| Excessive exposure to fluoride has adverse effects on nervous system and other organs,but the underlying mechanisms remain unclear.Brain-derived neurotrophic factor(BDNF)/tyrosine kinase receptor(TrkB)pathway and its upstream signaling extracellular signal-regulated protein kinase 1/2(ERK1/2)are necessary for the establishment and maintenance of synapses during development and in adults,but the effects of fluoride on synapses,BDNF/TrkB pathway and ERK1/2 signaling needs to be further studied.Part 1 Involvement of synaptic impairment,BDNF/TrkB pathway in fluoride-induced developmental neurotoxicityObjective: This study aimed to investigate the effects of fluoride exposure on the ability of learning and memory,morphology and function of synapse,and BDNF/TrkB pathway to provide a basis for the mechanism of fluoride-induced developmental neurotoxicity.Methods: A total of 40 Sprague-Dewley rats(200 ± 20 g,female: male = 1:1)were randomLy divided into four groups: a control group(drinking water with fluorine concentration less than 1.0 mg/L),and three sodium fluoride(NaF)-treated groups(drinking water with 10,50 and 100 mg/L NaF solution).After 2 months of exposure,female and male rats(1:1)in each group were randomLy mated overnight.The pregnant rats were housed in separate cages with the same environmental and exposure conditions till the weaning of pups.The female pups were given the same concentration of NaF as their parents did for 6 months after birth.At the end of NaF administration,Morris water maze(MWM)task was conducted to detect the learning and memory ability of offspring rats,Golgi-Cox staining was performed to observe the morphological changes of dendrite and the density of dendritic spines in hippocampal CA1 area.The expression level of presynaptic protein synaptophysin(SYN),postsynaptic marker post-synaptic density-95(PSD-95)and BDNF/TrkB pathway proteins phospho-ERK1/2(p-ERK1/2),phospho-cyclic adenosine monophosphate response element binding protein(p-CREB),BDNF and TrkB in the hippocamp of offspring rats were determined using western blot analysis.The expression level and location of PSD-95,SYN,BDNF and TrkB proteins in different hippocampal areas were determined using immunohistochemistryResults: The results of MWM showed that the escape latency and the swimming distance of the 50 and 100 mg/L NaF group rats were significantly longer than that of the control group rats on the third day of training(P < 0.05).The 50 mg/L NaF group rats significantly spent more time and swam longer in the target quadrant(where the platform was hidden)than the control group rats(P < 0.05).The result of Golgi-Cox staining showed decrease in dendritic branches and dendritic spines in the CA1 area of hippocamp following NaF exposure.The density of dendritic spines were significantly decreased in the 50 and 100 mg/L groups as compared to the control(P < 0.05).Moreover,significantly decreased expression levels of SYN and PSD-95 were detected in the 50 and 100 mg/L groups as compared to the control(P < 0.05).The levels of p-ERK1/2 were significantly increased,while the level of TrkB was significantly decreased in 50 and 100 mg/L NaF group as compared to the control(P < 0.05).The levels of BDNF were significantly increased in NaF treated groups,and the level of p-CREB was significantly increased in 100 mg/L NaF group as compared to the control(P < 0.05).The results of immunohistochemical staining showed that the number of SYN positive particles was decreased in CA3-CA1 and DG area,and the number of PSD-95 positive cells was decreased in CA3-CA1 area of hippocampus in 100 mg/L NaF group.Besides,the number of BDNF positive cells was increased in CA3-CA1 and DG area,and the number of TrkB positive cells was decreased in CA3-CA1 area of hippocampus in 100 mg/L NaF group.Conclusions: Excessive exposure to fluoride resulted in learning and memory deficit of offspring rats.In addition,excessive exposure to fluoride induced morphological changes and decreased expression level of synaptic related proteins of neurons,and affected the expression levels of BDNF/TrkB proteins and its upstream regulator p-ERK1/2/p-CREB signaling of neurons,which is necessary for the morphology and function of synapse.In summary,morphological and functional changes of synapse and disturbance of BDNF/TrkB pathway were involved in the developmental neurotoxicity of fluoride.Part 2 The role of ERK1/2-mediated changes of BDNF/Trk B pathway in fluoride-induced synaptic impairment of SH-SY5 Y cellsObjective: To investigate the role of BDNF/Trk B pathway and its upstream regulator p-ERK1/2 in fluoride-induced synaptic impairment of SH-SY5 Y cells,therefore providing a basis for exploring the mechanism of fluoride-induced neurotoxicityMethods: The SH-SY5 Y cells in logarithmic growth phase were divided into four groups: a control group and three Na F-treated groups with different concentrations(20,40 and 60 mg/L)of Na F for 24 h.Western blot analysis was performed to detect the expression level of synaptic related proteins and BDNF/Trk B pathway proteins.Immunofluorescence was performed to study the morphology of synapse,the expression level of PSD-95 in dendrite and the nuclear translocation of p-ERK1/2.To inhibit the phosphorylation of ERK,cells were pretreated with 10 ?M mitogen-activated extracellular signal-regulated kinase(MEK)inhibitor-PD98059 for 2 h,and then exposed to Na F for 24 h.Western blot analysis was performed to detect the expression level of PSD-95 and BDNF/Trk B pathway proteins.Immunofluorescence was conducted to study the morphology of synapse and the expression level of PSD-95 in dendrite.Results: The results of western blot analysis showed that the levels of SYN were significantly decreased in the 40 and 60 mg/L Na F group when compared with the control group(P < 0.05).The level of PSD-95 was significantly decreased in the 60 mg/L Na F group as compared to the control group(P < 0.05).In addition,the levels of p-ERK1/2,were significantly increased,while the level of Trk B was decreased in the 60 mg/L Na F group when compared with the control group(P < 0.05).The levels of p-CREB and BDNF were significantly increased in the 60 mg/L Na F group as compared to the control group(P < 0.05).Immunofluorescent staining results showed that fluoride treatment reduced the number of dendritic branches and the aggregation of PSD-95 dot especially located in synapses.Besides,fluoride treatment increases the aggregation of p-ERK1/2 dot in nuclear.Intriguingly,compared with the Na F group,the expression level of PSD-95 and Trk B proteins were significantly increased,while the expression level of BDNF protein was significantly reduced in Na F+PD98059 group(P < 0.05).Moreover,PD98059 effectively rescued the aberrant synaptic morphology and decreased aggregation of PSD-95 dot in synapses of Na F-treated cells.Conclusions: Excessive exposure to fluoride resulted in morphological changes and decreased expression level of synaptic related proteins,and affected the expression levels of BDNF/Trk B proteins and its upstream regulator p-ERK1/2/p-CREB signaling proteins in SH-SY5 Y cells.Additionally,fluoride treatment could stimulate the nuclear translocation of p-ERK1/2 to promote the expression of p-CREB.Importantly,PD98059 significantly attenuated the effects of Na F on the expression levels of BDNF/Trk B pathway proteins and PSD-95.Furthermore,PD98059 rescued the impairment of synaptic morphology and the decreased level of dendritic PSD-95 in Na F treated SH-SY5 Y cells.Collectively,these data suggest that p-ERK1/2-mediated changes of BDNF/Trk B pathway causes synaptic impairment,and inhibition of p-ERK1/2 can protect SH-SY5 Y cells from Na F-induced synaptic impairment. |
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