The Effect And Mechanism Of MK801 And Taurine Protect Against 1-BP Induced Neurotoxicity

1 BackgroundChlorofluorocarbons(CFCs)has been implicated in the depletion of the stratospheric ozone layer,which can also increase the incidence of cataract formation and skin cancer.As an alternative to ozone-depleting solvents(ODS),1-bromopropane(1-BP;CAS No.106-94-5)is widely used in industry,such as spray adhesives,cleaning metal or electronic components cleaning,and as dissolving fats,waxes,or resins.With the expanded application,the adverse effects of 1-BP exposure have gained more attention.Studies in experimental animals and occupational workers revealed the serve toxic effects of 1-BP in central nerve system(CNS).We recently reported that 1-BP poisoning in rat result in learning and memory deficits and hippocampal neuron damage.Occupational workers with exposure to 1-BP displayed depression,anxiety,reduced short-term memory,headaches,confusion and loss of consciousness.Cognitive impairments occur in 1-BP-posioned patients,which are gradually recognized to be key factors to decrease their life quality.However,the therapeutic strategies aimed at blocking 1-BP-induced cognitive impairments and related neurodegeneration remain to be investigated.Accumulating evidence suggests that the activation of NMDA receptors is involved in the cognitive impairments in neurodegenerative diseases.The purpose of this study is aimed to investigate whether inhibition of NMDA receptors by MK801 protects against 1-BP-induced cognitive dysfunction.According to the levels of five amino acids including Asp,Tau,GABA,Glu and Gly in hippocampus and cerebral cortex of 1-BP-intoxicated rats,we choose Taurine to observe its reverse effects of 1-BP induced central nervous system toxicity and mechanism.2 Methods2.1 MK801 protect against 1-BP-induced neurotoxicology2.1.1 Animal treatmentAfter 5 days of acclimatization to the new environment,the rats were randomly divided into four groups(n=12 animals in each group)as follows:control group,1-BP group,MK801+1-BP group,MK801 group.1-BP group were gavage at dose of 800mg/kg body weight(dissolved in corn oil)for consecutive 14 days.In MK801+1-BP group,rats were injected intraperitoneally(i.p.)with MK801(0.1mg/kg body weight)60min prior to given 1-BP.2.1.2 Morris water MazeThe rats were tested the cognitive dysfunction by Morris Water Maze(MWM)test from experimental days 14-20.Eight rats in each group were chosen randomly to sacrifice by euthanasia.The cerebella were dissected out,then the cerebral cortex and hippocampus were immediately separated on the ice,frozen in liquid nitrogen and stored at-80℃ until use.The remaining rats in each group were performed perfusion fixation for morphologic detection.2.1.3 Analysis of amino acids:HPLC was used to test the alteration of brain levels of amino acids includingγ-amino butyric acid(GABA),aspartate(Asp),Glutamate(Glu),glycine(Gly),taurine(Tau)in cerebral cortex and hippocampus.2.1.4 Histological and ImmunohistochemistryAnalysis Brain frozen coronal slices(40μm)including cerebral and hippocampus were cut on a freezing microtome and mounted to the Thermo super frost plus slide.2.1.4 For each rat,in the same part of cerebral cortex and hippocampus,every two series of adjacent coronal sections per rat were selected for Thionin staining,NeuN and microglia immunostaining.2.1.5 Western BlottingThe expression of NMDAR subunits including GluNl,GluN2A,GluN2B;and inflammation related proteins including NLRP3,caspasel,IL-1β in rats’ brains were investigated by Western Blotting.2.2 Taurine protect against 1-BP-induced neurotoxicology2.2.1 Animal treatmentAfter 5 days of acclimatization to the new environment,the rats were randomly divided into five groups(n=12 animals in each group)as follows:control group,1-BP group,Taurine+l-BP group(100mg/kg.bw),Taurine+l-BP group(200mg/kg.bw),Taurine group(200mg/kg.bw).1-BP group were gavage at dose of 800mg/kg body weight(dissolved in corn oil)for consecutive 14 days.2.2.2 Behaviors testsThe rats were tested the cognitive dysfunction by Morris Water Maze(MWM)test from experimental days 14-20.Eight rats in each group were chosen randomly to sacrifice by euthanasia.The cerebella were dissected out,then the cerebral cortex and hippocampus were immediately separated on the ice,frozen in liquid nitrogen.The remaining rats in each group were performed perfusion fixation for morphologic detection.2.2.3 The level of taurineHigh performance liquid chromatography(HPLC)was used to test the alteration of brain level of taurine(Tau)in cerebral cortex and hippocampus.2.2.4 Histological and Immunohistochemistry AnalysisBrain frozen coronal slices(40μm)including cerebral and hippocampus were cut on a freezing microtome and mounted to the Thermo super frost plus slide.For each rat,in the same part of cerebral cortex and hippocampus,every two series of adjacent coronal sections per rat were selected for Thionin staining,NeuN immunostaining.2.2.5 The level of MDAThe level of MDA in rats’ brains were tested by MDA assay Kit.2.2.6 Western Blot AnalysisThe expression of ASK1,P-ASK1,P38,P-P38,and apoptosis related proteins including Bcl-2,BAX,caspase-3 in rats’ brains were investigated by Western Blotting.2.3 Statistical AnalysisAll group data were presented as mean ± SD and analyzed statistically using SPSS 23.0 statistical software.The data obtained from MWM test and body weight were analyzed by repeated-measures analysis of variance(ANOVA).Other data were compared using one way-ANOVA,following by LSD post hoc test.P<0.05 was considered statistically significant.3 Results3.1 MK801 protect against 1-BP-induced neurotoxicologyWe found that rats exposed to 1-BP displayed reduced spatial learning and memory abilities,which was associated with neurodegeneration in both hippocampus(especially CA1 and CA3 sub-regions)and cortex.Taurine ameliorated 1-BP-induced cognitive impairments and loss of hippocampal and cortical neurons.Mechanistically,MK801 abrogated 1-BP-induced disruption of excitatory and inhibitory amino acids balance and expressions of NMDA receptors subunits including GluN1,GluN2A and GluN2B in hippocampus and cortex of rats,indicating inhibition of NMDA receptors by MK801.Subsequently,MK801 was shown to be able to inhibit microglial activation and release of proinflammatory cytokines in 1-BP-treated rats.Finally,NLRP3 inflammasome activation and related IL-1β production were recognized to be critical in bridging the cross-talk between NMDA receptors and microglial activation.3.2 Taurine protect against 1-BP-induced neurotoxicologywe found that rats exposed to 1-BP displayed reduced spatial learning and memory abilities,which was associated with neurodegeneration in both hippocampus(especially CA1 and CA3 sub-regions)and cortex.Taurine ameliorated 1-BP-induced cognitive impairments and loss of hippocampal and cortical neurons.Mechanistically,taurine abrogated 1-BP-induced decrease of taurine,and decrease 1-BP-induced high level of MDA in brains,and decrease the expressions of P-ASK1,P-P38 in hippocampus and cortex of rats,and increase the level of anti-apoptosis protein Bcl-2,decrease the level of BAX and cleaved-caspase3.4 Conclusions1.1-BP induce disruption of excitatory and inhibitory amino acids balance and expressions of NMD A receptors subunits including GluNl,GluN2A and GluN2B in hippocampus and cortex of rats.2.MK801 protected against 1-BP-induced cognitive dysfunction by inhibit microglial activation and release of proinflammatory cytokines in 1-BP-treated rats.3.Taurine protected against 1-BP-induced cognitive dysfunction by inhibit the oxidative stress and ASK/p38MAPK induced apoptosis.