氟对大鼠海马神经细胞磷酸化-NMDAR蛋白表达的影响

Effect of fluoride on expression of phosphorylated NMDAR protein in rat hippocampal neurons

目的 观察磷酸化-N-甲基-D-天门冬氨酸受体(P-NMDAR)蛋白在氟中毒大鼠海马神经细胞的表达情况,探讨氟中毒引起神经细胞损伤的机制。 方法 选择初生24 h内的SD大鼠(16只),处死后分离其大脑海马组织,体外培养原代神经细胞。倒置显微镜下观察细胞形态,采用免疫荧光染色鉴定神经细胞。在培养的第7天,将神经细胞分为对照组、低氟组、高氟组、拮抗组、低氟拮抗组、高氟拮抗组6组。对照组加入与实验组等体积的培养液,低氟组和高氟组加入氟化钠(NaF)含量分别为0.2、2.0 mmol/L,拮抗组加入10.0 μmol/L NMDAR拮抗剂(MK-801),低氟拮抗组和高氟拮抗组分别加入0.2 mmol/L NaF + 10.0 μmol/L MK-801、2.0 mmol/L NaF + 10.0 μmol/L MK-801,培养时间为24 h。采用蛋白免疫印迹(Western blot)法检测NMDAR亚基的磷酸化蛋白(P-NMDAR1、P-NMDAR2A、P-NMDAR2B)表达水平。 结果 倒置显微镜下,体外培养2 - 3 d的原代细胞胞体变大,并向外伸出许多凸起,呈小蜘蛛状;6 - 7 d,细胞突触长而纤细,呈网络样相互交错。荧光显微镜下,可见带有红色荧光的神经元核抗体阳性细胞(神经细胞),细胞纯度超过80%。P-NMDAR1蛋白的表达在对照组、低氟组、高氟组、拮抗组、低氟拮抗组和高氟拮抗组间比较差异无统计学意义(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、P-NMDAR2B蛋白表达在各组间比较差异有统计学意义(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均< 0.05),且P-NMDAR2A和P-NMDAR2B蛋白在高氟组的表达高于对照组(P均< 0.05),在高氟拮抗组的表达低于高氟组(P均< 0.05)。 结论 过量氟可以引起海马神经细胞中P-NMDAR2A和P-NMDAR2B蛋白表达增强,加入MK-801与NaF共培养后,可拮抗氟对神经细胞的损伤。

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 μmol/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.