染氟对人脑神经母细胞瘤细胞线粒体融合蛋白1和分裂蛋白1表达与膜电位的影响

Reactions of mitochondrial mitofnsion 1 and fission 1 proteins and membrane potential of human neuroblastoma cells to fluoride exposure

目的评估氟对体外培养的人脑神经母细胞瘤细胞株（SH．SY5Y细胞）线粒体膜电位和线粒体融合蛋白1（Mfn1）、分裂蛋白1（Fis1）表达的影响。方法在SH．SY5Y细胞中加入0．0（对照）、0．4、2．0、4．0mmol／L氟化钠（NaF），培养6、12、24、48h；利用线粒体膜电位检测试剂盒（JC-1法）对SH．SY5Y细胞线粒体膜电位进行染色；使用蛋白免疫印迹法（Westem Blot）检测线粒体Mfn1和Fis1蛋白的表达。结果NaF作用6、12、48h时，与对照组（1．63±0．18、1．13±0．15、1．30±0．02）比较．2．0、4．0mmol／L NaF处理组SH-SY5Y细胞线粒体膜电位的红绿荧光比值（1．01±0．10、0．80±0．04，0．75±0．13、0．62±0．10，0．82±0．01、O．56±0．04）明显降低（P均〈0．05）；NaF作用6、12、48h时，与对照组（O．93±0．03、1．05±0．07、1．17±0．04）比较，0．4、2．0、4．0mmol／L NaF处理组Mfn1蛋白的表达（0．75±0．02、0．65±0．05、0．57±0．06，0．83±0．06、0．79±0．06、0．69±0．06，0．98±0．05、0．73±0．07、0．62±0．09）明显降低（P均〈0．05）；NaF作用12h时，与对照组（0．90±0．05）比较，2．0、4．0mmol／L NaF处理组Fis1蛋白表达（1．14±0．06，1．23±0．06）明显升高（P均〈0．05）。结论氟中毒损伤可引起神经细胞线粒体融合分裂水平改变并致线粒体膜电位下降，其改变可能与高氧化应激发病学说相关。

Objective To evaluate the influence of fluoride on mitochondrial membrane potential of neuroblastoma SH-SY5Y cells, and on the expression levels of mitochondrial proteins mitofusion 1 （Mfn1） and fission 1 （Fis1）. Methods A stable and feasible culture method of SH-SY5Y cells in vitro was established with different concentration of sodium fluoride [0.0 （control）, 0.4, 2.0 and 4.0 mmol/L], and various periods exposure of 6, 12, 24, 48 h; the mitochondrial membrane potential of SH-SY5Y cells was detected by mitochondrial membrane potential assay kit （JC-1）; and the expression levels of Mfnl and Fisl proteins were detected by Western blotting. Results Compared with the control group （1.63 ± 0.18, 1.13 ±0.15, 1.30 ± 0.02） for various periods exposure （6, 12, 48 h）, the red/green fluorescence ratios of the mitochondrial membrane potential of SH-SY5Y cells exposed to 2.0 and 4.0 mmol/L of sodium fluoride were decreased significantly （1.01 ± 0.10, 0.80±0.04; 0.75 ± 0.13, 0.62 ± 0.10; 0.82 ± 0.01, 0.56 ± 0.04, P 〈 0.05）; compared with the control group （0.93 ± 0.03, 1.05 ± 0.07, 1.17 ± 0.04） for various periods exposure, the expression levels of mitochondrial Mfnl protein were decreased significantly in 0.4, 2.0, 4.0 mmol/L sodium fluoride groups （6, 12, 48 h： 0.75±0.02, 0.65 ± 0.05, 0.57±0.06; 0.83 ± 0.06, 0.79 ±0.06, 0.69 ±0.06; 0.98± 0.05, 0.73 ± 0.07, 0.62 ± 0.09, P 〈 0.05）. Compared with the control group （0.90 ± 0.05） for exposure time 12 h, the expression levels of Fisl protein were increased significantly in 2.0, 4.0 mmol/L sodium fluoride groups （1.14 ± 0.06, 1.23 ± 0.06, P 〈 0.05）. Conclusions The mitochondrial membrane potential and the expression levels of mitofusion 1 and fission 1 of SH-SY5Y ceils treated with fluoride are abnormal, which might be associated with the theory of nerve cell damage from high oxidative stress.