Effects Of Histone Acetylation Regulatory Enzymes On Learning And Memory Impairment Induced By Fluoride In Mice

Objective In this study,a subchronic fluoride exposure mouse model was established to investigate the effects of fluoride poisoning on the levels of histone acetylation regulatory enzymes E1A-associated protein p300(P300)/CREB-binding protein(CBP),Histone deacetylases 2(HDAC2),Histone deacetylases 3(HDAC3),Histone deacetylases 8(HDAC8),synaptic marker Postsynaptic Density 95(PSD95)and Brain Derived Neurotrophic Factor(BDNF)in the hippocampus of mice from the perspective of epigenetic histone acetylation and deacetylation modification.And the effect on Phosphatidylinositol-3-kinase(PI3K)/ Protein kinase B(AKT)signaling pathway,to explore the potential mechanism of fluorine-induced learning and memory impairment in mice,and to provide experimental data and theoretical support for the prevention and treatment of fluorosis.Methods Early weaned mice were randomly divided into 4 groups: control group,low,medium and high concentration group.After one week of adaptive feeding,the mouse began to be poisoned,and drank distilled water with different concentrations of 0,25,50 and 100 mg/L Sodium fluoride(Na F),respectively,and were infected with subchronic fluoride for 20 weeks.The appearance of dental fluorosis,body weight and the coefficients of whole brain and hippocampal organs were observed and recorded.Fluoride ion selective electrode was used to detect the blood and urine fluoride levels of mice.Morris water maze and new object recognition experiments were used to detect the effects of fluoride exposure on learning and memory ability of mice.The intact brains of mice in each group were extracted and the hippocampal tissue was isolated.The morphological changes of hippocampal neurons were observed by H-E staining.The m RNA expression levels of P300/CBP,HDAC2,HDAC3 and HDAC8 in the hippocampus of mice were detected by Real time RT-PCR.The protein expression levels of P300/CBP,HDAC2,HDAC3,HDAC8,BDNF,PSD95,PI3 K and p-AKT in the hippocampus of mice were detected by Western Blot.Results 1.All fluorinated mice had dental fluorosis of different degrees,and with the increase of the concentration of fluoride,the characteristics of dental fluorosis in mice became more obvious;Compared with the control group,the weight of mice in the poisoned group decreased(P < 0.05),and the weight of mice in the poisoned group decreased more obviously with the increase of the poisoned concentration.2.Compared with the control group,the whole brain and hippocampal organ coefficients of mice in fluorine-infected group were significantly increased(P < 0.05),and increased with the increase of the concentration of fluoride exposure,with positive correlation(r=0.9876,P < 0.05 and r=0.9988,P < 0.05).3.Compared with the control group,the blood and urine fluoride levels of fluorineinfected mice were significantly increased(P < 0.05),and increased with the increase of the concentration of fluorine-infected mice,with positive correlation(r=0.9895,P < 0.05 and r=0.9957,P < 0.05).4.The results of Morris water maze experiment showed that,compared with the control group,the escape latency of mice in the infected group was significantly prolonged in the positioning navigation experiment stage(P < 0.05).In terms of swimming distance,mice in the poisoned group showed an increasing trend,and the medium and high concentration groups were higher than the control group,the differences were statistically significant(P < 0.05).In the experimental stage of space exploration,the residence time of target platform in the exposed group showed a decreasing trend,and the high concentration group was less than the control group(P < 0.05).In terms of platform crossing times,the infected group showed a decreasing trend,and the low and high concentration groups were significantly less than the control group,the differences were statistically significant(P < 0.05).5.The results of the new object recognition experiment showed that there was no significant difference in the position preference index of mice among all groups.Compared with the control group,the new object recognition index of the infected group showed a downward trend,and the high concentration group was significantly lower than the control group(P < 0.05).6.HE staining showed that in the infected group,the arrangement of nerve fibers was disordered,the number of nerve cells was significantly reduced,the nucleolysis and deep staining were significantly increased,the area of cytoplasmic staining was significantly increased,the free cells became more and dispersed,and the pathological damage changes were obvious.7.Real-time RT-PCR results showed that the m RNA expression level of P300 in the infected group showed a decreasing trend,and the medium and high concentration groups were lower than the control and low concentration groups,with statistical significance(P < 0.05);The m RNA expression level of HDAC2 in the infected group showed an increasing trend,and the high concentration group was higher than that in the control,low and medium concentration groups,the differences were statistically significant(P < 0.05).The m RNA expression levels of HDAC3 and HDAC8 in the infected group showed an increasing trend,and those in the low,medium and high concentration groups were higher than those in the control group,with statistical significance(P < 0.05).8.Western Blot results showed that the expression level of P300 protein in the infected group showed a decreasing trend,and the medium and high concentration groups were lower than the control group,the differences were statistically significant(P < 0.05);The expression level of HDAC2 protein in the infected group showed an increasing trend,and the medium and high concentration groups were higher than the control group,the differences were statistically significant(P < 0.05).The protein expression levels of HDAC3 and HDAC8 in the infected group showed an increasing trend,and the high concentration group was higher than the control group and the low concentration group,the differences were statistically significant(P < 0.05).The protein expression levels of PSD95,PI3 K and p-AKT in the infected group were decreased,and the medium and high concentration groups were lower than those in the control group and low concentration group,with statistical significance(P < 0.05).The expression level of BDNF protein in the infected group showed a decreasing trend,and the medium concentration group was lower than the control group and the low concentration group,the differences were statistically significant(P < 0.05).Conclusions 1.Subchronic fluoride exposure can lead to the decline of learning and memory ability of mice,and with the increase of fluoride exposure concentration,the learning and memory impairment of mice becomes more obvious.2.Subchronic fluoride exposure can decrease the m RNA and protein expression levels of P300 in hippocampus of mice,increase the m RNA and protein expression levels of HDAC2,HDAC3 and HDAC8,interfere with the dynamic coordination and balance between HATs and HDACs,and may cause changes in the acetylation levels of factors related to synaptic plasticity.The decreased protein expression levels of PSD95 and BDNF may be affected by it,and then affect the learning and memory ability of mice.3.Subchronic fluoride exposure can reduce the protein expression levels of PI3 K and p-AKT in the hippocampus of mice.Histone acetylation regulatory enzymes may act through the PI3K/AKT signaling pathway to regulate the expression of learning and memory related factors,which together constitute the potential molecular mechanism of learning and memory impairment in mice with fluorosis.