目的 探讨脂氧素对脂多糖诱导的脐静脉内皮细胞氧化应激反应的影响.方法 切取正常妊娠剖宫产手术4 h内的新生儿脐带,从中分离出脐静脉内皮细胞并进行传代培养,将培养的脐静脉内皮细胞分成4组:空白对照组,脂多糖处理组(10 μg/ml脂多糖),脂多糖+脂氧素处理组(10μg/ml脂多糖+100 nmol/L脂氧素),脂氧素处理组(100 nmol/L脂氧素).分别作用12 h和24 h后进行检测.采用免疫细胞荧光法检测核因子E2相关因子2(Nrf2)的表达及核转位情况、内皮细胞特异性Ⅷ因子的表达;采用逆转录(RT)PCR检测Nrf2、血红素加氧酶(HO)1和还原型辅酶Ⅰ醌类氧化还原酶(NQO1)mRNA的表达.结果 (1)荧光显微镜下观察到脐静脉内皮细胞胞质内有黄绿色荧光反应,证实有内皮细胞特异性Ⅷ因子抗原存在.(2)免疫细胞荧光染色显示,空白对照组内皮细胞中Nrf2大部分表达在胞质,胞核内基本不表达.12 h后,脂多糖处理组Nrf2胞核内的表达增加,出现了由胞质向胞核的转位;但24 h后,Nrf2在胞质和胞核内的表达都明显下降.脂多糖+脂氧素处理组在12 h和24 h,Nrf2在胞质和胞核中的表达均明显高于脂多糖处理组;脂氧素处理组Nrf2大部分表达在胞质中.(3)脂多糖处理组和脂多糖+脂氧素处理组在12 h后,Nrf2和HO-1 mRNA表达水平分别为0.581±0.019、0.081±0.009及0.692±0.048、0.136±0.018,均明显高于空白对照组,分别比较,差异均有统计学意义(P＜0.05);脂多糖处理组在12 h后,NQO1 mRNA表达为0.381±0.009,虽高于空白对照组的0.355±0.044,但差异无统计学意义(P＞0.05),而脂多糖+脂氧素处理组NQO1 mRNA表达为0.574±0.034,与空白对照组比较,差异有统计学意义(P＜0.05);脂多糖处理组24 h后,Nrf2和NQO1 mRNA的表达水平分别为0.180±0.017、0.472±0.064,与空白对照组比较,差异均有统计学意义(P＜0.05);脂多糖+脂氧素处理组Nrf2、HO-1、NQO1 mRNA的表达均较脂多糖处理组明显增加,差异均有统计学意义(P＜0.05);脂氧素处理组Nrf2、HO-1、NQO1 mRNA表达水平无论12 h还是24 h,与空白对照组比较,差异均无统计学意义(P＞0.05).结论 脂氧素通过激活脐静脉内皮细胞胞质内的Nrf2,使其向胞核内转位,进而上调Nrf2下游对细胞有保护作用的抗氧化酶,如HO-1、NQO1,抑制脂多糖诱导的氧化应激,从而保护脐静脉内皮细胞免受损伤.%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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