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Abstract：
BACKGROUND: Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multipotent protein that plays essential roles in cellular responses to oxidative stress.
METHODS: To examine the role of APE1/Ref-1 in ischemia-reperfusion (I/R) injuries and hydrogen peroxide (H2O2)-induced renal tubular apoptosis, we studied male C57BL6 mice and human proximal tubular epithelial (HK-2) cells treated with H2O2 at different concentrations. The colocalization of APE1/Ref-1 in the proximal tubule, distal tubule, thick ascending limb, and collecting duct was observed with confocal microscopy. The overexpression of APE1/Ref-1 with knockdown cell lines using an APE1/Ref-1-specific DNA or small interfering RNA (siRNA) was used for the apoptosis assay. The promotor activity of nuclear factor kappa B (NF-κB) was assessed and electrophoretic mobility shift assay was conducted.
RESULTS: APE1/Ref-1 was predominantly localized to the renal tubule nucleus. In renal I/R injuries, the levels of APE1/Ref-1 protein were increased compared with those in kidneys subjected to sham operations. The overexpression of APE1/Ref-1 in HK-2 cells enhanced the Bax/Bcl-2 ratio as a marker of apoptosis. Conversely, the suppression of APE1/Ref-1 expression by siRNA in 1-mM H2O2-treated HK-2 cells decreased the Bax/Bcl-2 ratio, the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38, c-Jun N-terminal kinase (JNK) 1/2, and NF-κB. In HK-2 cells, the promoter activity of NF-κB increased following H2O2 exposure, and this effect was further enhanced by APE1/Ref-1 transfection.
CONCLUSIONS: The inhibition of APE1/Ref-1 with siRNA attenuated H2O2-induced apoptosis through the modulation of mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 and the nuclear activation of NF-κB and proapoptotic factors.
METHODS: To examine the role of APE1/Ref-1 in ischemia-reperfusion (I/R) injuries and hydrogen peroxide (H2O2)-induced renal tubular apoptosis, we studied male C57BL6 mice and human proximal tubular epithelial (HK-2) cells treated with H2O2 at different concentrations. The colocalization of APE1/Ref-1 in the proximal tubule, distal tubule, thick ascending limb, and collecting duct was observed with confocal microscopy. The overexpression of APE1/Ref-1 with knockdown cell lines using an APE1/Ref-1-specific DNA or small interfering RNA (siRNA) was used for the apoptosis assay. The promotor activity of nuclear factor kappa B (NF-κB) was assessed and electrophoretic mobility shift assay was conducted.
RESULTS: APE1/Ref-1 was predominantly localized to the renal tubule nucleus. In renal I/R injuries, the levels of APE1/Ref-1 protein were increased compared with those in kidneys subjected to sham operations. The overexpression of APE1/Ref-1 in HK-2 cells enhanced the Bax/Bcl-2 ratio as a marker of apoptosis. Conversely, the suppression of APE1/Ref-1 expression by siRNA in 1-mM H2O2-treated HK-2 cells decreased the Bax/Bcl-2 ratio, the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38, c-Jun N-terminal kinase (JNK) 1/2, and NF-κB. In HK-2 cells, the promoter activity of NF-κB increased following H2O2 exposure, and this effect was further enhanced by APE1/Ref-1 transfection.
CONCLUSIONS: The inhibition of APE1/Ref-1 with siRNA attenuated H2O2-induced apoptosis through the modulation of mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 and the nuclear activation of NF-κB and proapoptotic factors.
摘要：
■无嘌呤/无嘧啶核酸内切酶1/氧化还原因子-1（APE1/Ref-1）是一种多能蛋白，在细胞对氧化应激的反应中起重要作用。然而,APE1/Ref-1在肾脏损伤中的作用尚未确定.
■为了研究APE1/Ref-1在缺血再灌注（I/R）损伤和过氧化氢（H2O2）诱导的肾小管凋亡中的作用，我们研究了用不同浓度的H2O2处理的雄性C57BL6小鼠和人近端肾小管上皮细胞（HK-2）。APE1/Ref-1在近端小管的共定位，远端小管，粗大的上升肢体，共聚焦显微镜观察收集管。使用APE1/Ref-1特异性DNA或小干扰RNA(siRNA)用敲低细胞系过表达APE1/Ref-1用于细胞凋亡测定。评估核因子κB（NF-κB）的启动子活性，并进行电泳迁移率变化测定。
■APE1/Ref-1主要定位于肾小管核。在肾I/R损伤中，与接受假手术的肾脏相比,APE1/Ref-1蛋白水平升高.APE1/Ref-1在HK-2细胞中的过表达增强了Bax/Bcl-2比率，作为凋亡的标志物。相反，在1-mMH2O2处理的HK-2细胞中通过siRNA抑制APE1/Ref-1表达降低了Bax/Bcl-2比率,细胞外信号调节激酶（ERK）1/2，p38，c-JunN末端激酶（JNK）1/2和NF-κB的磷酸化。在HK-2细胞中，NF-κB的启动子活性在H2O2暴露后增加，APE1/Ref-1转染进一步增强了这种作用。
■用siRNA抑制APE1/Ref-1通过调节ERK介导的丝裂原活化蛋白激酶途径来减弱H2O2诱导的细胞凋亡，JNK,p38和核激活NF-κB和促凋亡因子。
■为了研究APE1/Ref-1在缺血再灌注（I/R）损伤和过氧化氢（H2O2）诱导的肾小管凋亡中的作用，我们研究了用不同浓度的H2O2处理的雄性C57BL6小鼠和人近端肾小管上皮细胞（HK-2）。APE1/Ref-1在近端小管的共定位，远端小管，粗大的上升肢体，共聚焦显微镜观察收集管。使用APE1/Ref-1特异性DNA或小干扰RNA(siRNA)用敲低细胞系过表达APE1/Ref-1用于细胞凋亡测定。评估核因子κB（NF-κB）的启动子活性，并进行电泳迁移率变化测定。
■APE1/Ref-1主要定位于肾小管核。在肾I/R损伤中，与接受假手术的肾脏相比,APE1/Ref-1蛋白水平升高.APE1/Ref-1在HK-2细胞中的过表达增强了Bax/Bcl-2比率，作为凋亡的标志物。相反，在1-mMH2O2处理的HK-2细胞中通过siRNA抑制APE1/Ref-1表达降低了Bax/Bcl-2比率,细胞外信号调节激酶（ERK）1/2，p38，c-JunN末端激酶（JNK）1/2和NF-κB的磷酸化。在HK-2细胞中，NF-κB的启动子活性在H2O2暴露后增加，APE1/Ref-1转染进一步增强了这种作用。
■用siRNA抑制APE1/Ref-1通过调节ERK介导的丝裂原活化蛋白激酶途径来减弱H2O2诱导的细胞凋亡，JNK,p38和核激活NF-κB和促凋亡因子。
