ABSTRACT
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 culture medium. 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-1: 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.
ABSTRACT
OBJECTIVE: To explore the effect of zinc oxide nanoparticles(ZnO NPs) on the oxidative damage in human alveolar type Ⅱ epithelial cell line A549.METHODS: The A549 cells in logarithmic growth phase were incubated with ZnO NPs solution at dose of 0,10,20 and 40 mg/L as 4 dose groups.The levels of reactive oxygen species(ROS) were measured by flow cytometer after 4 hours of exposure.The malondialdehyde(MDA) content and super oxide dismutase(SOD) activity were measured by microplate reader after 8 hours of exposure.RESULTS: The ROS levels in A549 cells exposed to 10,20,40 mg/L ZnO NPs were significantly increased compared with control group(P<0.05).The level of ROS increased with the exposure dose of ZnO NPs in A549 cells(P<0.01).The activities of SOD in A549 cells exposed to 10,20,40 mg/L ZnO NPs were significantly decreased compared with control group(P<0.05).The level of MDA and the ratios of MDA/SOD increased compared with control group(P<0.05).The activity of SOD in A549 cells decreased with the increase of ZnO NPs exposure dose(P<0.01),and the level of MDA and the ratios of MDA/SOD increased with the increase of exposure(P<0.01).CONCLUSION: ZnO NPs could induce lipid peroxidation in A549 cells with a dose-effect relationship.