The Mechanism Of NLRP3 Inflammasome Activation Induced By ZnO Nanoparticle In Microglia Cells

Background:With the development of nanotechnology,the application of nanomaterials is increasing.ZnO nanoparticles have special physical and chemical properties,so they are widely used in food industry,cosmetics or other fields.Once ZnO nanoparticles enter the body,they can pass through the blood brain barrier and finally enter the brain.Thus,the exposure of ZnO nanoparticles may lead to inflammation of the nervous system and even cause neurodegenerative diseases.Inflammasome are thought to be associated with a variety of inflammatory diseases,including neurodegenerative diseases.At present,studies about the activation of inflammasome by ZnO nanoparticles are relatively few.Therefore,it is of great significance for the safety of nanoparticles to explore the effect of ZnO nanoparticles on the activation of inflammasome.In this study,microglia was used to analyze the effect of ZnO nanoparticles on the activation of NLRP3 inflammasome.We also explore its possible mechanism in order to provide research basis for the safe use of ZnO nanoparticles.Objective:1.To analyze the size,polydispersity index and zeta potential of ZnO nanoparticles.2.To study the effect of ZnO nanoparticles on the cell viability,LDH release and inflammatory factor secretion of BV2 cells,and to explore the effect of ZnO nanoparticles on the expression of NLRP3 inflammasome related proteins.3.To study the role of TLR4/My D88/NF-κB signaling pathway in the activation of NLRP3inflammasome induced by ZnO nanoparticles.4.To investigate the damage of lysosomes induced by ZnO nanoparticles and the effect of lysosomal damage induced by ZnO nanoparticles on the activation of NLRP3 inflammasome.Methods:1.Transmission electron microscope was used to analyze the morphology of ZnO nanoparticles.Malvern particle size meter was used to measure the average hydration particle size,polydispersity index and zeta potential of ZnO nanoparticles.2.After treatment of BV2 cells with ZnO nanoparticles（0,2.5,5.0,10.0,15.0,20.0μg/m L）for24h,the cell viability was measured by MTT.The level of LDH in cell culture supernatant was detected.The secretion levels of IL-1βand IL-18 in the supernatant of cell culture were detected by enzyme linked immunosorbent assay（ELISA）.The expression of NLRP3,pro-caspase-1,ASC and caspase-1 proteins were measured by Western blot.3.The expression of TLR4,My D88,NF-κB p65 and its phosphorylated protein in BV2 cells treated with ZnO nanoparticles（0,2.5,5.0,10.0,15.0μg/m L）was determined by Western blot.Setting control group,BAY11-7082 inhibitor group,LPS treatment group,LPS+Bay 11-7082 group,ZnO nanoparticle group,ZnO nanoparticle+Bay 11-7082 group,LPS+ZnO nanoparticle group,LPS+Bay 11-7082+ZnO nanoparticle group,the levels of IL-1βand IL-18 in the cell culture supernatant of each group were measured by ELISA,the expression levels of NF-κB p65 and its phosphorylated protein,NLRP3,pro-caspase-1 and ASC proteins in each group were measured by Western blot.4.After treatment of BV2 cells with ZnO nanoparticles（0,2.5,5.0,10.0,15.0μg/m L）for 24h,Lyso-Tracker Red was used to label lysosomes,and the staining results were observed by fluorescence microscope.The protein levels of LAMP1 and LAMP2 in each group were determined by Western blot.The protein expression of Cathepsin B in each group was determined by Western blot.Control group,CA-074 Me inhibitor group,ZnO nanoparticle group,ZnO nanoparticle+CA-074 Me group were established.The levels of IL-1βand IL-18 in the supernatant of cells in each group were determined by ELISA.The expression of caspase-1 in each group was determined by Western blot.Results:1.The images of TEM showed that the dispersion of ZnO nanoparticles was good.The average hydrated particle size of ZnO nanoparticles was（178.13±11.23）nm,zeta potential was（-8.23±2.12）MV,and PDI was 0.37±0.08.2.（1）Compared with the control group,the viability of BV2 cells decreased significantly in ZnO nanoparticles treated groups at the concentration of 15.0μg/m L and 20.0μg/m L,respectively（P<0.05）.The cell viability decreased with the increase concentration of ZnO nanoparticle（r=-0.814,P<0.05）.（2）Compared with the control group,the LDH of cell culture supernatant increased significantly in ZnO nanoparticles treated groups at the concentration of5.0μg/m L,10.0μg/m L and 15.0μg/m L,respectively（P<0.05）.The LDH increased with the increase concentration of ZnO nanoparticle（r=0.962,P<0.05）.（3）Compared with the control group,the levels of IL-1βand IL-18 in cell culture supernatant increased significantly in ZnO nanoparticles treated groups at the concentration of 2.5μg/m L,5.0μg/m L,10.0μg/m L and 15.0μg/m L,respectively（P<0.05）.There was a dose-response relationship between the levels of IL-1βand IL-18and the concentration of ZnO nanoparticles(r _IL-1β=0.919,P<0.05;r _IL-18=0.994,P<0.05).（4）Compared with the control group,the proteins expression of NLRP3 and caspase-1 increased significantly in ZnO nanoparticles treated groups at the concentration of 5.0μg/m L,10.0μg/m L and15.0μg/m L,respectively（P<0.05）.Compared with the control group,the proteins expression of ASC and pro-caspase-1 increased significantly at the concentration of 10.0μg/m L and 15.0μg/m L,respectively（P<0.05）.The proteins expression levels of NLRP3,pro-caspase-1,ASC and caspase-1increased with the increase of ZnO nanoparticles concentration(r _NLRP3=0.897,P<0.05;r_{pro-caspase-1}=0.767,P<0.05;r _ASC=0.751,P<0.05;r_capase-1=0.885,P<0.05).3.（1）The expression of NF-κB p65 phosphorylated protein and TLR4 protein was significantly increased（P<0.05）at the concentration of 10.0μg/m L and 15.0μg/m L.The expression of My D88protein was significantly increased（P<0.05）at the concentration of 2.5μg/m L,5.0μg/m L,10.0μg/m L and 15.0μg/m L.There were dose-response relationship between the protein expression of TLR4,My D88 and NF-κB p65 phosphorylated protein and the concentration of ZnO nanoparticles(r _TLR4=0.798,P<0.05;r _{My D88}=0.826,P<0.05;r_pp65=0.758,P<0.05).（2）The levels of IL-1βand IL-18,NF-κB p65 phosphorylation protein,NLRP3,ASC and pro-caspase-1 protein decreased after pre-treatment with BAY 11-7082（P<0.05）.4.（1）Compared with the control group,the number of lysosomes labeled by fluorescent probes decreased and the fluorescence intensity decreased in ZnO nanoparticles treated groups.（2）Compared with the control group,the expression of LAMP1 protein decreased significantly in ZnO nanoparticles treated groups at the concentration of 5.0μg/m L,10.0μg/m L and 15.0μg/m L,respectively（P<0.05）.The expression of LAMP2 protein decreased significantly at the concentration of 15.0μg/m L（P<0.05）.The protein expression level of LAMP1 and LAMP2 decreased with the increase of ZnO nanoparticles(r _LAMP1=-0.943,P<0.05;r _LAMP2=-0.791,P<0.05).（3）Compared with the control group,the expression of Cathepsin B protein decreased significantly in ZnO nanoparticles treated groups at the concentration of 10.0μg/m L and 15.0μg/m L,respectively（P<0.05）.The level of Cathepsin B increased with the increase of ZnO nanoparticles(r _{Cathepsin B}=0.801,P<0.05).（4）Compared with the ZnO nanoparticle group,the secretion of IL-1βand IL-18 and the expression of caspase-1 protein decreased in the ZnO nanoparticle group pre-treated with inhibitor CA-074 Me（P<0.05）.Conclusions:1.The ZnO nanoparticles can lead to the decrease of BV2 cell viability,the increase of LDH release and the increase of IL-1βand IL-18 secretion.The ZnO nanoparticles can activate NLRP3inflammasome.2.The ZnO nanoparticles can activate TLR4/My D88/NF-κB signaling pathway,and affect the activation of NLRP3 inflammasome through this pathway.3.The ZnO nanoparticles can induce lysosomal damage and release Cathepsin B in BV2 cells,and affect the activation of NLRP3 inflammasome through the release of Cathepsin B.