| Objective: Fluorine is a kind of protoplasm toxin and can enter the brain tissue through the blood-brain barrier.Epidemiological survey showed that the type of drinking water fluorosis may lead to mental decline in children.The hippocampus is an important brain area that produces learning and memory,while synaptic plasticity is considered to be the basis of learning and memory.Glutamate receptor Glu R2 and NMDAR2β receptors play an important role in the learning and memory and also in the induction of LTP.PSD-95 and SYP belong to synaptic proteins,playing an importment role in synaptic plasticity,more closely related to some degenerative diseases.Earlier research had found that Fluoride can activate microglia,releasing inflammatory factors.TREM2/DAP12 is a protein specifically expressed in microglia,and an accelerator for inflammation.MAPK signaling pathway vitally important to regulate microglia mediated inflammatory factors,the activation of microglial can cascade MAPK’s reaction,How does activated microglia affect synaptic plasticity? So does fluoride activate the microglia to release inflammatory factors,Cause neuronal injury,affecting PSD-95 and SYP the expression of and altering synaptic structure and morphology? Therefore,the research is to observe the changes of neural behavior in rats exposed to subchronic fluoride exposure,the microglias’ activation in rat brain,and detection of activation of TREM2/DAP12 protein and MAPK signaling pathway in microglia,secretion of inflammatory factors and expression of ionotropic glutamate AMPA receptor and synaptic protein PSD-95 and SYP,To investigate the mechanism of fluoride exposure on learning and memory in rats.Methods:1.Behavioral methods:The water maze was used.2.Determination of LTP:In vivo Electrophysiological Experimental in rats.3.Determination of blood and urine fluoride: Fluoride ion selective electrode assay.4.Pathomorphological observation:HE stain.5.Detection methods of apoptosis:TUNEL.6.Methods for detecting synapse associated proteins:(1)Westernblot(2)IHC.7.Determination of microglia:Immunofluorescence.8.Detection of DAP12/TREM2 and MAPK signaling pathways:Double labeled immunofluorescence method.Results:1.General status of rats exposed to subchronic fluoride exposure:With the increase of fluoride exposure time,dental fluorosis symptoms gradually worsened,white and yellow,and the emergence of chalk stripe type,However,after the 9 weeks,rats from120 ppm fluoride group were observed to have lower body weight.There were no differences in the brain organ coefficient between the groups at exposure 4 weeks,at 12 weeks the 120 ppm fluoride treated rats had significantly higher coefficients than rats in the control group at exposure 12 weeks,due to the decrease of body weight in 120 ppm fluoride exposed rats(P<0.05).2.Fluoride content in blood and urine in rats with fluorosis:When fluoride is exposed to 4w and 12 w blood and urine fluorine levels were markedly increased with fluoride exposure in rats(P<0.05).3.Pathological changes of brain tissue in rats exposed to subchronic fluoride exposure:With the increase of fluoride concentration and the extension of fluoride exposure time,in the DG group,vacuolar degeneration of the brain cells increased and irregular arrangement occurred.4.Effects of subchronic exposure to fluoride on apoptosis in rat hippocampal neurons:TUNEL results show that:The positive fluorescence intensity of TUNEL in the hippocampus of rats exposed to 4w and 12 w fluoride increased with the increase of dose(P<0.05).5.Effects of subchronic exposure to fluoride on learning and memory in rats:In 5 days-training trial sessions of the MWM,the latency time of 120 ppm fluoride-treated 12 weeks group in the first and second days of training sessions was significantly longer than that of control group(P<0.05).6.Electrophysiological changes in the hippocampus of rats exposed to subchronic fluoride exposure:After high frequency stimulation,the PS wave of 4w and12 w rats in 120 ppm group was significantly lower than that of control group(P<0.05).There was no significant difference in PS wave between the 60 ppm group and the control group(P>0.05).Compared with the control group,the f EPSP of 4w and 12 w rats in group 60 ppm and 120 ppm were significantly decreased(P<0.05),there was no significant difference in f EPSP between group 60 ppm and group 120ppm(P>0.05).7.Expression of synaptophysin PSD-95 and SYP in hippocampus of rats exposed to subchronic fluoride exposure: Compared with the control group,with the increase of exposure,and the exposure time,in 4w and 12 w,60 and 120 ppm fluoride exposure group PSD-95 in the hippocampus of region decreased(P<0.05),and the result of Immunohistochemistry and Western was consistent.while SYP was no significant difference in the immunohistochemistry and western between the hippocampus.8.Expression of glutamate receptor Glu R2 and NMDAR2β in hippocampus of rats exposed to subchronic fluoride exposure: In 4w and 12 w rats,the protein contents of Glu R2 and NMDAR2β in hippocampus were gradually decreased with the amount of toxic agents.At 12 w,compared with the control group,the protein levels of Glu R2 and NMDAR2β in the 120 ppm fluoride exposure group were significantly decreased(P<0.05).9.The activition of microglia and dap12/trem2 in hippocampus of rats exposed to subchronic fluoride exposure:The expression level of OX-42 in hippocampus of 4w and 12 w 60ppm and 120 ppm fluoride exposed groups was increased,and the immunofluorescence optical density was higher than that of control group(P<0.05).Double immunofluorescence staining of DAP12 and OX-42 or TREM2 and OX-42 that The expression of DAP12 and TREM2 protein in rat hippocampal activation of microglial cells was increased in 120 ppm group(P<0.05).10.Secretion of inflammatory cytokines in rat hippocampus microglia exposed to subchronic fluoride exposure:The results of ELISA showed that the content of IL-1β in hippocampus of rats exposed to12 W 120ppm group was higher than that of the control group(P<0.05).In 120 ppm group,compared with the control group,the content of TNF-ɑ in hippocampus of rats exposed to 4w and 12 w increased(P<0.05).The doubled immunofluorescence of OX-42 and IL-1β in 4w and 12 w,60ppm and 120 ppm group,the expression level of IL-1β was significantly higher than the control group(P<0.05).The doubled immunofluorescence of OX-42 and TNF-ɑ in 4w,60 ppm and 120 ppm group,the expression level of TNF-ɑ was significantly higher than the control group(p<0.05).At 12 w,120ppm rats hippocampus microglia TNF-ɑ expression level increased significantly(P<0.05).11.The effects of MAPK signaling pathway in rats hippocampus microglia exposed to subchronic fluoride exposure,When exposed to 4w and 12 w,the expression of p-ERK protein in 120 ppm group was markedly increased(P<0.05),The expression level of p-p38 in microglia in120 ppm group exposed to 4w was markedly increased(P<0.05).At 12 w,the expression levels of p-p38 in hippocampus microglia of 60 and 120 ppm groups weremarkedly increased(P<0.05).The p-JNK expression level of microglia in fluoride exposed group was no difference between the groups(P>0.05).Conclusion:1.Subchronic exposure to fluoride in 4w and 12 w can reduce the acquisition capability of learning and memory and impair the spatial learning and memory ability of rats.2.The levels of synaptophysin PSD-95 express,glutamate receptor GluR2 and NMDAR2β protein in hippocampus of rats exposed to subchronic fluoride exposure were decreased,and LTP was suppression.3.The activation of microglia in hippocampus of rats exposed to subchronic fluoride exposure increased the expression of pro-inflammatory factor TNF-ɑ and IL-1β,and increased the DAP12/TREM2 protein and actived the P38,ERK signaling pathway. |
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