50 Hz磁场和双酚A暴露对大鼠脑组织和Neuro-2a细胞Tau蛋白磷酸化的影响

Effects of 50-Hz magnetic field and bisphenol A exposure on phosphorylation of Tau protein in rat brain tissue and Neuro-2a cells

目的研究50 Hz磁场、双酚A(bisphenol A,BPA)单独和联合暴露对神经细胞Tau蛋白磷酸化的影响。方法24只SD雄性大鼠,体质量(150±10)g,分为对照组(生理盐水灌胃+50 Hz磁场假性暴露)、50 Hz磁场暴露组(0.5 mT、4 h/d 50 Hz磁场暴露+生理盐水灌胃)、BPA暴露组(BPA20μg·kg^(-1)·d^(-1)灌胃+50 Hz磁场假性暴露)、50 Hz磁场和BPA复合暴露组(BPA 20μg·kg^(-1)·d^(-1)灌胃+0.5 mT、4 h/d 50 Hz磁场暴露)4组,每组6只。处理1次/d,6 d/周,连续12周。小鼠神经母细胞瘤细胞株(Neuro-2a),分为对照组、50 Hz磁场暴露组(1 mT、48 h)、BPA暴露组(100μmol/L、24 h)、50 Hz磁场+BPA复合暴露组(1 mT、48 h+100μmol/L,24 h)4组。大鼠暴露结束按标准方法取材,组织免疫荧光化学方法检测大脑皮层和海马脑组织Tau-Thr^(231)、Tau-Ser^(404)的表达以及细胞凋亡。细胞实验暴露后采用CCK-8法检测细胞活力,采用细胞免疫荧光化学和Western blot方法检测Tau-Thr^(231)、TauSer;、Caspase-3的表达。结果50 Hz磁场暴露12周后,大鼠皮层和海马脑区Tau-Thr^(231)位点磷酸化水平明显升高(P<0.05);BPA暴露或与50 Hz磁场复合暴露12周后,大鼠皮层和海马脑区Tau-Thr^(231)位点磷酸化水平、皮层Tau-Ser^(404)位点磷酸化水平明显升高(P<0.05),复合暴露组皮层和海马脑区细胞凋亡水平变化明显(P<0.05)。50 Hz磁场暴露、BPA单独暴露或与50 Hz磁场复合暴露48 h后,Neuro-2a细胞活力均明显低于对照组(P<0.05)。50 Hz磁场暴露48 h后,Neuro-2a细胞Tau-Ser^(404)位点磷酸化水平明显高于对照组(P<0.05);BPA单独暴露或与50 Hz磁场复合暴露48 h后,Neuro-2a细胞Tau-Thr^(231)位点和Tau-Ser^(404)位点磷酸化水平、Caspase-3的表达水平明显升高(P<0.05)。结论50 Hz磁场、BPA暴露可引起神经细胞Tau蛋白不同位点过度磷酸化,并且二者之间存在协同效应,可引起神经细胞损伤。

Objective To investigate the effects of 50-Hz magnetic field and bisphenol A(BPA)alone or combined exposure on the phosphorylation of Tau protein in nerve cells.Methods A total of 24 SD male rats(body weight 150±10 g)were randomly and equally divided into 4 groups:control group(normal saline gavage+magnetic field sham-exposure),50-Hz magnetic field exposure group(0.5 mT,50 Hz magnetic field exposure 4 h/d+normal saline gavage),BPA exposure group(BPA 20μg·kg^(-1)·d^(-1)gavage+magnetic field sham-exposure),and combined exposure group(BPA 20μg·kg^(-1)·d^(-1)gavage+0.5 mT,50 Hz magnetic field exposure 4 h/d).The magnetic field exposure was performed once per day,6 days per week,for consecutive 12 weeks.The protein expression of Tau-Thr^(231)and Tau-Ser^(404),as well as cell apoptosis in frozen slices of cerebral cortex and hippocampus were detected by immunohistofluorescence assay and TUNEL assay,respectively.For cell experiments,Neuro-2a cells were exposed to 50 Hz magnetic field(1.0 mT,48 h),BPA treatment(100μmol/L,24 h),as well as combined exposure(50 Hz magnetic field+BPA),respectively.Cell viability was tested by CCK-8 assay,and the protein expression of Tau-Thr^(231),TauSer;and Caspase-3 were measured using immunofluorescence assay and Western blotting.Results As compared with the control group,the phosphorylation of Tau-Thr^(231)was significantly increased in cerebral cortex and hippocampus of rats after 12 weeks of 50 Hz magnetic field exposure(P<0.05).The Tau-Thr^(231)phosphorylation in rat cerebral cortex and hippocampus and the Tau-Ser^(404)phosphorylation in cerebral cortex were remarkably elevated after 12 weeks of BPA exposure alone or in combination with 50 Hz magnetic field(P<0.05).The co-exposure also induced obvious neuronal apoptosis in cerebral cortex and hippocampus of rats(P<0.05).For cell experiments,the Neuro-2a cell viability was greatly lowered after exposure to 50 Hz magnetic field,BPA alone or combined treatment for 48 h(P<0.05).The Tau-Ser^(404)phosphorylation was notably up-regulated(P<0.05)after 50 Hz magnetic field exposure;the phosphorylation of Tau-Thr^(231)and Tau-Ser^(404),as well as the expression of Caspase-3 were also enhanced(P<0.05)after BPA exposure or coexposure.Conclusion Exposure to 50 Hz magnetic field and/or BPA can induce hyperphosphorylation of Tau protein at different amine acid site,and there are synergistic effects between the 2 exposures,which may cause nerve cell damage.