| Background and Objective Widespread use of zinc oxide nanoparticles(Zn O-NPs)increases exposure opportunities of occupational populations in the process of production,transportation,and usage.Zn O-NPs can result toxic damage in target tissues through a variety of routes(inhalation,skin,oral,etc.)and undergo migration and accumulation in vessels in vivo.Vascular endothelial dysfunction can lead to the development of a variety of cardiovascular diseases,whether Zn O-NPs can cause oxidative stress or inflammation in vascular endothelial cells remains controversial and further studies are needed.Heme oxygenase-1(HO-1)can protect cells from oxidative stress by catalyzing heme degradation into bilirubin,ferrous ions,and carbon monoxide.It has been found that HO-1 has anti-oxidation,anti-apoptosis and anti-inflammatory functions in specific cells.However,the role of HO-1 in inflammatory endothelial cells induced by Zn O-NPs has not been studied yet,and further explorations are needed.To investigate the inflammatory response of endothelial cells induced by Zn O-NPs and the role of HO-1 in inflammatory response,this study intends to explore the potential toxicity of Zn O-NPs induced oxidative stress and inflammatory in EA.hy 926 cells,and identify the role of HO-1 in the inflammatory response by activation and inhibition HO-1 expression for providing a theoretical basis of the cardiovascular toxicity of Zn O-NPs.Methods 1.EA.hy 926 cells were treated with Zn O-NPs with concentrations of 0,2.5,5,10,15,20,25,30,and 40 ?g/m L,and the effect of Zn O-NPs on the activity of EA.hy 926 cells was determined by cell counting kit-8(CCK 8)..2.EA.hy 926 cells grown to confluence were exposed to 0,2.5,5,10,15 ?g/m L Zn O-NPs.The intracellular reactive oxygen species(ROS)levels,the activity of superoxide dismutase(SOD)and malondialdehyde(MDA)levels in the cells were measured by the detection assay kit of ROS,SOD and MDA,respectively.Levels of IL-6,IL-1?,VEGF,MCP-1,HO-1 m RNA were measured by RT-PCR,the levels of IL-1?,VEGF in cell culture supernatant were measured by ELISA,and the levels of HO-1 in cells were measured by Western Blot.3.EA.hy 926 cells pretreated with Co PPIX or Zn PPIX were exposed to 15 ?g/m L Zn O-NPs,and the level of HO-1,ROS,SOD,MDA,IL-1? and VEGF were measured by corresponding methods.Results 1.Cell viability of EA.hy 926 cells when treated with Zn O-NPs Cell viability decreased when the concentration of Zn O-NPs reached 10 ?g/m L,and with the increase of Zn O-NPs concentration,the activity of cells decreased gradually.The cell viability was 63.18 % when the concentration of Zn O-NPs was 15 ?g/m L,and cell viability reduced to 46.07 % at 20 ?g/m L.2.The lipid peroxidation effects of Zn O-NPs on EA.hy 926 cells The expression levels of ROS in EA.hy 926 exposed to 5,10,15 ?g/m L Zn O-NPs were significantly increased compared with control group(P < 0.05).The activities of SOD in EA.hy 926 cells exposed to 5,10,15 ?g/m L were significantly decreased compared with control group(P < 0.05),the levels of MDA and the ratios of MDA/SOD increased with the increasing Zn O-NPs exposure dose(P < 0.05).With the increase of the concentration of Zn O-NPs,intracellular level of ROS and MDA increased,SOD activity gradually decreased,MDA/SOD ratio increased,and the cells had lipid peroxidation.3.The inflammatory effects induced by Zn O-NPs in EA.hy 926 cells The expression levels of IL-1?,IL-6,VEGF and MCP-1 m RNA in cells treated with Zn O-NPs for 12 h were increased compared with the control group,and the differences were statistically significant(P < 0.05).4.The expression of HO-1 in EA.hy 926 cells induced by Zn O-NPs The expression levels of HO-1 m RNA and protein present low levels in normal conditions,and expression of HO-1 in cells increased with the increase of Zn O-NPs dose.Compared with the control group,the expression of HO-1 m RNA and HO-1 protein were significantly increased in the 10 and 15 ?g/m L Zn O-NPs-treated groups(P < 0.05).Relative expression level of HO-1 in the 15 ?g/m L group was significantly higher compared with the 10 ?g/m L group(P < 0.05). 5.Inflammatory effects of different HO-1 levels 5.1 Activation of HO-1 After being treated with Co PPIX,intracellular HO-1 gene and protein expression levels increased significantly.ROS level,MDA content,and MDA/SOD ratio of cells in the Co PPIX + Zn O-NPs group significantly decreased compared with Zn O-NPs group,while SOD activity was increased and the differences were statistically significant(P < 0.05).In addition,IL-1? levels decreased and VEGF levels increased in Co PPIX + Zn O-NPs group,and the differences were statistically significant(P < 0.05).5.2 Inhibition of HO-1 After being treated with Zn PPIX,intracellular HO-1 gene and protein expression levels decreased significantly.The cells in the Zn PPIX + Zn O-NPs group had significantly increasing on ROS level,MDA content,and MDA/SOD ratio compared with Zn O-NPs group(P< 0.05).However,no significant difference was found in the SOD activity.In addition,significantly increased IL-1? levels and decreased VEGF levels in Zn PPIX + Zn O-NPs group was found(P< 0.05).Conclusions 1.Zn O-NPs(10 or 15 ?g/m L)could induce lipid peroxidation and inflammatory in EA.hy 926 cells.2.Activation of HO-1 could against the inflammatory response of EA.hy 926 cells induced by Zn O-NPs. |
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