Low-dose Nanoparticles Induce ROS Production In Vascular Endothelial Cells And Its Effect On The Integrity Of The Connection Of Endothelial Cells

Objective:The exposure of engineered nanoparticles is increasing due to the widespread R&D and applications of nanotechnology.With the increase in the exposure of nanoparticles,safety issues have attracted the attention of government regulators,scientific community,business community and the public.Studies have shown that when the exposure dose reaches a certain limit,nanoparticles could mediate the production of reactive oxygen species(ROS)in cells,causing oxidative stress to activate downstream signaling pathways,leading to apoptosis.On the wall of blood vessels,there is an endothelial layer composed of endothelial cells connecting each other,which is the most important biological barrier between blood and tissues to maintain the vascular function and homeostasis.The junctions of endothelial cells are involved in cell localization,proliferation,apoptosis and signaling.In particular,nanoparticles can enter into circulating blood not only by inhalation,but also by intravenous administration when used as therapeutics or contrasts,thereby gaining access to vascular endothelial cells.Also,in our previous research,silver nanoparticles(Ag-NPs)were taken up by endothelial cells,which upregulated the intracellular ROS and reduced the adherens junction.Hence the aim of this work is to figure out the effects of several common nanoparticles on the junctions of vascular endothelial cells and the intracellular ROS at low doses.The relationship between the intracellular ROS and vascular endothelial cell junctions was explored as well,which provide experimental research basis for the establishment of safety evaluation methods for nanoparticles.Methods:In terms of physical and chemical properties,the size and morphology of nanoparticles were observed by transmission electron microscopy(TEM),and the Zeta potential of nanoparticles was measured by dynamic light scattering(DLS).In the cell research,human umbilical vein endothelial cells(HUVECs)were used as the research object,and the cell viability of the nanoparticles after exposure for 24 h was determined by the CCK-8 method;The uptake of different nanoparticles by endothelial cells was observed by transmission electron microscopy.The effect of nanoparticles on the level of ROS in endothelial cells was observed by flow cytometry and fluorescent microscopy with 2,7,-Dichlorodihydrofluorescein diacetate(DCFH-DA)probe.CAT kit was used to analyze the effect of nanoparticles on the level of antioxidant enzymes in cells;Labeling endothelial cells with VE antibody,we observed changes in cell junctions by laser scanning confocal microscopy.Antioxidant N-acetylcysteine(NAC)was used to analyze the relationship between intracellular ROS levels and cell-cell junctions.Image J software was used to analyze the gap widths between cell junctions.By means of cross-monolayer movement of fluorescein isothiocyanate(FITC)-dextran,the changes of endothelial cell permeability were observed by laser scanning confocal microscopy.Finally,we used western blot to analyze the effects of different nanoparticles on the expression of VE-Cadherin and acetylation level of microtubules.Results:(1)In addition to the tubular structure of MWCNTs,several other nanoparticles were spherical particles and uniform in size.(2)Different nanoparticles had different effects on endothelial cell activity.Even with the same kind of nanoparticles,the effects of different size and surface modification on the toxicity of endothelial cells were also different.In the experimental concentration range,MWCNTs,Au-NPs and Dex-Fe2O3 had little effect on the vascular endothelial cell toxicity compared with the control group.The cell toxicity of Pt-NPs,SiO2-NPs,TiO2-NPs,P25-NPs and DMSA-Fe2O3 showed a significant concentration dependent effect.(3)The nanoparticles can be taken up by endothelial cells.Low dose exposure induced intracellular ROS production and increased catalase activity,resulting in the down-regulation of VE-Cadherin and tubulin acetylation.These effects eventually led to endothelial leakage.(4)When treated with antioxidant NAC,the intracellular ROS levels induced by nanoparticles were reduced,the destructive effect on cell junctions was significantly weakened,and the cell junctions became intact.Conclusion:It was found out that the nanoparticles of low dose exposure induced the ROS production and increased the catalase activity at the same time,which led to the downregulation of VE-Cadherin and acetylation of microtubules.These effects resulted in the gap formation among the endothelial cells and induced endothelial leakiness.In vivo application of nanoparticles,in addition to using conventional methods to detect its impact on cell activity,we should also focus on the role of reactive oxygen species and intercellular cell junctions in vascular endothelial cells.