Study On The Mechanism Of Taste Disorder Caused Via Inflammatory Induced By Tongue-Brain Transport Of ZnO Nanoparticles Into Brain

BackgroundIn recent years,zinc oxide nanoparticles(ZnO NPs)have been widely used in dental field due to their excellent biological characteristics.However,ZnO NPs can be transported into the brain by oral administration,intranasal inhalation and intraperitoneal injection,which has potential central nervous toxicity,and its toxic effect has gradually attracted people’s attention.Studies have shown that intranasal inhalation or intranasal drip of ZnO NPs can be transported into the brain via olfactory-brain pathway,causing tissue inflammation,resulting in olfactory dysfunction.Previous studies in our group confirmed that ZnO NPs could be transported into the brain through the tongue-brain pathway after tongue instillation,causing inflammation in brain tissue.Thus,after long-term exposure to oral tissues,ZnO NPs are transported into the brain via tongue-brain pathway,causing inflammation damage of brain tissue,will it lead to taste disorder?There is no report yet.ObjectionZnO NPs is widely used in the field of stomatology,and its biological safety evaluation is particularly important.In this study,different doses of ZnO NPs suspension were dripped into the tongue to explore whether ZnO NPs could be transported into the brain through the tongue-brain pathway,causing brain inflammation,leading to taste disorder in rats,and to elucidate the mechanism of taste disorder in rats induced by ZnO NPs.The level of inflammation damage in brain tissue was preliminarily assessed at molecular,gene and protein levels.Animal behavioral experiments were used to analyze rat taste learning and taste recognition.Depend on the results of inflammatory factor protein chip,and combined the in vivo and in vitro experiments,the mechanism of ZnO NPs induced inflammatory reaction and taste disorder caused by inflammatory reaction were deeply analyzed.It provides a reference for the safe application of ZnO NPs in dental field.Materials and Methods1.ZnO NPs were characterized by Transmission Electron Microscope(TEM),Energy Dispersive Spectromete(EDS),X-Ray Diffraction(XRD),Raman spectroscopy,Zeta pontential and Dynamic light scattering(DLS).2.Different doses of ZnO NPs suspension were dripped into the tongue for 30 days,and the animal model of ZnO NPs transported into the brain via tongue-brain pathway was successfully constructed.Inductively coupled plasma mass spectrometry(ICP-MS)was used to detect the content of elements in tongue,nerve and brain tissues of rats.TEM was used to observe the ultrastructure of brain tissues and analyze the distribution of ZnO NPs in brain tissues.3.Animal behavioral experiments were conducted to analyze the effects of ZnO NPs transported into the brain on taste behavior and whether there were taste disorders in rats.Two-bottle preference test(TBP)was used to analyze the sensitivity of rats to acid,sweet,bitter and salty taste,and to evaluate their taste cognitive function.Conditional taste aversion(CTA)test was used to analyze the learning ability of taste aversion in rats and evaluate their taste learning function.Preliminary assessment of brain inflammation injury level was made by screening brain inflammatory factor protein chip.4.Combining in vivo and in vitro experiments,by immunohistochemistry,TEM observation,ELISA,qRT-PCR and Western blot detection,we can accurately assess the level of inflammation damage in rat brain tissue,deeply analyze the relationship between inflammation of brain tissue and JAK-STAT signaling pathway,and clarify the mechanism of inflammation induced by nano-zinc oxide.5.Combining in vivo and in vitro experiments,the expression level of synaptophysin was evaluated by Immunofluorescence and Western blot.Combined with the expression of c-fos,JAK-STAT signaling pathway was further analyzed to regulate neurexin 1-PSD95-Neuroligin 1 signaling pathway,and to elucidate the mechanism of taste disorder caused by inflammation induced by ZnO NPs.Results1.Characterization results show that ZnO NPs suspension has high purity,no impurities and 50 nm particle size.The prepared ZnO NPs suspension has uniform dispersion and no obvious agglomeration or precipitation.2.The animal model of ZnO NPs transported into brain via tongue-brain pathway was successfully constructed.TEM results showed that ZnO NPs transported into the brain was scattered around synapses in the cerebral cortex,which may cause synaptic damage.3.The sensitivity of rats to acid,sweet,bitter and salty taste decreased in high-dose ZnO NPs group and medium-dose ZnO NPs group.The learning ability of taste aversion of rats in high dose ZnO NPs group,middle dose ZnO NPs group and low dose ZnO NPs group decreased significantly.Inflammatory factor protein microarray results showed that taste disorder in rats may be related to brain inflammation,and JAK-STAT signaling pathway may play an important role in it.4.Immunohistochemical staining showed slight damage to the morphology and structure of rat cerebral cortex,decrease of neurons,increase of expression of inflammatory factors TNF-α and IL-1β.TEM was used to observe the changes of cerebral cortex ultrastructure.ELISA and qRT-PCR showed that inflammation occurred in cerebral cortex and PC 12 cells,and inflammatory factors TNF-α,IL-1β,IL-4,IL-6,IL-13 and MCP-1 were released.Western blot analysis showed that the inflammation of cerebral cortex and PC 12 cells was related to the activation of JAK-STAT signaling pathway.Inhibiting this pathway could significantly inhibit the release of inflammatory factors,suggesting that ZnO NPs was an inflammatory response induced by activating JAK-STAT signaling pathway.Western blot was used to detect the expression of JAK-STAT signaling pathway protein in cerebral cortex and PC 12 cells.It was found that the phosphorylation levels of JAK2 and STAT3 increased,suggesting that JAK2-STAT3 signaling pathway was activated.In addition,inhibiting this pathway could significantly inhibit the release of inflammatory factors,suggesting that the inflammatory response caused by ZnO NPs was induced by activating JAK-STAT signaling pathway.5.Western blot was used to detect the expression of Neurexin 1-PSD95-Neuroligin 1 signaling pathway in cerebral cortex.Neurexin 1,PSD95 and Neuroligin 1 were down-regulated,suggesting that Neurexin 1-PSD95-Neuroligin 1 signaling pathway was down-regulated in inflammatory brain tissue.Cell immunofluorescence showed that PC 12 cells stimulated by LPS and treated with ZnO NPs showed swelling,morphological changes,and down-regulation of PSD95 expression in cytoplasm.Western blot results showed that the expression of Neurexin 1,PSD95 and Neuroligin 1 in PC12 cells was down-regulated after LPS-induced inflammation and ZnO NPs treatment,suggesting that the expression of Neurexin 1-PSD95-Neuroligin 1 signaling pathway was related to inflammation induced by JAK2-STAT3 signaling pathway.Inhibiting JAK2-STAT3 signaling pathway could significantly alleviate the release of inflammatory factors,thereby alleviating the down-regulation of Neurexin 1-PSD95-Neuroligin 1 expression and promoting the expression of immediate early gene c-fos.It further suggested that the down-regulation of Neurexin 1-PSD95-Neuroligin 1 expression was related to the taste disorder caused by ZnO NPs.ConclusionZnO NPs were dripped into the tongue of Wistar rats for 30 days.It could be transported into the brain through the tongue-brain pathway and deposited in the brain tissue,which has potential central neurotoxicity.Tongue-brain transport of ZnO NPs can cause impairment of taste learning and taste cognitive function in rats,which may be related to brain inflammation caused by ZnO NPs.ZnO NPs can induce histopathological changes of cerebral cortex,neuron cell damage,brain inflammation damage,and changes in ultrastructure of brain tissue in rats.It is suggested that ZnO NPs has central neurotoxicity.Furthermore,ZnO NPs can activate JAK-STAT signaling pathway in rat brain tissues and PC 12 cells,and cause a large number of inflammatory factors to release,and then down-regulate the expression of Neurexin 1-PSD95-Neuroligin 1,resulting in synaptic dysfunction,impaired taste and cognitive function,eventually leading to taste disorders.Inhibiting JAK-STAT signaling pathway can effectively alleviate inflammation induced by ZnO NPs,thereby alleviating down-regulation of Neurexin 1-PSD95-Neuroligin 1 expression,promoting immediate early gene expression of taste and improving taste disorder.