gingivalis was extracted by phenol-water methods, and then purified, The preparation was confirmed by SDS-PAGE with silver staining and by infrared spectropescopy. Tachypleus Amebocyte Lysate kit was use to test the bio-activity of purified Pgingivalis-LPS.HUVECs were cultured in vitro, Pgingivalis-LPS from different fimA genotypes of P. gingivalis were added to co-cultured with HUVECs monolayer. The expression of IL-1β, IL-6, TNF-α, IL-8 and MCP-1 protein levels in culture supernatant was determined by enzyme linked immunosorbent assay at different time intervals (2h,6h, and 24h). HUVEC without stimulation of P.gingivalis were used as control group. The cells were harvested for the extraction of total RNA, reverse transcription polymerase chain reaction (RT-PCR) was used to detect the level of IL-1β, IL-6, TNF-α, IL-8, MCP-1, ICAM-1 and VCAM-1 mRNA. And the expression of ICAM-1 and VCAM-1 on HUVECs were detected by FCM.Result:(1) Under the enviroments of our experiment, the preparation extracted from the P.gingivalis was not only the bacterial endotoxin, but also had a similar structure with Ecoli-LPS. The extracted of P.gingivalis-LPS was biologically active (≥15ng/ml) and could be used in the following experiment.(2) When the HUVECs were stimulated with P.gingivalis-LPS, the production of IL-1βprotein was higher than the negative control. The regulation effects on IL-1βexpression by typeⅡP.gingivalis-LPS were more than typeⅠP.gingivalis-LPS.The up-regulation effects on IL-6 expression by P.gingivalis-LPS were in a dose-dependent manner, which happened on the transcription and post-transcription levels. and the stimulation of typeⅠP.gingivalis-LPS, which IL-6 protein level in HUVEC culture superntant was lower than that of typeⅡandⅣP.gingivalis-LPS. The regulation effects on IL-6 mRNA expression by typeⅡP.gingivalis-LPS was stronger than other fimA types.The up-regulation effects on TNF-αexpression by P.gingivalis-LPS were in a dose-dependent manner, which happened on the transcription and post-transcription levels. The protein level reached its peak at 24h, and the regulation effects TNF-αprotein level by typeⅡP.gingivalis-LPS was stronger than other fimA types in some conditions. With high dose (5,10μg/ml) of typeⅡP.gingivalis-LPS induce much more TNF-αmRNA than other fimA types.It indicated that the difference of fimA types may lead different ability of P.gingivalis-LPS to induce HUVECs to express pro-inflammatory cytokine, and the secreation of IL-1β, IL-6 and TNF-αby HUVECs may contribute to atherosclerosis.(3) The up-regulation effects on IL-8, MCP-1 expression by P.gingivalis-LPS were in a dose-dependent manner, which happened on the transcription and post-transcription levels. The level of MCP-1 mRNA reached its peak at 6 h, and down to a normal level at 24h. With the dose of 5μg/ml, the regulation effects on IL-8 mRNA expression by typeⅡP.gingivalis-LPS was stronger than other fimA types at 6h. The regulation effects on MCP-1 mRNA expression by typeⅠP.gingivalis-LPS was stronger than typeⅡP.gingivalis-LPS.It indicated that the difference of fimA genotypes may lead different ability of P.gingivalis-LPS to induce HUVECs to express chemokines, and the secretion of IL-8 and MCP-1 by HUVECs may contribute to atherosclerosis.(4) Under the enviroments of our experiment, the expression of VCAM-1 mRNA induced by P.gingivalis-LPS was no significant difference compare to the negative control. With high dose (5,10μg/ml) of either typeⅠor typeⅡP.gingivalis-LPS induce much more ICAM-1 mRNA than the negative control did at 6h. The regulation effects on ICAM-1 mRNA expression by typeⅡP.gingivalis-LPS was stronger than other fimA types. We analyzed ICAM-1, VCAM-1 by flow cytometry at 2h,6h and 24h, we observed that P.gingivalis-LPS failed to stimulate ICAM-1 or VCAM-1 expression on HUVEC.Conclusion:Based on these findings, we concluded that P.gingivalis-LPS could active HUVECs by express pro- inflammatory cytokine, chemokines, which is likely in part mediates the hose inflammatory response responsible for initiation of atherosclerosis.