The Role Of Mitophagy In The Activation Of NLRP3 Inflammasome Induced By Nano-Zinc Oxide

Background and ObjectiveZinc oxide nanoparticles(ZnO-NPs)is a new type of multifunctional inorganic material,which has many excellent special properties not available in macroscopic materials.Therefore it is widely used in many fields of industry and life,such as ceramics,textiles,cosmetics,paints and coatings,chemical raw materials and other industries.The wide application of ZnO-NPs increases the exposure opportunities of population greatly.Respiratory inhalation is the main exposure route.Due to its small particle size,the main target organ after exposure to ZnO-NPs through the respiratory tract is the lung.It can enter the deep part of the alveoli and cause a series of damage to the lung,including oxidative stress and inflammatory reactions.NLRP3 inflammasome is a intracellular protein complexe that play a critical role in the inflammatory response process.The activation of NLRP3 inflammasome can induce the maturation and secretion of inflammatory factors such as IL-1?,and play an important role in the inflammation process.Mitophagy maintains intracellular homeostasis by degrading damaged mitochondria.It can affect mitochondrial signaling pathways and regulate oxidative stress and inflammatory responses.We aimed to investigate the effect of ZnO-NPs on mitophagy and the activation of NLRP3 inflammasome in A549 cells,and the relationship between mitophagy and the activation NLRP3 inflammasome.We explored the effects of different concentrations of ZnO-NPs on mitophagy and the activation of NLRP3 inflammasome in A549 cells.And we explored the relationship between mitophagy and the activation NLRP3 inflammasome by using the inhibitor of mitophagy and NLRP3 inflammasome.It is expected to provides an in-depth theoretical basis for studying the toxicity of respiratory system induced by ZnO-NPs.Method1.A549 cells were stimulated with ZnO-NPs suspensions with different concentrations(0,5,10,15,20,25,30,35,40?g/mL)for 24 hours,and the activity of A549 cells was detected with the CCK-8.The exposure concentration was determined according to the result of CCK-8.2.A549 cells were stimulated with ZnO-NPs suspensions with different concentrations(0,5,10,15,20 ?g/mL)for 4,8,12 h.The intracellular reactive oxygen species(ROS)were detected with DCFH-DA fluorescent probes.And the mitochondrial membrane potential(MMP)were measured using rhodamine 123 dye.3.A549 cells were treated with ZnO-NPs suspensions at different concentrations(0,5,10,15,20 ?g/mL)for 24 h.The expression levels of mitophagy-related proteins,such as LC3B,PINK1,and Parkin,were detected by Western Blot.Transmission electron microscopy was used to observe the occurrence of mitophagy in A549 cells after stimulation with 20 ?g/mL ZnO-NPs suspension.The proteins expression levels of NLRP3 and Caspase-1 p20 were detected by Western Blot.The expression level of inflammatory cytokine(IL-1?)in A549 cell supernatant was detected by Enzyme Linked Immuno-sorbent Assay(ELISA).4.We used inhibitors to explore the relationship between mitophagy and the activation of NLRP3 inflammasome induced by ZnO-NPs.Before treated with 20?g/mL ZnO-NPs,cells were pretreated with ROS scavenger NAC,mitophagy inhibitor cyclosporine(CsA),and the inhibitor of NLRP3 inflammasome glibenclamide(GEL)for 1 h,respectively.Detect the expression levels of mitophagy-related proteins(LC3B,PINK1,and Parkin),NLRP3-related proteins(NLRP3 and Caspase-1 p20)and inflammatory cytokine(IL-1?)after 24 h.Results1.The effect of ZnO-NPs on cell viability of A549 cells The cell viability decreased with the exposure concentration of ZnO-NPs.When the ZnO-NPs concentration reached 20 ?g/mL,the cell viability decreased to 54.60%.2.The effect of ZnO-NPs on oxidative stress in A549 cells When the exposure dose was the same,the expression levels of ROS were increased with the exposure times,and the differences were statistical significance(P<0.05).At the same exposure time,the expression levels of ROS were increased with the exposure dose,and the differences were statistical significance(P<0.05).3.The effect of ZnO-NPs on MMP in A549 cells When the exposure dose is the same,the MMP was decreased with the exposure time,and the differences were statistical significance(P<0.05).At the same exposure time,MMP was decreased with the exposure dose,and the differences were statistically significant(P<0.05).4.The effect of ZnO-NPs on mitophagy and activation of NLRP3 inflammasome in A549 cells4.1 The effect of ZnO-NPs on mitophagy in A549 cells Compared with the control group,the expression levels of LC3-?,PINK1 and Mito-Parkin proteins were increased with the the exposure dose(P<0.05)after stimulating with different concentrations(0,5,10,15,20 ?g/mL)of ZnO-NPs for 24 hours.After stimulation with 20?g/mL ZnO-NPs,transmission electron microscopy revealed that the number of swelling mitochondria and mitophagy lysosomes were increased.4.2 The effect of ZnO-NPs on the activation of NLRP3 inflammasome in A549 cells Compared with the control group,the expression levels of NLRP3 and Caspase-1 p20 proteins were increased with the the exposure dose(P<0.05)after stimulating with different concentrations(0,5,10,15,20 ?g/mL)of ZnO-NPs for 24 hours.When the concentration of ZnO-NPs reached 5?g/mL,the level of released IL-1? in the supernatant increased significantly in comparison with the control group(P<0.05).5.The relationship between mitophagy and the activation of NLRP3 inflammsome The cells were divided into eight groups including blank control group,ZnO-NPs group,NAC group,CsA group,GEL group,ZnO-NPs+NAC group,ZnO-NPs+CsA group,and ZnO-NPs+GEL group.The examinations were conducted after stimulated for 24 h.5.1 The effects of NAC on mitophagy and the activation of NLRP3 inflammasome Compared with the control group,the levels of mitophagy and activation of NLRP3 inflammasome were significantly increased,and the expression of IL-1? in the cell culture supernatant was increased in the ZnO-NPs group.Compared with the ZnO-NPs group,the level of mitophagy and NLRP3 the activation of NLRP3 inflammasome were significantly reduced,and the expression of IL-1? was decreased(P<0.05)in the ZnO-NPs+NAC group.5.2 The effect of CsA on mitophagy and the activation of NLRP3 inflammasome Compared with the control group,the level of mitophagy and the activation of NLRP3 inflammasome were significantly increased,and the expression of IL-1? in the cell culture supernatant was increased in the ZnO-NPs group.Compared with the ZnO-NPs group,the level of mitophagy and the activation NLRP3 inflammasome were significantly reduced,and the expression level of IL-1? was decreased(P<0.05)in the ZnO-NPs+CsA group.5.3 The effect of GEL on mitophagy and the activation of NLRP3 inflammasome Compared with the control group,the level of mitophagy and the activation of NLRP3 inflammasome were significantly increased,and the expression of IL-1? in the cell culture supernatant was increased in the ZnO-NPs group.Compared with the ZnO-NPs group,the activation of NLRP3 inflammasome was significantly reduced,and the expression of IL-1? was reduced(P<0.05)in the ZnO-NPs+GEL group.But the level of mitophagy was not significantly changed.Conclusion1.ZnO-NPs can induce mitophagy and the activation of NLRP3 inflammasome in A549 cells.2.Eliminating intracellular ROS can inhibit the activition of mitophagy and NLRP3 inflammasome induced by ZnO-NPs.3.Mitophagy-related signals may play a regulatory role upstream of NLRP3 inflammasome.