摘要
目的探讨血红素加氧酶（HO-1）对纳米氧化锌（ZnO-NPs）致人脐静脉血管内皮细胞株（EA．hy926细胞）活性氧水平改变的影响。方法取对数生长期EA．hy926细胞，分别采用0．0、2．5、5．0、10．0和15．0mg／LZnO．NPs溶液进行染毒，4h后采用流式细胞仪检测细胞内活性氧水平，计算平均荧光强度（MFI），24h后采用WesternBlot检测细胞内HO-1蛋白表达水平。将t5．0mg／L浓度的ZnO-NPs溶液刺激细胞设定为ZnO—NPs组，同时设定空白对照组。另外使用终浓度为10μmol／L的HO-1抑制剂锌原卟啉（ZnPPIx）及HO-1激活剂钴原卟啉（CoPPIx）分别预处理细胞，设置为ZnPPIX、CoPPIx组。以终浓度为10μmol／L的ZnPPIX、CoPPIx预处理EA．hy926细胞1h后，于培养基中加入15．0mg／LZnO—NPs进行培养，分别设定为ZnPPIX＋ZnO-NPs组、CoPPIx＋ZnO．NPs组。各组均于染毒后4h后检测活性氧水平，计算平均荧光强度（MFI），24h后检测HO．1表达水平。结果ZnO．NPs染毒剂量增加，EA．hy926细胞内活性氧与HO-1水平增加（0．0、2．5、5．0、10．0和15．0mg／LZnO．NPs染毒组的MFl分别为22627．22±718．27、24726．474-568．52、31141．75-4-1312．24、39824．824-4774．74、50569．034-1497．63，HO．1相对表达量分别为0．16±0．01、0．19＋0．02、0．16＋0．01、0．23＋0．02、0．92＋0．06），P值均〈0．001。15．0mg／LZnO-NPs组HO-1灰度值高于ZnPPIx＋ZnO-NPs组（1．124-0．01比1．05±0．05，P〈0．051，且活性氧MFI水平较低（53654．53±2229．01比62683．95±2589．59，P〈0．001）；CoPPIx＋ZnO—NPs组HO-1灰度值高于15．0mg／LZnO-NPs组（1．744-0．11比0．22-0．03，P〈0．001），且活性氧水平较低（32845．04±993．48比53654．534-2229．01，P〈0．001）。结论ZnO．NPs可致EA．hy926细胞发生氧化应激，呈剂量．效应关系，HO．1可调控ZnO-NPs诱发的氧化应激水平。
Objective To explore the effect of heme oxygenase-1 （HO-1） on level of reactive oxygen species （ROS） induced by zinc oxide nanoparticles （ZnO-NPs） in Human umbilical vein endothelial cells line EA.hy926. Methods The EA.hy926 cells in logarithmic growth phase were incubated with 0.0, 2.5, 5.0, 10.0 and 15.0 mg/L ZnO-NPs respectively. The ROS level, reflected by mean fluorescence intensity （MFI）, was examined by flow cytometer after 4 hours exposure, the protein expression of HO-1 which was determined by Western Blot after exposed to ZnO-NPs for 24 hours. Cells incubated with 15.0 mg/L were set as the ZnO-NPs group; a blank control group was set at the same time. Cells were pretreated with HO-1 inhibitor zinc protoporphyrin （ZnPPIx） and HO-1 activator cobalt protoporphyrin （CoPPIx）, they were classified as ZnPPIx group and CoPPIx group. 15 mg/L ZnO-NPs was chosen to conduct the experiment of HO-1 activation and inhibition. Cells were classified as ZnPPIX＋ ZnO-NPs group and CoPPIx＋ ZnO-NPs group after pretreated with 10 μmol/L ZnPPIx or CoPPIx for 1 h, added 15 mg/L ZnO-NPs to cell culturemedium. In all groups ROS levels were detected after exposed to ZnO-NPs for 4 hours, the protein expression of HO-1 was detected after exposed to ZnO-NPs for 24 hours. Results With the increased dose of ZnO- NPs, levels of ROS and HO-1 in EA.hy926 cells were clearly elevated （the MFI of 0.0, 2.5, 5.0, 10.0 and 15.0 mg/L ZnO-NPs incubated groups was 22 627.22±718.27, 24 726.47±568.52, 31 141.75± 1 312.24, 39 824.82±4 774.74, 50 569.03±1 497.63 respectively, and HO-1 relative expression were 0.16±0.01, 0.19± 0.02, 0.16 ± 0.01, 0.23 ±0.02, 0.92±0.06 respectively）. HO-1 expression in ZnPPIx pretreatment group decreased compared with ZnO-NPs group （1.05± 0.05 vs. 1.12 ± 0.01, P〈0.05）, meanwhile ROS level enhanced （62 683.95±2 589.59 vs. 53 654.53±2 229.01, P〈0.05）. However, CoPPIx pretreatment had higher HO-1 level and lower level of ROS compared with ZnO-NPs group （HO-h 1.74±0.11 vs. 0.22±0.03, P〈0.05; ROS： 32 845.04 ＋ 993.48 vs. 53 654.53 ± 2 229.01, P〈0.05）. Conclusions Exposure to ZnO-NPs significantly induced ROS generation in EA.hy926 cells in a dose-dependent manner. HO-1 regulated ZnO- NPs-induced oxidative stress.
作者
乔亚梅
梁肖
路亚柯
卓来宝
武佳佳
王惠欣
姚武
燕贞
Qiao Yamei;Liang Xiao;Lu Yake;Zhuo Laibao;Wu Jiajia;Wang Huixin;Yao Wu;Yan Zhen.(Department of Occupational and Environmental Health Sciences,School of Public Health,Zhengzhou University,Zhengzhou,Henan 450001,China)
出处
《中华预防医学杂志》
CAS
CSCD
北大核心
2018年第11期1177-1181,共5页
Chinese Journal of Preventive Medicine
基金
郑州大学优秀青年教师发展基金（1521329034）
关键词
血红素加氧酶.1
细胞
活性氧
纳米氧化锌
Heme oxygenase-1
Cells
Reactive oxygen species
Zinc oxide nanoparticles
