氟对原代培养大鼠海马神经元一氧化氮和钙及钙调激酶的影响

Effects of fluoride on levels of NO,calcium and CaM kinase Ⅱ in cultured primary hippocampal neurons

目的探讨氟对原代培养海马神经元中NO含量、钙离子水平及磷酸化钙调蛋白激酶蛋白表达水平的影响。方法将原代培养的新生SPF级SD大鼠海马神经元分别暴露于含终浓度为0(对照)、0.1、0.2、0.4、0.8、1.6 mg/L氟化钠的培养基中,于37℃、5%CO_2培养箱中继续培养6、12、24 h。检测海马神经元的活性、NOS活力、NO含量及海马神经元中钙离子水平、磷酸化钙调蛋白激酶Ⅱ(pCaMKⅡ)蛋白的表达水平。结果与对照组比较,0.4、0.8、1.6 mg/L NaF染毒6、12、24 h大鼠海马神经元的细胞存活率均较低,而NOS的活力和NO的含量以及钙离子的水平均升高,差异有统计学意义(P<0.05);与染毒6 h比较,仅0.4、0.8、1.6 mg/L NaF染毒24 h大鼠海马神经元的细胞存活率均较低,而NOS的活力和NO的含量以及钙离子的水平均升高,差异有统计学意义(P<0.05)。与对照组比较,0.4、0.8、1.6 mg/L Na F染毒6 h及0.1、0.2、0.4、0.8、1.6 mg/L Na F染毒12、24 h大鼠海马神经元中pCaMKⅡ蛋白的表达水平均较高,差异有统计学意义(P<0.05);与染毒6 h比较,不同浓度Na F染毒12、24 h大鼠海马神经元中pCaMKⅡ蛋白的表达水平均较高,差异有统计学意义(P<0.05)。随着氟染毒浓度的升高和染毒时间的延长,原代培养大鼠海马神经元的存活率呈下降趋势,而NOS的活力、NO的含量和钙离子的水平及pCaMKⅡ蛋白的表达水平均呈上升趋势。结论氟对中枢神经系统的损伤可能是通过增加信号转导通路中重要的第二信使以及神经递质表达所致。

Objective To understand the effects of fluoride exposure on the levels of NO,Ca^2＋ and protein expression of phospho-CaM kinase Ⅱ（pCaMK Ⅱ） in the cultured primary hippocampal neurons of rats. Methods The cultured primary hippocampal neurons from neonatal SPF grade SD rats were exposed to sodium fluoride in the medium with the final concentrations of 0.0（control）,0.1,0.2,0.4,0.8 and 1.6 μg/ml and cultured at 37 ℃,5% CO2 in incubator for 6,12 and 24 h,respectvely. The activity of NOS,the content of NO in the supernatant of the hippocampus and the level of Ca^2＋,the protein expression of phosphorylated protein kinase Ⅱ（pCaMKⅡ） were detected in the hippocampal neurons. Results Compared with the control group,the viabilities of hippocampal neurons in 0.4,0.8 and 1.6 μg/ml sodium fluoride groups reduced,the activities of NOS,the contents of NO and Ca^2＋ increased significantly（P〈0.05）. Compared with the group exposed to sodium fluoride for 6 h,the survival rates of hippocampal neurons treated with 0.4,0.8 and 1.6 μg/ml sodium fluoride for 24 h reduced,the activities of NOS and NO and the contents of Ca^2＋ increased significantly（P 0.05）. Compared with the control group,0.4,0.8,1.6 μg/ml groups exposed to sodium fluoride for 6 h and 0.1,0.2,0.4,0.8,1.6 μg/ml groups exposed to sodium fluoride for 12 and 24 h,the expression levels of p Ca MK Ⅱ protein in hippocampal neurons significantly increased after different durations of sodium fluoride exposure（P〈0.05）. Compared with sodium fluoride exposure for 6 h,the expression levels of pCaMKⅡ protein in hippocampal neurons exposed to different concentrations of sodium fluoride for 12 and 24 h increased significantly（P 0.05）. The obvious dose-time dependent manner was seen in fluoride exposure and research indexes in the study. Conclusion Fluoride may cause central nervous system damage through increasing the expression of important second messengers and neurotransmitters in the signal transduction pathway.