Effects Of Supernatant Of Porphyromonas Gingivalis On Inflammatory Cytokines Expression In Human Umbilical Vein Endothelial Cells

Objective:The aim of this study is to observe the effects of supernatant of Porphyromonas gingivalis on proliferation and inflammatory cytokines expression in human umbilical vein endothelial cells. The results may provide laboratory evidence of the relationship between periodontal disease and atherosclerosis.Methods:. Human umbilical vein endothelial cells were obtained by Enzyme digest method, and the source of cells was identified. Porphysomanas gingivalis were gained by anaerobic culture method. Proliferation of HUVECs infected by different concentrations of supernatant of P.g were investigated by MTT, according to the results of MTT, the concentration of 0.5×107CFU/ml of P.g supernatant was chosen to stimulate HUVECs, the ultrastructure changes in cells were observed under the transmission electron microscopy (TEM), and the amount of Interleukin-1βand Interleukin-6 protein production in cell supernatant secreted by HUVECs were detected with enzyme-linked immunosorbent assay(ELISA) kit after cultured respectively 6h,12h,18h,24h, at the same time, their mRNA were measured by reverse transcription-polymerase chain reaction (RT-PCR). The data was statistically analyzed by SPSS 11.0 software package.Results:(1) Under the enviroments of our experiment, the HUVECs had a cobblestone appearance with a strict monolayer growth,Ⅷfactor in the HUVECs showed positive reaction by immunohistochemistry.(2) The proliferation of HUVECs was obviously inhibited when the concentration of supernatant of P.g was above 0.5×107CFU/ml (P<0.05), while there are no significant difference as the concentration was less or equal to 0.5x10'CFU/ml (P>0.05). (3) TEM showed that there are some minor morphological damages after 24 hours of stimulation with the concentration of 0.5×107CFU/ml supernatant of P.g compared with the control group, including mitochondria swelling, cristae dissolution, and rough surface endoplasmic reticulum(RSER) were increased in some cells.(4) The results of ELISA showed that HUVECs itselves can express small amounts of IL-1βand IL-6. After stimulation with the concentration of 0.5×107 CFU/ml supernatant of P.g, the protein expression of IL-1βwas obviously higher than the control group at 18h,24h (P<0.05), and the protein expression of IL-6 was obviously higher than the control group at 12h,18h,24h (P<0.05), while there are no significant difference in control groups (P>0.05)(5) The RT-PCR results showed that the mRNA of IL-1p and IL-6 could be detected at every time period in the HUVECs stimulated with the concentration of 0.5×107CFU/ml supernatant of P.g, the expression of IL-1βwas obviously higher than the control group at 12h,18h (P<0.05), and the expression of IL-6 was obviously higher than the control group at 12h (P<0.05), while there are no significant difference in control groups (P> 0.05)Conclusion:The experiment shows that high concentration of P.g supernatant had a significant inhibibating action on the proliferation of HUVECs, and there are some minor morphological damages of ultrastructure of cells with the concentration of 0.5×107CFU/ml supernatant of P.g; it can also promote the expression of protein and mRNA of IL-1βand IL-6, which suggests that P.g infection could increase the release of inflammatory cytokines, and subseguently lead to the dysfunction of endothelial cells, and thus may play an important role in the development and progression of atherosclerosis.