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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.
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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.
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Objective To investigate protective effects and mechanisms of lipoxinA4(LXA4)on human umbilical vein endothelial cells(HUVEC)under hypoxia in vitro.Methods The HUVEC culture were divided into groups as followed:added M199 cudture medium as normal contml groups,added CoCl2 to mimic hypoxia in vitro as hypoxia group and added different concentrations of LXA4(1,10,100 nmol/L)were added to the induced hypoxiai HUVEC as agents intervention group.Morphological changes of HUVEC were observed by using inverted phase contrast mieroscope.The influence of LXA4 on cell survival was investigated by methyl thiazolyl tetrazolium(MTT) assaying method after the treatment with different concentrations of LXA4 and 100 nmol/L lipoxinA4 according to different time(4,8,12 and 24 hours).The expression of von-willebrand factor (vWF) was detected by immunocytoehemistry method. The changes of cytosolic Ca2+ were measured by laser scanning confocal microscope. Results ( 1 ) Morphological changes:the cells under hypoxia lost its normal shapes and showed necrosis, while the cells cocuhured with 100 nmol/L LXA4 were normal appropriately. (2)Survival rate: the survival rates of HUVEC under hypoxia was (40. 1±3.9) % and increased to ( 52. 9 ± 1.4) %, (64. 1 ± 3. 3 ) %, ( 76. 6 ± 1.6) % respectively when added with LXA4 with concentration of 1,10, 100 nmol/L into culture medium. There was significant different survival rate when compared with that of hypoxia group. (3) The level of vWF: The expression of vWF was decreased with the increasing concentrations of LXA4 added into culture medium, the gray values were 203.9 ±0. 7 in 1 nmol/L,204.6 ±0. 9 in 10 nmoL/L,191.8 ±0. 5 in 100 nmol/L respectively, which reached statistical difference in comparison with that of hypoxia groups (P<0. 05). (4) Confocai analysis:the intracellular free Ca2+ concentrations of HUVEC were intensified with LXA4 treatment. Conclusions LXA4 plays an important role in keeping the normal shape of HUVEC under hypoxia, can enhance survival of hypoxial HUVEC and decrease the level of vWF in cytoplasm. The protective mechanism might be via decreasing mitochondria Ca2+ overload and increasing cytoplasm Ca3+ by nucleus Ca2+ transference.
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Objective To investigate the effects of lipoxin A4 on store-operated calcium channel (SOC) and production of reactive oxygen species in macrophages induced by hpopolysaccharide (LPS).Method Macrophages were randomly assigned Io one of the following six groups:control group,LPS group,Thapsigargin group,lipoxin A4+LPS group,lipoxin A4+Thapsigargin group,2-Aminoethoxydiphenylborate+Thapsigargin group.The intracellular[Ca2+]iwas analyzed by eonfoeal laser microscopy.The production of reactive oxygen specips(ROS) was assayed by flow cytometry.Results LPS increased intracellular[Ca2+]i and reactive oxygen species in a dose-dependent manner.Lipoxin A4 suppressed approximately 75% of the Ca2+ ertry signal induced by thapsigargin and suppressed approximately 93% of the Ca2+ entry signal induced by LPS.The increase in intracellular[Ca2+]i was associated with increased ROS production which was abolished in the presence of lipoxin A4.Conclusions These findings indicate that the LPS-indueed intracellular[Ca2*]i increase depends on the Ca2+entry through SOC channel,and lipoxin A4 inhibits Ca2+ influx and ROS production through SOC channel in ratine maerophages induced by LPS.
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In order to investigate the expression of cyclooxygenase-2 (COX-2) in human lower segments of myometrium obtained from women in labor and those not in labor and identify the splicing variant of COX-2, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression of COX-2. The primers were designed and synthesized according to the sequence of rat COX-2 splice variant which was discovered firstly by us. Then the splicing variant of COX-2 in human myometrium from woman in labor was identified, cloned into vector and sequenced. The results showed that the expression of COX-2 mRNA was lower in human myometrium obtained from women who were not in labor than that in labor women and a new band of COX-2 was obtained in myometrium from labor woman. The fragment included an unspliced intron, which pitched between exons 7 and 8. It was suggested that COX-2 gene was not only expressed highly in human myometrium from woman in labor, but also produced splicing variant by alternative splicing.
Subject(s)
Amino Acid Sequence , Base Sequence , Cyclooxygenase 2/biosynthesis , Cyclooxygenase 2/genetics , Labor Onset/metabolism , Molecular Sequence Data , Myometrium/enzymology , Myometrium/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Sequence AnalysisABSTRACT
To explore the pharmacological effect of 3,4-dihydroxyacetophenone (DHAP) on the apoptosis of RAW264. 7 macrophage cells and the mechanism, RAW264. 7 macrophage cells were treated with 100 or 500 mg/L lipopolysaccharide (LPS), with or without 10-5 mol/L DHAP for 24h. Trypan blue dye exclusion assay was used to assess cell viability. Cell apoptosis was morphological studied and flow cytometric assay was used. Tumor necrosis factor-α (TNF-α) level was measured by ELISA methods. IκB protein was determined by Western blotting. Our results showed that in 100 mg/L LPS-stimulated macrophages, DHAP enhanced the cell apoptosis while in 500 mg/L LPS-stimulated macrophages, DHAP significantly inhibited the cell apoptosis. In both groups,DHAP increased the level of IκB but decreased the level of TNF-α. It is concluded that DHAP has dual effect on the apoptosis of RAW 264.7 cells treated with different concentrations of LPS. This effect may be due to the inhibition of activation of NF-κB and autocrine production of TNFα. Our study suggests that DHAP may have anti-inflammatory effect on LPS-activated macrophages.
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In order to investigate the expression of cyclooxygenase-2 (COX-2) in human lower segments of myometrium obtained from women in labor and those not in labor and identify the splicing variant of COX-2, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression of COX-2. The primers were designed and synthesized according to the sequence of rat COX-2 splice variant which was discovered firstly by us. Then the splicing variant of COX-2 in human myometrium from woman in labor was identified, cloned into vector and sequenced. The results showed that the expression of COX-2 mRNA was lower in human myometrium obtained from women who were not in labor than that in labor women and a new band of COX-2 was obtained in myometrium from labor woman. The fragment included an unspliced intron, which pitched between exons 7 and 8. It was suggested that COX-2 gene was not only expressed highly in human myometrium from woman in labor, but also produced splicing variant by alternative splicing.
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BACKGROUND:Cervical cancer is one of the most frequent malignancies in women worldwide, and its occurrence and development is closely related to cyclooxygenase-2 (COX-2).OBJECTIVE: To examine the expression of COX-2 alternative splicing variants in human cervical carcinoma tissue and understand its possible implications.DESIGN: Non-randomized controlled experiment.SETTING: Key Laboratory of Biochemistry and Molecular Pharmacology,Department of Obstetrics and Gynecology, First Affiliated Hospital,Chongqing Medical University.PARTICIPANTS: Carcinoma tissue and normal tissue were obtained from 13 cervical carcinoma patients admitted during March 2002 to April 2002in the Department of Obstetrics and Gynecology, First Affiliated Hospital,Chongqing Medical University.METHODS: A pair of specific primers were designed for reverse transcription-polymerase chain reaction (RT-PCR) to obtain the mRNA of COX-2 in human cervical carcinoma tissues. The resultant band on electrophoresis was cloned, sequenced and analyzed.MAIN OUTCOME MEASURES: ① Agarose gel electrophoresis result of the PCR product of carcinoma and normal tissues; ② Sequencing result of the electrophoresis band from carcinoma and normal tissues.RESULTS: No COX-2 band (252 bp) was found in electrophoresis for normal tissues, while 2 bands appeared for cervical carcinoma tissues, including a new electrophoresis band of 534bp besides the COX-2 band. Cloning and sequencing revealed that this new band contained not only exons 7and 8 of COX-2 gene but also a reserved intron of 282 bp intron between exons 7 and 8. Analysis of the predicted amino acid sequence indicated that an in-frame stop codon occurred in the 48-50 bp of the intron retained in the mRNA.CONCLUSION: The presence of COX-2 alternative splicing mRNA variant (Genbank accession number:BU493602)is confirmed in human cervical carcinoma tissue, which codes for a protein possibly smaller than COX-2.
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AIM: To investigate the effect of epinephrine on LPS-induced pro-inflammatory mediators (TNF-?, NO and COX-2) and anti-inflammatory mediators (HO-1 and IL-10) production in murine macrophage RAW264.7 cells, and to determine whether these effect is due to the influence of epinephrine on NF-?B activation. METHODS: RAW264.7 cells were cultured in vitro with 10 ?g/L LPS in the absence or presence of epinephrine at variant concentrations (1, 5, 10, 50 ?mol/L) for 24 hours, then the supernatants was collected for measuring TNF-? and IL-10 by ELISA and Griess reagent was used to measure NO (NO_2-/NO_3-) concentration. At the same time point, cells were harvested and COX-2, HO-1 and I?B-? was detected by Western blotting. RESULTS: 10 ?g/L LPS significantly induced the production of TNF-?, NO (NO_2-/NO_3-), COX-2, HO-1 and IL-10. When epinephrine was added into the medium together with LPS, the pro-inflammatory mediators production was decreased in a dose-dependent manner, however, anti-inflammatory mediators HO-1 and IL-10 expression was enhanced by epinephrine. Epinephrine has no significant effect on I?B-? degradation in LPS-activated RAW264.7 cells. CONCLUSION: Epinephrine down-regulates LPS-induced pro-inflammatory mediator expression while promotes anti-inflammatory mediator production in murine macrophages. These effect seems to be independent of NF-?B activation.