Zinc Oxide Nanoparticles Disrupt Autophagy And Induce Toxicity In Human Keratinocytes By Perturbation Of NRF2 Signal Pathway

Object: Zinc oxide nanoparticles(ZnO NPs)are widely used in cosmetics and sunscreens.Increasing evidences indicate ZnO NPs induces unignorable harmful effects to human skin,but the underlying molecular mechanisms of the toxic effect of ZnO NPs remain poorly understood.Previous studies have indicated that oxidative stress and autophagy are involved in ZnO NPs-induced toxicity.Nuclear factor-E2-related factor 2(NRF2)is a key nuclear transcriptional factor for intracellular antioxidant reaction.NRF2 pathway has been recognized as an important toxicity pathway mediating chemical induced skin injury.The current study aims to investigate the possible involvement of the NRF2 mediated antioxidant pathway in human keratinocytes toxicity by ZnO NPs.Methods:1.The Shape and particle size of ZnO NPs was observed by transmission electron microscopy.The hydrodynamic diameter and Zeta potential of ZnO NPs were measured by dynamic light scattering;2.CCK-8 kit and LDH kit were used to detect the survival rate and LDH leakage rate of HaCaT cells exposed to ZnO NPs for 24 h;Flow cytometry was used to detect the effect of ZnO NPs on reactive oxygen species(ROS)and mitochondrial membrane potential(MMP)of HaCaT cells;The mRNA expression of P62 and LC3 II/I related to autophagy in HaCaT cells was detected by Real-time qPCR,and the protein expression of P62 and LC3 II/I was quantitatively analyzed by Western blot technology;3.The expression of downstream genes HO-1,GCLC and GCLM of NRF2 in HaCaT cells treated with ZnO NPs was detected by Real-time qPCR,and the expression of NRF2 and KEAP1 protein was detected by Western Blot technology;4.HaCaT cells were transduced by lentiviral transfection system to establish HaCaT cells with stable transfection and knockdown of NRF2,and the expression level of NRF2 protein was verified by Western Blot technology;5.Scramble and NRF2-KD Ha CaT cells were treated with ZnO NPs,then cell viability and the changes of ROS and MMP were detected.Quantitative analysis of autophagy-related gene expression in Scramble and NRF2-KD HaCaT cells by Real-time qPCR;6.Quantitative analysis of the mRNA of HO-1,GCLC and GCLM,the downstream antioxidant genes of NRF2 in Scramble and NRF2-KD HaCaT cells treated with ZnO NPs by Real-time qPCR;7.The ultrastructure of HaCaT cells treated with ZnO NPs in Scramble and NRF2-KD was examined by transmission electron microscopy,and the changes of autophagic lysosomes were observed;8.Cells were pretreated with 3-Methyladenine(3-MA),followed by treatment with ZnO NPs.Autophagy-related protein expression,cell survival rate,and intracellular ROS expression were detected.Results:1.Treatment of HaCaT cells with different concentrations of ZnO NPs for 24 h can induce cell viability reduction and LDH leakage.ZnO NPs affect cell viability in a doseand time-dependent manner,significantly increase ROS levels and cause mitochondrial membrane potential loss;ZnO NPs treatment increased NRF2 protein expression in HaCaT cells,and its downstream antioxidant genes were activated,such as HO-1,GCLC and GCLM;2.We showed NRF2 knockdown in NRF2-KD HaCaT cells measured by Western Blot,The cells viability of NRF2-KD Ha CaT cells treated by ZnO NPs was lower than Scramble cell.Compared with Scramble cells,NRF2-KD cells have significantly increased ROS production and decreased MMP after ZnO NPs exposure;TEM images of HaCaT cells treated with ZnO NPs for 6 h showed that a significant increase in the number of autophagic vacuole was observed in NRF2-KD cells compared with Scramble cells;3.ZnO NPs induced autophagy in Ha CaT cells,and the autophagy-related proteins LC3 II/I and P62 were increased.After adding the autophagy inhibitor 3-MA,the expression of autophagy-related proteins decreased.3-MA attenuated ZnO NPs-induced cytotoxicity but did not affect ROS accumulation in HaCaT cells.Conclusion:1.Exposure of HaCaT cells to ZnO NPs promoted the accumulation of ROS and depolarization of MMP,along with the nuclear translocation of NRF2 and reduction of KEAP1 protein levels,and increase of autophagy markers of LC3 II/I and P62.2.NRF2 silencing resulted in the cells more sensitive to ZnO NPs exporsure.NRF2 silencing sensized the ZnO NPs-induced oxidant damage and mitochondrial dysfunction and significantly enhance autophagy levels in HaCaT cells.Pre-treatment with the autophagy inhibitor 3-MA can reduce the cell death induced by ZnO NPs.3.Perturbation of the NRF2 pathway can regulate the toxic effects,oxidative stress and autophagy response of ZnO NPs-induced Ha CaT cells.