Zinc Oxide Nanoparticle Induces Inflammatory Response Via P38 JNK MAPK And JAK2/STAT3 Signaling In BV2 Microglia

With the development of nanotechnology,zinc oxide nanoparticles have been widely used in various fields due to their excellent physicochemical properties,and the chances of human exposure to ZnO NPs has greatly increased.Because of its small size and unique physicochemical properties,nanoparticles are easily to cross multiple biological barriers.ZnO NPs enteres to the brain through the olfactory bulb-cerebral transport pathway and the blood-brain barrier,which accumulates in the brain and may lead to Nerve cells damage and permanent brain damage.Microglia thought to be a "macrophage" in the central nervous system and are considered as the resident immune cells of the central nervous system(CNS).Microglia are particularly sensitive to the change of their microenvironment and readily activated by outside stimulation or injury on brain.The activated microglial cells present antigens and secretory cytokines,which form the first line of defense against the invasion of pathogens in the central nervous system.Therefore,it is important to evaluate the inflammatory response of microglia and study its inflammatory mechanism.It will be a critical role in biological safety assessment of ZnO NPs.In this study,BV2 macroglia were exposure to ZnO NPs and the inflammatory response and relative mechanisms of inflammation were explored in vitro.All of these results will provide sensitive indicators and theory evidence for ZnO NPs evaluation.The experimental scheme is constructed with three parts as follows.Part I Characterization of Nano-zinc oxidePurposesCharacterization of the nano-zinc oxide to provide a basis for the following study of ZnO NPs.MethodsThe particle size and shape of ZnO NPs particles were observed by TEM;the Zeta potential and the hydration particle size of nano-particle suspensions were measured using a Malvern particle size analyzer.ResultsThe TEM observation shows that the ZnO NPs has a spherical shape with uniform dispersion with some particles agglomerate.The average particle size is about 61.84±18.51 nm.The average hydrated particle size of the dispersed ZnO NPs in the medium was220.83±9.11 nm,the Zeta potential was-17.87±0.58 mV,and the dispersion coefficient PDI was 0.11±0.074.Part II Effect of Nano-Zinc Oxide on Cytokines Secretion of Microglial cellsPurposesTo investigate the effects of ZnO NPs on inflammatory cytokines expression.MethodsBV2 microglia were treated with different concentrations of ZnO NPs(5.0,10.0,15.0,20.0,30.0,50.0,75.0μg/mL)for 24 h.Control group was treated without intervention.Cell viability was assessed by MTT assay at the different ZnO NPs concentration.BV2 microglia was intervened with ZnO NPs(5.0,10.0,15.0,20.0μg/mL).The lactate dehydrogenase was detected by colorimetry method to examine integrity of BV2 membrane.Inflammatory response of BV2 cells to ZnONPs exposure was further evaluated by the secretion of typical pro-inflammatory cytokine TNF-α,IL-1β and IL-6.The secretion level of pro-inflammatory cytokines TNF-α,IL-1β and IL-6 was examined using specific ELISA kits.Results1.ZnO NPs treatment caused a significant decrease the viability of BV2 cells in BV2microglia(P<0.05).2.ZnO NPs treatment caused a significant increase in the secretion of lactate dehydrogenase in BV2 microglia(P<0.05).3.The results of enzyme-linked immunosorbent assay demonstrated that with increasing doses of ZnO NPs the secretion of inflammatory mediator THF-α,IL-1β and IL-6 increased(P<0.05).Part III Effect of Nano-Zinc Oxide on p38 JNK MAPK,JAK2/STAT3 Signaling PathwayPurposeTo investigate the effects of p38,JNK MAPK and JAK2/STAT3 signaling pathways in the secretion of inflammatory cytokines in BV2 macroglia by ZnO NPsMethods1.The protein levels of phosphorylation of mitogen-activated protein kinases p-38,JNK,JAK2 and STAT3 of different concentrations of ZnONPs(0,5.0,10.0,15.0,20.0μg/mL)and different periods(0,0.5,1,1.5,3h)in same concentration was detected by Western Blotting.2.Image J was used to analyze the gray value of the protein band.The relationship between the secretion of inflammatory factors and the level of phosphorylation of mitogen-activated protein kinases p-38,JNK,JAK2,and STAT3 protein was analyzed by correlation analysis.3.The experimental groups divided into Control group(without any treatment),inhibitor control group,ZnO NPs treatment group,SB203580 inhibitor + ZnO NPs co-treatment group,SP600125 inhibitor group + ZnO NPs co-treatment group and AG490 +ZnO NPs co-treatment group.Western Blot assay was used to determine the changes of p38 MAPK,p-p38 MAPK,JNK,p-JNK,JAK2,STAT3,p-JAK2,and p-STAT3 proteins in each group.Results1.The photos of WB show that compared to the control phosphorylation of mitogen-activated protein kinases p38 MAPK,JNK,JAK2,and STAT3 was increased by nano-ZnO treatment.The gray ratios of p-p38MAPK/p38 MAPK,p-JNK/JNK,p-JAK2/JAK2,and p-STAT3/STAT3 were gradually increased(P<0.05)and have significant difference in comparison to control group,and the expression of phosphorylation of p38 MAPK,JNK,JAK2,and STAT3 was consistent with proinflammatory mediators secretion.2.The results of WB show that with the increase of exposure time,the protein levels of the phosphorylation of p38 MAPK,JNK,JAK2,and STAT3 increased in a certain ammount of time(P<0.05).Additionally,the expression level of p-JNK was the most obvious at 1.5 h.The phosphorylation of p38 MAPK began to change at 1.5 h.The expression levels of p-JAK2 and p-STAT3 proteins began to change after nano-zinc oxide intervention for 1 h,and the expression level of the phosphorylation of JAK2 was the most obvious at 1.5 h,compared with the control group,by STATistical analysis,all P< 0.05,significant difference.3.Comepared with ZnONPs treatment group,SB203580 and SP600125 can effectively inhibit the protein level of the phosphorylation of p38 MAPK and JNK induced by nano-zinc oxide,respectively,with STATistical significance(P<0.05);JAK2 inhibitor(AG490)can inhibit p-JAK2 protein in BV2 macroglia induced by ZnONPs as well as can inhibit the expression of p-STAT3 in its downstream pathway.4.SB203580,SP600125 and AG490 can inhibit the secretion of inflammatory cytokines in BV2 cells induced by ZnO NPs(P<0.05).Conclusion:ZnO NPs induced the increase of inflammatory cytokines and the expression of mitogen-activated protein kinases p38 MAPK,JNK,JAK2,and STAT3 phosphorylation in BV2 microglia.The p38 MAPK,JNK,and JAK2/STAT3 signaling pathways play an important role in the secretion of inflammatory cytokines in BV2 cells induced by ZnO NPs.