过氧化物酶体增生物激活受体γ在牙龈卟啉单胞菌刺激血管内皮细胞氧化应激中的作用

摘要

目的:明确牙龈卟啉单胞菌( Porphyromonas gingivalis, P. gingivalis)刺激人血管内皮细胞时造成氧化应激的损伤程度,探索过氧化物酶体增生物激活受体( peroxisome proliferator-activated receptor, PPAR)γ在此过程的作用. 方法:研究设4组,对照组为人血管内皮细胞系EA. hy926(美国标准生物品收藏中心,美国)加培养基,P. gin-givalis刺激组为P. gingivalis W83刺激EA. hy926细胞,PPARγ激活组为加入PPARγ的激动剂15d-PGJ2 (10 μmol/L)的条件下P. gingivalis 刺激EA. hy926细胞,PPARγ抑制组为加入PPARγ的拮抗剂GW9662(10 μmol/L)的条件下P. gingivalis 刺激EA. hy926细胞,在0、0. 5、1、1. 5、2、4、8、12 h分别留取细胞培养上清液,通过酶联免疫吸附实验检测不同组各时间点培养基中氧化应激产物谷胱甘肽过氧化物酶( glutathione peroxidase ,GSH-PX)、丙二醛( ma-londialdehyde,MDA)浓度,利用荧光探针2 ' ,7 '-二氯荧光素二乙酸酯( 2 ' , 7 '-dichlorofluorescin diacetate, DCFH-DA)检测不同组各时间点细胞内活性氧( reactive oxygen species, ROS)量. 结果:在P. gingivalis刺激组,抗氧化应激的产物GSH-PX(5.56 ±0.97) μmol/L和MDA(0.84 ±0.18) nmol/L较对照组[GSH-PX(4.71 ±0.64) μmol/L, MDA(0.59 ±0.18)nmol/L]显著升高(P<0.05). GSH-PX和MDA水平在PPARγ激活组[GSH-PX(5.38 ±0.84)μmol/L, MDA(0.84 ±0.22) nmol/L]与抑制组[GSH-PX(5.37 ±0.76) μmol/L, MDA(0.85 ±0.14) nmol/L]显著高于对照组(P<0. 05). PPARγ激活组,GSH-PX水平在0. 5和8 h显著高于1. 5至4 h水平(P<0. 05),MDA水平各时间点差异无统计学意义. PPARγ抑制组,各时间点GSH-PX和MDA水平差异无统计学意义. P. gingivalis刺激组细胞内的活性氧水平显著高于对照组(10 108. 65 ± 1 805. 18 vs. 6 049. 06 ± 1 199. 19,P<0. 05),PPARγ激活组(7 120. 94 ± 1 447. 30)和PPARγ抑制组(6 727. 35 ± 1 483. 68)细胞内的活性氧水平与对照组差异无统计学意义. 结论:P. gingivalis刺激人血管内皮细胞可引起氧化应激反应,PPARγ参与调节此过程.%Objective:To detect the degree of oxidative stress in the process when Porphyromonas gin-givalis ( P. gingivalis) stimulates human vascular endothelium, And to investigate the effect of peroxi-some 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, PPARγactivated group and PPARγblocked group. In con-trol 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 PPARγblocked group, PPARγ was pre-activated or blocked by a representative PPARγ agonist(15d-PGJ2 10μmol/L) or antagonist ( GW966210μmol/L) 30 minutes before the cells were stimulated by P. gingiva-lis. At 0, 0. 5, 1, 1. 5, 2, 4, 8, and 12 h, the culture medium was collected individually and centri-fuged, 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 PPARγactivated group [GSH-PX (5. 38 ± 0. 84) μmol/L, MDA (0. 84 ± 0. 22) nmol/L] and in PPARγblocked 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 be-tween PPARγ activated group (7 120. 94 ± 1 447. 30) or PPARγblocked 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.