The Role Of Autophagy In The Inflammatory Response Of A549 Cells Induced By Zinc Oxide Nanopaticles

Background and ObjectiveThe wide application of zinc oxide nanoparticles(ZnO-NPs)greatly increases the exposure opportunities of occupational employees.ZnO-NPs can enter cells and destroy the cell structure even induce cellular inflammatory reactions through respiratory system.Autophagy is a way for cells to maintain homeostasis,which can protect cells by degrading damaged proteins,organelles.To investigate whether ZnO-NPs can initiate cell autophagy we stimulated A549 cells with ZnO-NPs in different concentrationss.We regulated autophagy with pharmaceutical chemistry to investigate the role of autophagy in ZnO-NPs-induced inflammation.Therefore we can have a solide foundation on respiratory toxic effects caused by ZnO-NPs.Method1.A549 cells were treated with ZnO-NPs at concentrations of 0,5,10,20,40 mg/L.The activity of A549 cells were determined by CCK-8 and the release of detected by lactate dehydrogenase(LDH)was detected with LDH kit.Inflammatory factors IL-6 and TNF-? in cell supernatants was determined by enzyme-linked immunosorbent assay kit(ELISA).2.A549 cells were treated with ZnO-NPs at different concentrations of 0,5,10,15,20,25,30 mg/L for 24 h,and the expression levels of autophagy-related proteins LC3 B and P62 were detected by Western Blot.The expression levels of LC3 B and P62 were determined after treated with 20mg/L ZnO-NPs for 0,4,8,12,24,and 48 h.The morphology of autophagosomes in cells was observed by transmission electron microscopy(TEM)after stimulation with 20 mg/L ZnO-NPs for 24 h.3.Before treated with 20 mg/L ZnO-NPs cells were pretreated with autophagy inhibitor trimethyladenine(3-MA)and activator rapamycin(RAPA)for 1 h.Detect autophagy-related protein LC3 B,P62 expression levels,cell activity,LDH release and IL-6,TNF-? expression levels after 24 h.Results1.The effect of ZnO-NPs on cell viability of A549 cellsThe cell viability decreased depended on the ZnO-NPs dose.When the concentration of ZnO-NPs was smaller than 10 mg/L,there was no significant change in the viability of A549 cells.When the concentration of ZnO-NPs reached 20 mg/L,the cell viability decreased to 63.38 %.2.The effect of ZnO-NPs on inflammation level of A549 cellsWith the increased ZnO-NPs dose,the release of cytokine in the cell supernatant showed increasing trend.When the concentration of ZnO-NPs was thiner than 5 mg/L,there was no significant change in the release of cytokine in A549 cells.When the concentration of ZnO-NPs reached 10 mg/L,the level of released LDH and IL-6 in the supernatant increased significantlycompared with the control group(P<0.05).While the expression of TNF-? in the supernatant increased significantly when the concentration of ZnO-NPs heavier than 20 mg/L.3.Effect of ZnO-NPs on autophagy level of A549 cellsThe level of basal autophagy in the cells was low in normal condition.After stimulation with 20 mg/L,25 mg/L and 30 mg/L ZnO-NPs,the level of LC3 BII was significantly higher than that of the control group(P<0.05).Compared with the control group,LC3 II protein was higher in the 24 h group and the 48 h group increased significantly(P<0.05).Transmission electron microscopy showed that the number of autophagy bodies increased significantly after stimulation with 20 mg/L ZnO-NPs.4.Different effects of different autophagic levelsThe cells were devided into four groups including blank control group,ZnO-NPs group,pharmaceutical control group and pharmaceutical + ZnO-NPs group.The examinations were conducted after treated for 24 h.4.1 Inhibition of autophagyCompared with the control group the level of autophagy in the ZnO-NPs and 3-MA+ZnO-NPs group was significantly higher.Compared with the ZnO-NPs group,the cell viability of the 3-MA+ZnO-NPs group increased while the release of LDH,IL-6 and TNF-? decreased significantly(P<0.05).4.2 Activation of autophagyCompared with the control group,the autophagy level of ZnO-NPs group and RAPA + ZnO-NPs group were significantly higher.Compared with the ZnO-NPs group,IL-6 of RAPA + ZnO-NPs group increased significantly(P<0.05).While cell viability,the release of LDH,TNF-? did not change significantly.Conclusion1.ZnO-NPs can induce inflammatory and autophagy in A549 cells.2.Inhibition of autophagy can attenuate the inflammatory response of A549 cells induced by ZnO-NPs while activation of autophagy cannot influence the ZnO-NPs induced inflammation.