脂氧素通过Nrf2信号通路对LPS诱导脐静脉内皮细胞氧化应激的抑制作用  

The Inhibition Effect of Lipoxin on Oxidative Stress of Human Umbilical Vein Endothelial Cells(HUVEC) Stimulated by LPS through Nrf2 Signaling Pathway

作　　者：龚建明[1] 黄引平[1] 

机构地区：[1]温州医学院附属第一医院妇产科,325000

出　　处：《医学研究杂志》2011年第1期97-101,共5页Journal of Medical Research

基　　金：高等学校博士学科点专项科研基金(200803430002)

摘　　要：目的研究脂氧素对脂多糖(LPS)刺激的脐静脉内皮细胞引起的氧化应激的抑制作用及其机制。方法以LPS10μg/ml诱导脐静脉内皮细胞氧化应激,以不同浓度脂氧素(lipoxin A4,LXA4)进行干预,以2,7-二氢二氯荧光素二乙酸酯(DCFH-DA)为荧光探针检测细胞内活性氧(reactive oxygen species,ROS)水平;细胞免疫荧光检测转录因子NF-E2相关因子2(Nrf2)转位变化;RT-PCR检测Nrf2及其下游抗氧化酶mRNA的表达;化学显色法测定脐静脉内皮细胞内超氧化物歧化酶(SOD)活性和谷胱甘肽过氧化物酶(GSH-PX,GPX)活性。结果细胞内ROS测定显示,脂氧素能够有效地降低脐静脉内皮细胞内由LPS刺激引起的ROS升高;细胞免疫荧光结果显示,LPS组Nrf2主要在胞质中表达。而加入脂氧素干预后胞核可见较强的Nrf2的表达;其中加入200nmol/L脂氧素作用后胞核中的Nrf2的表达明显增强。RT-PCR结果显示,LPS组Nrf2、血红素氧合酶-1(HO-1)、SOD、GPX mRNA表达水平明显降低,加入脂氧素干预后Nrf2、HO-1、SOD、GPX mRNA表达水平均增加。化学显示法结果显示,LPS组脐静脉内皮细胞的SOD、GSH-PX的活性明显降低,使用100nmol/L和200nmol/L脂氧素干预后均能明显减少细胞的氧化损伤。结论脂氧素通过活化转录因子Nrf2来促进多种抗氧化酶的表达,进而减轻细胞内的氧化应激损伤。Objective To explore the inhibition effect of lipoxinA4 on oxidative stress of HUVEC stimulated by LPS and study its mechanisms.Methods Oxidative stress in HUVEC was induced by LPS（10μg/ml） and HUVEC were treated with different concentrations of LXA4.The intracellular formation of reactive oxygen species（ROS） was detected by 2,7-dichlorofluorescein diacetate（DCFH-DA） as a fluorescent probe.Nrf2 location was identified with cell immunofluorescence histochemistry method.The expression of Nrf2 and its downstream antioxidant enzymes genes were detected by RT-PCR.The levels of SOD and GSH-PX in HUVEC were detected by detection kit.Results The results showed that treatment of HUVEC with lipoxinA4 reduced LPS-stimulated ROS formation.Immunofluorescence histochemistry results showed that Nrf2 was predominatly cytoplasmic in HUVEC in LPS group,while Nrf2 expression could be seen clearly in nucleus in HUVEC after treatment with lipoxinA4 and 200nmol/L lipoxinA4 could significantly increase the location of Nrf2 in nucleus.RT-PCR results showed that the expression of Nrf2,HO-1,SOD,GPX mRNA was lower than that in control group.Chromatometry results showed that the levels of SOD and GSH-PX in HUVEC were obviously lower than those in control group.Whereas,lipoxinA4 at 100nmol/L and 200nmol/L concentration could significantly reduce the cell oxidative damage.Conclusion These findings suggest that lipoxinA4 promotes the expression of various antioxidant enzymes through the activation of Nrf2,and then reduces intracellular oxidative stress.

关 键 词：脐静脉内皮细胞 脂氧素 NRF2 氧化应激 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]