ABSTRACT
Objective To investigate the effect and mechanism of lipoxin A4 (LXA4) on uric acid (UA) induced oxidative stress of human umbilical vein endothelial cells (HUVECs).Methods The HUVECs was treated with uric acid to constructing the model of oxidative stress,and intervene the model with LXA4 and xylene based iodine (DPI),rotenone.Reactive oxygen species (ROS) of HUVEC were detected by a fluorescence probe 2',7'-dichlorofluorescin diacetate (DCFH-DA).The activity of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and p47phox protein was measured by Lucigenin enhanced chemiluminescence and Western blotting among control,uric acid (UA),LXA4 and UA +LXA4 groups,respectively.All the results were described by the relative expression of the control group,repeated measure variance analysis and least significant difference test (LSD) were used for statistical analysis.Results UA could stimulate HUVEC to generate ROS with different concentrations and times (F=7.286,F=4.532,P＜0.05).Compared with the control group(100±l 1),the ROS production of group with 80 mg/L UA (177±18),120 mg/L (226±29) and 160 mg/L (225±16) increased significantly (t=4.127,t=7.591,t=7.236,P＜0.05).Compare with baseline(100±8),the ROS production increased significantly (t=3.688,t=3.513,t=4.526,t=8.269,t=3.829,P＜ 0.05) at 3 h(143±16),6 h(140±17),12 h(183±20),24 h(240±29) and 48 h(160±22).LXA4 could inhibit ROS generation at different concentrations and times (F=4.008,F =4.497,P ＜0.05).Compared with LXA4 concentration of 0 nmol/L,the LXA4 concentrations of 10 nmol/L (162±16) and 100 nmol/L (132±15) could significantly inhibit ROS generation(t=3.712,t=4.083,P＜0.05).Compared with pretreatment (269±39),the ROS generation decreased significantly (t=6.373,t=6.426,t=7.125,t=6.981,P＜0.05).with LXA4 pretreated for 15 min (160±16),30 rain(158±21),1 h (136±13) and 2 h(140±13).Compared with the UA group(252±31),LXA4 and DPI could significantly inhibited ROS generation (145±29,154±27;t=6.356,t=5.853,P＜0.05),but Rot was not significantly intervented (241±32;t=1.027,P＞0.05).The NADPH oxidase activity in the UA group was significantly higher than that in the control group (144±16,100±13;t=3.659,P＜0.05),but the group of LXA4+ UA was significantly lower than that of the UA group (119±14;t=3.124,P＜0.05).The cytoplasmic expression of NADPH oxidase subunit p47phox of UA group was significantly lower than that in the control group (47±6,100±8;t=7.562,P＜0.05),but the LXA4+UA group was significantly increased compare with the UA group (83±6,t=5.386,P＜0.05).The cytomembrane expression of p47phox of UA group was significantly higher than that in the control group (328±36,100±4,t=12.817,P＜0.05),but the LXA4+UA group was significantly decreased compared with the UA group (183±30,t=5.129,P＜0.05).Conclusion LXA4 inhibits UA induced ROS production in HUVECs.This mechanism might be through inhibiting p47phox trafficing from cytoplasm to cytomembrane,results in inhibiting the activation of NADPH oxidase.