RESUMEN
Objective To explore whether lipoxin A4 (LXA4)could prevent lipopolysaccharide (LPS)-induced human umbilical vein endothelial cells (HUVEC) monolayer hyperpermeability and its possible mechanism. Methods Human umbilical cords were obtained from women with normal pregnancy immediately after delivery from Tongji Hospital Affiliated of Tongji Medical College. Primary HUVEC were isolated from umbilical veins and subcultured, then, HUVEC were divided into four groups:control group;LPS group (10 mg/L of LPS); LPS + LXA4 group(10 mg/L of LPS and 100 nmol/L of LXA4); LPS +LXA4 + BOC-2 group [10 μmol/L of BOC-2, an effective antagonist of formyl peptide receptor like 1 (FPRL-1)]. All expriments were performed after cells were treated for 24 hours. Endothelial permeability was measured by fluorescein isothiocyan-ate labelled bovine serum albumin (FITC-BSA) clearance across the monolayer; tumor necrosis factor α(TNF-o) mRNA and secretion were detected by reverse transcriplase (RT) -PCR and ELISA assay respectively, and nuclear factor κB(NF-κB) protein change was determined by western blot. Results (1) LPS induced a significant increase in the permeability [Pa value of LPS group was (183.1 ±1.7)%], while co-administrating with LXA4 obviously attenuated this LPS-induced hyperpermeability, Pa value of LPS + LXA4 group was (103.1 ±2.2)%, LPS + LXA4 + BOC-2 group was (162.2 ± 2.8)%, control group was 100%, the permeability of HUVEC monolayer was significantly increased by LPS which was (83.1 ± 1.7)% of control (P <0.01), however, it was notably inhibited by LXA4 (P<0.05); the blockade of FPRL-1 could attenuate the effect of LXA4, that is, there was no difference between the LPS + LXA4 + BOC-2 group and the LPS group. (2) After treatment with different concentration of LPS(0,0.1, 1,10 mg/L), the mRNA expressions of TNF-α were increased (1.11 ±0.11,1.27 ± 0.03, 1.60 ± 0.06, 1.82 ± 0. 04, respectively), compared with the control group, at the concentration of 1,10 mg/L LPS, the difference was statistically significant (P<0. 05). (3) The increased levels of NF-κB and inflammatory mediator TNF-α in the LPS group were both inhibited by LXA4. Levels of NF-κB protein and TNF-o mRNA secretion in LPS treated group (0.53 ±0.06 and 0.81 ±0.09 ,respectively)were both inhibited by LXA4 (0.19 ± 0.05 and 0.41 ± 0.07, respectively, and both had significant difference, P<0.05). (4) Levels of TNF-α in HUVEC culture medium of LPS group [(31.94 ±0.01)ng/L] was significantly higher than the control group [(18.17 ± 0.03) ng/L, P<0.05], LPS + LXA4 group [(15.72 ± 0.07) ng/L] was significantly lower than the LPS group (P<0.05). Conclusion Our findings demonstrated that LXA4 could prevent the endothelial cell hyperpermeability induced by LPS in HUVEC under which the possible mechanism was through inhibiting the expression of NF-κB and its related cytokines through receptor-dependent.
RESUMEN
Objective To explore the effects of lipoxin A4 ( LXA4 ) on lipopolysaccharide ( LPS)-induced oxidative stress in human umbilical veins endothelial cells(HUVEC) and the possible mechanism.Methods Neonatal umbilical cords were obtained from normal term pregnant women with cesarean section within 4 hours and then were used to isolate HUVEC for subculture.HUVEC were divided into four groups:control group; LPS group ( 10 μg/ml of LPS); LPS + LXA4 group ( 10 μg/ml of LPS and 100 nmol/L of LXA4); LXA4 group (100 nmol/L of LXA4) All expriments were performed after cells treated for 12 and 24 hours respectively.Immunofluorescence was used to detect the expression of Ⅷ foctor and nuclear translocation of nuclear factor-erythroid-2-related factor 2 ( Nrf2 ); the mRNA expression of Nrf2, heme oxygenase 1 (HO-1) and reduced form of nicotinamide-adenine dinucleotide quinone oxidoreductase-1(NQO1) were evaluated by reverse transcription-PCR .Results (1)The flavovirens fluorescence was observed in the cytoplasm under fluorescence microscope, which confirmed the existence of Ⅷ factor which specifically expressed in endothelial cells, especially in HUVEC.(2)Immunofluorescent results showed that in control group, Nrf2 protein expressed in the cytosol rather than in the nucleus.In LPS group, the expression of Nrf2 protein obviously increased in the nucleus while decreased in the cytosol after 12 hours.However, after LPS treatment for 24 hours, Nrf2 expression reduced in the cytosol and nucleus.In cotreatment with LPS and LXA4 group,the expression of Nrf2 protein was much higher than that in LPS group after 12 hours or 24 hours.Furthermore, Nrf2 protein also mostly expressed in the cytosol in LXA4 group.(3) After stimulation for 12 hours, compared with control group, the gene expression of Nrf2 and HO-1 were significantly enhanced in LPS group (0.581 ± 0.019 and 0.081 ±0.009, P ＜ 0.05 ) and in LPS + LXA4group(0.692 ±0.048 and 0.136 ± 0.018, P ＜ 0.05 ), the level of NQO1 mRNA in LPS group and LPS +LXA4 group were 0.381 ± 0.009 ( P ＞ 0.05 ) and 0.574 ± 0.034 ( P ＜ 0.05 ).After treatment for 24 hours,compared with control goup, the gene expressions of Nrf2 and NQO1 were down-regulated in LPS group (0.180±0.017 and 0.472 ±0.064, P＜0.05).But in LPS + LXA4 group the expression of Nrf2 and NQOI were upregulated (0.532 ± 0.051 and 0.830 ± 0.068, P ＜ 0.05, compared with treatment for LPS group).The mRNA expressions of Nrf2, HO-1 and NQO1 were increased in LPS + LXA4 group compared with LPS group ( P ＜ 0.05 ).In addition, there was no markedly difference in the expressions of Nrf2, HO1 and NQO1 between control and LXA4 group after 12 hours and 24 hours ( P ＞ 0.05 ) .Conclusion Through activating nuclear translocation of Nrf2 protein from cytoplasm, LXA4 upregulates the Nrf2downstream enzymes, such as NQO1 and HO-1 to protect HUVEC against the oxidative stress induced by LPS.