Nrf-2在纳米二氧化硅致人支气管上皮细胞氧化损伤中的作用
Effects of Nrf-2 on Oxidative Damage of Human Bronchial Epithelial Cells Induced by Silica Nanoparticles
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摘要:
目的 探讨核因子E2相关因子2(Nrf-2)在纳米二氧化硅(nano-SiO2)致人支气管上皮细胞(16HBE)氧化应激损伤中的作用。 方法 分别以终浓度为1.0、2.5、5.0、10.0、25.0和50.0μg/mL nano-SiO2处理人支气管上皮细胞24 h,检测细胞活性。根据检测结果,确定本实验nano-SiO2 (粒径15 mm)的暴露浓度为2.5、5.0和10.0μg/mL,分别设定为低、中、高浓度组。检测各暴露浓度组细胞内活性氧(ROS)、丙二醛(MDA)、总超氧化物歧化酶(T-SOD)和谷胱甘肽过氧化物酶(GSH-Px)等氧化应激相关指标。Western Blotting 检测细胞总蛋白和核蛋白中Nrf-2的表达水平,同时采用实时荧光定量聚合酶链反应检测Nrf-2 基因mRNA 表达水平。 结果 中、高浓度nano-SiO2 组16HBE 胞内ROS 水平明显增高(P<0.01),同时T-SOD和GSH-Px 含量降低(P<0.05);各浓度nano-SiO2 组16HBE胞内MDA含量均明显增高(P<0.05)。与对照组相比,随着nano-SiO2 暴露浓度的增高,细胞总Nrf-2蛋白和Nrf-2 基因的表达水平呈先增高后降低的趋势,而核内Nrf-2蛋白表达水平均明显增高。 结论 15 nm粒径的SiO2 可诱导人支气管上皮细胞发生氧化应激性损伤,而Nrf-2的激活和核转位在调节细胞内氧化-抗氧化体系中起重要作用。 Abstract:Objective To examine the effect of nuclear factor E2-related factor 2 (Nrf-2) on silica nanoparticle (nano-SiO2)-induced oxidative damage to human bronchial epithelial cells. Methods Human bronchial epithelial cells (16HBE) were exposed to 1.0, 2.5, 5.0, 10.0, 25.0, and 50.0 μg/mL nano-SiO2 for 24 h to determine cell viability. According to the results of cell viability assay, the cells in the following tests were then exposed to 2.5, 5.0, and 10.0 μg/mL nano-SiO2 (low, medium, and high dose groups, respectively), and the tests included cellular reactive oxygen species (ROS), malondialdehyde (MDA), total superoxide dismutase (T-SOD), and glutathione peroxidase (GSH-Px). Western Blotting was used to estimate the expression levels of Nrf-2 in total and nuclear proteins, and real-time quantitative polymerase chain reaction was used to assess the mRNA expression level of Nrf-2. Results The cellular ROS level significantly increased (P<0.01), while T-SOD and GSH-Px levels decreased (P<0.05) in the medium and the high dose groups. The MDA contents significantly increased in the low, medium, and high dose groups (P<0.05). The total Nrf-2 protein and the Nrf-2 mRNA expression levels increased in the low dose group, while decreased in the medium and the high dose groups; and the nuclear Nrf-2 protein expression level significantly increased in the low, medium, and high dose groups. Conclusion In the current study settings, nano-SiO2 with 15 nm in size can induce oxidative damage in human bronchial epithelial cells, and Nrf-2 activation and nuclear translocation play important roles in the regulation of intracellular oxidative-antioxidative system.
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