关键词: 牙龈卟啉单胞菌, 氧化性应激, PPAR&gamma, 内皮细胞

Abstract:

Objective：To detect the degree of oxidative stress in the process when Porphyromonas gingivalis (P. gingivalis) stimulates human vascular endothelium, And to investigate the effect of peroxisome proliferator-activated receptor（PPAR）γ on oxidative stress during this process.Methods:Human vascular endothelial cells (HVECs) line EA.hy926 (American Type Culture Collection ,United States) was cultured in high glucose Dulbecco’s modified eagle medium (DMEM). Four groups were designed: control group, P. gingivalis infected group, PPARγ activated group and PPARγ blocked group. In control group HVECs were cultured with only DMEM. In P. gingivalis infected group, HVECs were time-dependently stimulated by P. gingivalis W83 from 0 to 12 h. In PPARγ activated group or PPARγ blocked group, PPARγ was pre-activated or blocked by a representative PPARγ agonist(15dPGJ2 10 μmol/L) or antagonist (GW966210 μmol/L) 30 minutes before the cells were stimulated by P. gingivalis. At 0, 0.5, 1, 1.5, 2, 4, 8, and 12 h, the culture medium was collected individually and centrifuged, and the supernatant was stored for assay. Glutathione peroxidase (GSHPX) and malondialdehyde (MDA) were analysed by enzyme-linked immunosorbent assay. Cellular reactive oxygen species (ROS) were detected through 2’,7’-dichlorofluorescin diacetate (DCFA-DA) fluorescent probe at various time points of the different groups.Results: In P. gingivalis infected group, the levels of GSH-PX ［(5.56±0.97) μmol/L］ and MDA ［(0.84±0.18) nmol/L］ were significantly higher than those in control group ［GSH-PX(4.71±0.64) μmol/L, MDA (0.59±0.18) nmol/L)］. The levels of GSHPX and MDA in PPARγ activated group ［GSH-PX (5.38±0.84) μmol/L, MDA (0.84±0.22) nmol/L］ and in PPARγ blocked group ［GSH-PX (5.37±0.76) μmol/L, MDA (0.85±0.14) nmol/L］ were signi-ficantly higher than those in control group (P<0.05). In the PPARγ activated group, the levels of GSH-PX at 0.5 and 8 h were significantly higher than those from 1.5 h to 4 h (P<0.05), while no difference was observed on the MDA levels at  different time points. There was no significant difference at various time points for the levels of GSH-PX and MDA in PPARγ blocked group. The level of cellular ROS detected by DCFHDA in P. gingivalis infected group was significantly higher than that in control group (10 108.65 ± 1 805.18 vs. 6 049.06 ± 1 199.19，P<0.05). No difference was observed between PPARγ activated group (7 120.94±1 447.30) or PPARγ blocked group (6 727.35±1 483.68) and control group.Conclusion:Oxidative stress happens when P. gingivalis stimulates human vascular endothelium. PPARγ may involve in modulating oxidative stress during this process.

Key words: Porphyromonas gingivalis, Oxidative stress, PPAR gamma, Endothelial cells