| Background:Zinc Oxide nanoparticles (ZnO-NPs) have been produced in high tonnage worldwide. After inhaled they can induce lung inflammation and injury. In addition, ZnO-NPs can also penetrate air-blood barrier and enter blood circulation. Once pass through multiple protective barriers, such as blood-brain, blood-eye, and blood-testis barries, ZnO-NPs may cause impairment of crucial organs in human body. Epidemiological and toxicologic studies have demonstrated that exposure to ZnO-NPs could increase the permeability of human heart microvascular endothelial cells and induce inflammatory response and cell toxicity, leading to atherosclerosis-like alterations.The mechanisms underlying ZnO-NP-induced cardiovascular disorders, especially atherosclerotic alterations, remain unclear. It has been proposed that the pathogenesis of atherosclerosis is characterized by apoptosis of endothelial cells, and overexpression of platelet endothelial cell adhesion molecules-1 (PECAM-1) and heme oxygenase (HO-1). Therefore, this study mainly examined whether ZnO-NP could induce arterial endothelial cell damage and over-expression of HO-1 and PECAM-1 using co-culture of human alveolar epithelial cells (A549) and human coronary arterial endothelial cells (HCAEC) to resemble human air-blood barrier. In the co-culture studies, the inhibitors of gene transcription and phagocytosis were administrated, respectively, to determine their role in ZnO-NP-induced HO-1 and PECAM-1 expression in HCAEC. Taken together, the application of these two inhibitors were to determine the involvement of A549 in ZnO-NP-induced biological effects in HCEAC. The results fom this study will provide evidence for elucidation of ZnO-NP-induced endothelial cytotoxicity and underlying mechanisms, design of preventive and therapeutic measures, and establishment of hygegien standards. Objective:The aims of this study were to determine cell toxicity and expressions of HO-1 and PEC AM-1 in HCEAC exposed to ZnO-NP and underlying mechanisms, using co-culture of A549 and HCAEC, or HCAEC alone.Methods:1. Effect of ZnO-NPs on the co-culture model:(1) Co-culture model:A549 cells were plated on transwell filters with 0.4μm diameter pores, through the A549 cells could communicate with the basolateral HCAEC.(2) Determination of cytotoxicity:Upon confluence, A549 were treated with ZnO-NPs. Cell viability of A549 was determined using MTT assay.(3) Measurement of HO-1 and PEC AM-1 levels in culture medium:HCAEC were treated with ZnO-NPs 24 h. The medium in the basolateral chamber was collected. Levels of HO-1 and PEC AM-1 proteins were measured using ELISA.(4) Effects of transcription and phagocytosis inhibitors on ZnO-NP-induced HO-1 and PECAM-1 expression:A549 cells were pretreated with the transcription inhibitor Act D (1μg/ml) and phagocytosis inhibitor C B (10μg/ml) prior to ZnO-NP treatment. Levels of HO-1 and PECAM-1 in the basolateral chamber were determined as described previously.2. Direct effect of ZnO-NPs on HCAEC:HCAEC were treated with ZnO-NPs. Cytotocity, HO-1 and PECAM-1 levels were measured as described earlier.3. Determination of zinc levels:Zinc levels in culture medium and HEAEC were measured using flame atomic absorption spectrometry.4. The data were analysised by SPSS 12.0 using t-test and oneway-ANOVA to compare the difference of the two group and multi-groups; 2×2 Factorial Design Variance Analysis to compare the inhibitor effect. And the Significant level was a= 0.05.Results:Effect of ZnO-NPs on co-cultured HCAEC1. Cytotoxicity:ZnO-NPs treatment could inhibit A549 proliferation, and reduce their viability in a dose-dependent fashion. 2. Levels of HO-1 and PECAM-lin the supernatant:A549 cells were treated with 0-40μg/ml ZnO-NPs for 24 h. It was shown that levels of HO-1 and PECAM-1 in the basolateral chamber were significantly increased by ZnO-NP treatment compared to control (F=66.927, P<0.001; F=321.842, P<0.001).3. Zinc content:A549 cells were treated with 0-40μg/ml ZnO-NPs for 24 h. It was found that ZnO-NPs could increase both extracellular and intracellular zinc levels of HCAEC. At the doses of 20μg/ml and 40μg/ml, ZnO-NPs significantly increase zinc levels compared to control(F=93.884, P<0.001).4. Effect of Act D on ZnO-NP-induced expressions of HO-1 and PECAM-1: ZnO-NPs could significantly increase the expressions of HO-1 and PECAM-1 in the DMSO group (F=175.509, P<0.001; F=181.124, P<0.001). Pretreatment of A549 with Act D could significantly blocked ZnO-NPs-induced expressions of HO-1 and PECAM-1 in HCEAC compared to the ZnO-NPs in DMSO group (t=2.885, P=0.045; t=2.975, P=0.041).5. Effect of C B on expressions of HO-1 and PECAM-1:ZnO-NP stimulation could increase the expressions of HO-1 and PECAM-1 in the DMSO group (F=89.738, P<0.001; F=116.241, P<0.001). Pretreatment of A549 with C B markedly suppressed ZnO-NPs-induced HO-1 and PECAM-1 expressions in HCEAC compared to the ZnO-NPs in DMSO group (t=5.038, P=0.007; t=3.392, P= 0.027).Direct effect of ZnO-NPs on HCAEC1 Cytotoxicity:ZnO-NPs significantly inhibited HCAEC proliferation and cell viability in a dose-dependent fashion.2 HO-1 and PECAM-1 expression:HCAEC were treated directly with different concentrations (0μg/ml,10μg/ml,20μg/ml,40μg/ml) of ZnO-NPs for 24 h. It was shown that the expression levels of HO-1 and PECAM-1 were significantly increased by ZnO-NP stimulation in a dose-dependent fashion. At the doses of 20μg/ml and 40μg/ml, ZnO-NP stimulation significantly increased HO-1 and PECAM-1 production compared to control (F=145.228, P<0.001; F=295.535, P<0.001). 3 Zinc levels:Similarly, HCAEC were treated with different concentrations (0μg/ml,10μg/ml,20μg/ml,40μg/ml) of ZnO-NPs for 24 h. Levels of extracellular and intracellular zinc contents were significantly elevated compared to the control and in a dose-dependent pattern (F=12957.464, P<0.001; F=7021.460, P<0.001). The extracellular zinc levels were significantly higher than intracellular ones when the concentrations of ZnO-NPs were 10μg/ml and 20μg/ml (t=701.596,P<0.001;t=179.041,P<0.001), but not 40μg/ml(t=-0.411, P=0.702).Conclusions:1 ZnO-NP exposure could induce damage to both alveolar epithelial cells and arterial endothelial cells.2 ZnO-NP treatment could significantly increase the over-expression of HO-1 and PECAM-1, the biomarkers of atherosclerosis.3 ZnO-NPs might enter alveolar epithelial cells through phagocytosis, resulting in release of intermediates from A549 cells into the basolateral chamber and upregulation of HO-1 and PECAM-1 of HCAEC. This was supported by the evidence that ZnO-NP treatment could increase intracellular zinc levels, and that inhibition of transcription and phagocytosis of A549 cells significantly blocked ZnO-NP-induced HO-1 and PECAM-1 expression in HCAEC.4 Elevation of zinc levels in HCAEC might result from the penetration of ZnO-NPs through A549 cells, and the transpassed ZnO-NPs further entered into HCAEC. ZnO-NPs could increase the expressions of HO-1 and PECAM-1, and induce altheroslerotic lesions on the level of molecular biology.5 ZnO-NPs might induce atherosclerotic alterations through direct and indirect effect on HCAEC. |
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