| Fluorine can cross the blood-brain-barrier caused pathological changes in the hippocampus structure,disturbs the arrangement in the actin cytoskeleton,and damages to synaptic structure,thus further affect the function of nerve cells,but the precise mechanisms by which fluoride exerts its neurotoxic effects are poorly unclear.Furthermore,RhoA/ROCK signaling pathway is involved in regulating a variety of neural functions,such as actin cytoskeleton rearrangement,axon elongation and dendritic development.To explore the mechanism of RhoA/ROCK signaling pathway in neuronal cytoskeleton injury induced by fluoride from the following several aspects:1.Effects of fluoride on RhoA/ROCK signaling pathway in nerve cellsIn the present study,we tested the state of RhoA/ROCK signaling pathway in vivo and vitro fluorsis models,and to explore the effect of fluoride on RhoA/ROCK signaling pathway.（1）In vitro:Neuro-2A cells were divided into control group and NaF treatment groups（NaF concentration of 1,2,4and 6 m M）,and treatment for 24 h.Real-time PCR and western blot were used to detect the mRNA and protein expressions of RhoA,ROCK and（p）-cofilin in each group,which in order to investigate the expression of RhoA/ROCK signaling pathway proteins in nerve cells.（2）In vivo: A total of 60 ICR mice aged 8-10 weeks（The ratio of male to female is 1:3）were randomly divided into three groups,which were the control group and 50、100 m M NaF treatment groups,respectively.After one month of exposure,the males and females were caged and allowed to mate freely.The brains of the offspring mice were collected for follow-up experiments.Real-time PCR and western blot were used to detect the mRNA and protein expressions of RhoA,ROCK and（p）-cofilin in each group,which in order to investigate the expression of RhoA/ROCK signaling pathway proteins in the brain of offspring mice.Results:（1）The results of in vitro test showed that the protein and mRNA expressions of RhoA,ROCK and p-cofilin/cofilin in the NaF treatment group were increased,indicating that NaF activated RhoA/ROCK signaling pathway in nerve cells.（2）The results of in vivo test showed that the expression of RhoA,ROCK and p-cofilin/cofilin protein and mRNA in the brain of mice treated with NaF at different ages and concentrations with an increasing trend.These results indicated that NaF activates RhoA/ROCK signaling pathway in mouse brain.2.Effect of inhibition of RhoA/ROCK signaling pathway on neural cytoskeleton and synaptic function injury induced by fluorideIn order to further study the regulation effect of RhoA/ROCK signaling pathway on neural cytoskeleton and synaptic injury induced by fluoride.Neuro-2A cells were treated with Y-27632（ROCK inhibitor,10 μM）and different concentrations of NaF.（1）The morphology of nerve cells was observed by light microscope;（2）CCK-8 and CCK-F were used to detect the survival rate of nerve cells;（3）The LDH assay kit were used to detect the membranes permeability of nerve cells;（4）The glutamate assay kit were used to detect the content of neurotransmitter glutamate in cell culture supernatants;（5）Real-time PCR and western blot were used to detect the mRNA and protein expressions of RhoA,ROCK and（p）-cofilin in cells of each group;（6）The ultrastructure of nerve cells was observed by transmission electron microscope;（7）The arrangement of neural cytoskeleton was observed by F-actin staining;（8）The regulation of Y-27632 on actin cytoskeleton dynamics were verified by F-actin/G-actin double staining in fluoride exposed nerve cells;（9）Real-time PCR and western blot were used to detect the protein and mRNA expression levels of cytoskeleton related proteins MAP2,Dbn and synaptic function related proteins SYP,NMDAR and GABAR in each group.Results: In NaF treatment group,the number of cells and neurites were decreased,cell survival rate were decreased significantly（P < 0.01）,the concentrations of LDH were increased significantly（P < 0.01）,cell membrane permeability were increased and the contents of excitatory neurotransmitter glutamate were decreased extremely significantly（P < 0.01）.Ultrastructural observation showed that NaF caused the interruption of cell membrane integrity,the widening of mitochondrial cristae space and the expansion of rough endoplasmic reticulum.F-actin staining showed that NaF caused the number of synapses and dendritic branches were decreased,the axon length were shortened（P < 0.01）,F-actin/G-actin ratio were decreased significantly（P < 0.01）.Real-time PCR and western blot results showed tha NaF can activate the expression of key proteins in RhoA/ROCK signaling pathway,and the protein and mRNA expressions of cytoskeleton related proteins MAP2,Dbn and synaptic function related proteins SYP,NMDAR and GABAR were decreased.After adding Y-27632,the activation of RhoA/ROCK signaling pathway were inhibited,and the survival rate and glutamate content were significantly increased in NaF treatment group（P < 0.05）,the swelling of mitochondria and the expansion of rough endoplasmic reticulum were reduced,the number of synapses and branches were increased of nerve cells,and the length of axons were significantly extended（P < 0.01）,F-actin/G-actin ratio were significantly increased（P < 0.01）,the imbalance of actin homeostasis was partially recovered.The protein and mRNA expressions of MAP2,Dbn,SYP,NMDAR and GABAR were increased.These results suggest that Y-27632 can alleviate the cytoskeleton and synaptic function damage induced by NaF.3.Effects of inhibition of RhoA/ROCK signaling pathway on learning and memory ability decline induced by fluoride in miceThis study established a animal model to explore the effects of inhibiting RhoA/ROCK signal pathway on learning and memory ability,hippocampal structure and function of fluorosis mice.Male ICR mice aged 6-8 weeks were randomly divided into 6 groups: control group,50 m M NaF treatment group,15 mg/kg fasudil（ROCK inhibitor）treatment group,NaF+fasudil treatment groups（5,10 and 15mg/kg fasudil hydrochloride injection were injected intraperitoneally once a day,and 50 m M fluorinated water was given）,the mice in each group were exposed by free drinking water,and the experiment lasted for 2 months.（1）The contents of creatinine（CREA）,blood urea nitrogen（BUN）,alanine aminotransferase（ALT）and alkaline phosphatase（ALKP）in blood of mice were detected;（2）Y-maze test was used to detect the learning and memory ability of mice;（3）Real-time PCR and western blot were used to detect the mRNA and protein expressions of RhoA,ROCK and（p）-cofilin in each group;（4）HE staining was used to observe the histopathological changes of hippocampus;（5）Real-time PCR and western blot were used to detect the protein and mRNA expression levels of MAP2,Dbn,SYP,NMDAR and GABAR in each group.Results: The contents of CREA,BUN and ALKP in each group were all within the normal range,and ALT content in NaF+15 mg/kg fasudil treatment group decreased,but there was no clinical significance,indicating that the dose of fasudil is safe in this study.Y-maze results showed that NaF significantly reduced the learning and memory ability of the mice,the residence time and the total distance of movement in the food arm were decreased（P < 0.05）.HE staining showed that the demyelination of cerebral white matter neurons of mice were obvious in NaF treatment group,the cerebral vascular wall were edema,the number of neurons were decreased.Real-time PCR and western blot results showed that NaF activates key proteins of the RhoA/ROCK signaling pathway in the mouse brain,the protein and mRNA expression of MAP2,Dbn,SYP,NMDAR and GABAR were decreased.After treatment with different concentrations of fasudil,it inhibited the activation of RhoA/ROCK signal pathway induced by NaF,the time of mice staying in the food arm were significantly increased and the total distance of movement were prolonged（P < 0.05）,but the histopathological changes in hippocampus were not obvious.In addition,fasudil also increased the protein and mRNA expressions of MAP2 Dbn,SYP,NMDAR and GABAR.These results suggest that fasudil can improve the damage of cytoskeleton and synaptic function and ameliorate the cognitive dysfunction in mice induced by NaF.Conclusion: NaF can activate RhoA/ROCK signaling pathway of nerve cells,resulting in damage to cytoskeleton and synaptic structure,and inhibit nerve cell function.Inhibition of RhoA/ROCK signaling pathway can alleviate the damage of neurocytoskeleton and dysfunction of synapse and improve the learning and memory ability in mice induced by fluoride. |
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