| Our previous studies showed that the learning and memory abilities of rat offspring was declined in the high fluoride model,the structural of cerebral cortex and hippocampus were damaged,and increased apoptosis rate and DNA damage of neuron were found.However,the underlying mechanisms remain unclear.Whether the cytoskeleton has suffered damage?On the trigger of it,ICR mice and Neuro-2A cellwere used in this study to investigate the role of cytoskeleton-MAP2 in mechanisms of fluorosis.1 Fluoride-induced alterations of synapse-related proteins and cytoskeletal proteins in the cerebral cortex of ICR offspring miceThe animal model of fluoride exposure was created by providing mice daily water containing 0,50 or 100 mg/L F-for 1 month.After mating,the mice offspring on 0 d,10 d,20 d,30 d,60 d and 90 d were used to further study.The cognitive abilities of mice were tested using the Morris water maze test.The data indicated that fluoride exposure significantly prolonged the escape latency period and decreased the numbers of crossings for a particular zone.Histopathologic analysis revealed shrinkage and fragmentation of glial ceils in treatment groups,and fewer pyramidal cells in the cerebral cortices in fluoride-treated groups.The expression of MAP2,SYP and Dbn(both protein and mRNA)of the cerebral cortex were decreased signigicantly in fluoride-treated groups.In addition,glutamate receptor(NMDAR)expression was detected,which is consistent with reduced MAP2,SYP and Dbn expression.Therefore,fluoride-mediated reduction in cognitive dysfunction is likely caused by the disruption of the expression of these synapse-associated proteins,resulting in attenuated neuronal functioning2 The effects of fluoride on cytoskeleton damage and signal transmission of Neuro-2A cellsNeuro-2A cells were used in this study to investigate the effects of fluoride on the cytoskeleton.The Neuro-2A cells were exposed to 0,1,2,4 and 6 mM sodium fluoride(NaF)for 24 h.Cell viability and lactate dehydrogenase(LDH)release were examined.It was observed that exposure to NaF reduced cell viability,disrupted cellular membrane integrity,and high levels of LDH were released.The observed changes occurred in a dose response manner.Morphologic observations showed that cell became rounded and were loosely adherent following exposure to NaF.Axon spines and normal features disappeared with high-dose NaF treatment.The data of western blot showed that the expression of MAP2 and synaptophysin were decreased in Neuro-2A cells exposed to fluoride,which corroborate the F-actin staining observations.On the other hand,glutamate and NMDAR expression declined significantly in fluoride-treated cells compared with the controls.Finally,the ultrastructural changes were observed with increasing doses of NaF:disappearance of synapses,mitochondrial agglutination,vacuole formation,and cellular edema.3 Fluoride effects the RhoA/ROCK signalling pathway activity in the cerebral cortex of ICR offspring mice and Neuro-2A cellThe cerebral cortex of offspring mouse brain and Neuro-2A cells were used in this study to investigate the relations of cytoskeleton damage and RhoA/ROCK signalling pathway.The data of yeast two-hybrid experiment showed it have potential of functional-interaction between MAP2 and Myosin;The status of cytoskeleton were observed by immunofluorescents staining with phalloidin(green)and β-actin(red),which showed that the number of synapses and branches were decreased with the increasing dose of fluoride,microtubule and nicrofilament retracted to cell body,the vague outline of cell nucleus with shrinked;The expression of Tau mRNA were decreased in Neuro-2A treated with fluoride(P<0.05),however protein level of it were increased in low concentration groups(1 and 2 mM,P<0.01)but declined in high dosage groups(4 and 6 mM,P<0.01);Tau mRNA in mouse brain was increased slightly after fluoride exposure,but generally declined in protein level,except 10 d and 60 d of low fluoride group(P<0.05);Western blot results demonstrated that RhoA/ROCK signalling pathway were activitied in low concentration groups(1 and 2 mM,P<0.01)but decreased in high dosage groups(4 and 6 mM,P<0.05);The RhoA/ROCK signalling pathway were activated by fluoride in mouse brain.Y-27632 were used in this study to investigate the relations of cytoskeleton damage and RhoA/ROCK signalling pathway.Compared with NaF-treated group,Y-27632 can suppress the activation of RhoA/ROCK signalling pathway induced by NaF.The cell survival rate were increased after co-incubated with Y-27632 and NaF,meanwhile,synapses were abundant and axon extended,the inhibitory expression of MAP2 and Tau protein induced by NaF can counteract with Y-27632(P<0.05).the above data showed that activitied RhoA/ROCK signalling pathway were affected synapse plasticity and function of neurons via regulate the rearrangement of cytoskeleton. |
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