Effect of fluoride on expression of phosphorylated NMDAR protein in rat hippocampal neurons / 中华地方病学杂志

ABSTRACT

Objective To observe the expression of phosphorylated N-methyl-D-aspartate receptor (P-NMDAR) protein in hippocampal neurons of rats with fluorosis and explore the mechanism of neuronal damage caused by fluorosis.Methods Sixteen Sprague-Dawley rats within 24 h after birth were selected,and hippocampus of the brain was isolated after sacrifice.The primary neurons were cultured in vitro.Observation of the cell morphology under an inverted microscope,neurons were identified by immunofluorescence staining.In the 7th day of cultivation,the neurons were divided into control group,low fluoride group,high fluoride group,antagonist group,low fluoride antagonist group and high fluoride antagonist group.The control group was treated with the same volume of medium as the experimental group.The concentration of NaF was 0.2 and 2.0 mmol/L in the low fluoride group and the high fluoride group,respectively.The antagonist group was treated with 10.0 μ mol/L NMDAR antagonist (MK-801).The low fluoride antagonist group and the high fluoride antagonist group were treated with 0.2 mmol/L NaF + 10.0 μmol/L MK-801,2.0 mmol/L NaF + 10.0 μmol/L MK-801,respectively.The culture time was 24 hours.The expression levels of phosphorylated protein (P-NMDAR1,P-NMDAR2A,P-NMDAR2B) of NMDAR subunits were detected by Western blotting.Results Under inverted microscope,the primary cell body of the cultured in vitro for 2-3 days became larger,and many protrusions appeared outward,showing a small spider shape;6-7 days,the cells synaptic long and slender,a network-like interlaced form.Under fluorescent microscope,NeuN positive cells (neurons) with red fluorescence were observed,and the cell purity exceeded 80％.There was no significant difference in the expression of P-NMDAR1 protein between the control group,the low fluoride group,the high fluoride group,the antagonist group,the low fluoride antagonist group and the high fluoride antagonist group (0.44 ± 0.12,0.46 ± 0.06,0.46 ± 0.12,0.56 ± 0.10,0.70 ± 0.12,0.46 ± 0.09,F=2.75,P ＞ 0.05);P-NMDAR2A and P-NMDAR2B protein expressions were statistically significant between the six groups (0.75 ± 0.17,0.74 ± 0.08,1.13 ± 0.27,0.87 ± 0.15,0.67 ± 0.11,0.66 ± 0.09;0.68 ± 0.24,0.66 ± 0.12,1.46 ±0.27,0.74 ± 0.16,0.56 ± 0.13,0.91 ± 0.35,F =3.68,6.11,P ＜ 0.05),and the expressions of P-NMDAR2A and P-NMDAR2B protein in high fluoride group were higher than those in the control group (P ＜ 0.05);the expressions in the high fluoride antagonist group were lower than those in the high fluoride group (P ＜ 0.05).Conclusion Excessive fluoride can increase the expressions of P-NMDAR2A and P-NMDAR2B protein of hippocampal neurons,and co-culture of MK-801 and NaF can antagonize the damage of fluoride to neurons.