Proinflammation And Mechanisms Of ZnO Nanoparticles Transported Via Taste Nerve Pathway In Central Nervous System

BackgroundThe extensive biological applications of zinc oxide nanoparticles(ZnO NPs)in stomatology have created serious concerns about their biotoxicity,which provides a potential route for ZnO NPs to translocate into human body.In our previous study,ZnO NPs were confirmed to transfer to the central nervous system(CNS)via the taste nerve pathway after tongue instillation.Furthermore,NPs deposited into the brain induce oxidative damage and degenerate learning and memory,resulting in neurodegeneration.Increasing evidence shows that neurodegenerative diseases have close relationship with neuroinflammation.However,there are no relevant research to evaluate whether tongue-instilled NPs induce inflammatory responses in the CNS.ObjectiveTo evaluate whether tongue-instilled ZnO NPs induce inflammatory responses in the CNS and the molecular mechanisms underlying neuroinflammation were further elucidated by treating BV2 and PC 12 cells with ZnO NPs in vitro.Material and methods1.The characterization of ZnO NPs were determined by TEM,DLS,BET surface area measuring instrument and Raman spectrometry system.The levels of Zn in the blood or sub-brain regions were quantified by ICP-MS.Inflammatory responses in the rat brain were measured by qRT-PCR and ELISA.Morphological observation and immunohistochemical analysis of rat brain.2.MTT,LDH,TEM,qRT-PCR and ELISA were used to determin the toxic effect and inflammatory response in BV2 and PC 12 cells after treatment with ZnO NPs.Mechanism underlying ZnO NPs-induced inflammation was detected by calcium kit and western blot.3.A839977(a P2X7 receptor antagonist)and BAPTA-AM(an intracellular Ca2+chelator)were used to test the possibility that ZnO NPs-induced inflammation is mediated by calcium-dependent pathways.Results1.We obtained the model of tongue-instilled ZnO NPs and verified ZnO NPs can enter the rat brain via the taste nerve pathway and deposit in the sub-brain regions.2.ZnO NPs induced proinflammatory cytokines release,astrocytes and microglia activation,neuron damage,and cause neuroinflammation after transfer to rat brain.3.ZnO NPs induced Ca2+ influx,promoted the phosphorylation levels of NF-κB、ERK and p38,caused TNF-a and IL-1β release,which further contributed to the inflammatory response in BV2 and PC12 cells.4.BAPTA-AM decreased the concentration of Ca2+,prevented the NF-κB,ERK and p38 activation,proinflammatory gene upregulation,TNF-a and IL-1βrelease and cell viability decrease induced by ZnO NPs.The level of neuroinflammation was blocked by BAPTA-AM confirmed the Ca2+ increase is essential for ZnO NPs-induced neuroinflammation.ConclusionIn summary,this study demonstrated that ZnO NPs can be transported to the brain via the taste nerve after tongue instillation and induce glial cell activation and inflammatory responses in the CNS.Moreover,ZnO NPs induce neuroinflammation via the Ca2+-dependent NF-κB,ERK and p38 activation pathways was verified at the cytological level.