Stabilization Of Nrf2 Leading To Ho-1 Activation Protects Against Zinc Oxide Nanoparticles-induced Endothelial Cell Death

Objective : HUVEC(Human umbilical vein endothelial cells)and C57BL/6J mice were treated with Zn ONPs(Zinc oxide nanoparticles)to establish cell and animal oxidative stress models.Respectively,to explore the role and possible mechanism of Nrf2 degradation abnormality in HUVEC cell death induced by Zn ONPs,so as to better understand the related toxicity mechanism of Zn ONPs.Methods : HUVEC cells were treated with different concentrations of Zn ONPs(0,5,10,15,20 μg/m L).Respectively,The oxidative stress level of HUVEC cells was detected by flow cytometry,the survival rate of HUVEC cells was detected by MTT,and the expression level of Nrf2(NF-E2-related factor 2)was detected by Western blot to explore the effect of Zn ONPs treatment on HUVEC cells and the Nrf2 expression in cells.q RT-PCR(Quantitative Real-time Polymerase Chain Reaction),Western blot,si-RNA cell transfection,cell immunofluorescence,cytoplasmic and nuclear protein separation,and over-expression cell lines were used to explore the role and mechanism of Nrf2 degradation abnormality in HUVEC cell death induced by Zn ONPs.In the animal experiments,Zn ONPs(0.6 mg/kg)were intratracheally injected into mice in the experimental group,and PBS(Phosphate buffer saline)was intratracheally injected into mice in the control group.After 3 days,the abdominal aorta of mice was taken out,and the expression of Nrf2,HO-1(Heme oxygenase-1)and Ubiquitylation protein was detected by Western blot.Results : In the cell oxidative stress models,we detected a significant increase in the level of reactive oxygen species(ROS)in HUVEC cells,indicating that oxidative stress occurred in HUVEC cells,and activated the expression of Nrf2 and the antioxidant genes in the HUVEC cells,including HO-1,SLC7A11(recombinant Solute Carrier Family 7),NQO1(NADPH Quinone acceptor Oxidoreductase 1),GCLM(Glutamate-cysteine ligase),TXN(Thioredoxin).At the same time,Zn ONPs treatment also increased the ubiquitination level of HUVEC cells.In animal experiments,lung Zn ONPs exposure also induced the increase of Nrf2 and HO-1 expression and the increase of ubiquitination level.Through si RNA cell transfection,Western blot,MTT,cell immunofluorescence,flow cytometry and other experiments,we found that Nrf2 induced HO-1 activation played a key protective role in Zn ONPs induced HUVEC cell death,and Nrf2 activation may be due to the destruction of the ubiquitin-proteasome system by Zn ONPs,while the classical p62-Keap1 axis was not involved in the activation of Nrf2.