硒酸钠抑制铁诱导的tau过度磷酸化

Sodium selenate inhibits iron-induced tau hyperphosphorylation

目的探讨硒酸钠对过量铁诱导的tau过度磷酸化的影响及其机制。方法将人神经瘤细胞母细胞SHSY5Y细胞共设计4个不同的处理组:对照组;加入抗坏血酸;硫酸亚铁处理组:加入配置好的溶解于抗坏血酸的硫酸亚铁溶液(终浓度50μmol/L)处理24 h;硒酸钠预处理组:硒酸钠(终浓度为0.1μmol/L)预处理细胞12 h后,更换为配置好的硫酸亚铁溶液(终浓度50μmol/L)处理24 h;硒酸钠处理组:加入硒酸钠(终浓度为0.1μmol/L)预处理细胞12 h后,更换为抗坏血酸溶液处理24 h。活性氧试剂盒检测细胞氧化应激水平,免疫印迹法检测tau在Thr231、Ser396和Ser404等位点的磷酸化水平、蛋白磷酸酶2A(PP2A)和糖原合成酶激酶3β(GSK3β)的活性。结果各组细胞中总tau的表达水平无显著变化。与对照组比较,硫酸亚铁处理组tau在Thr231、Ser396和Ser404等位点的磷酸化程度分别增加了(77.40±26.62)%、(116.80±30.90)%和(78.80±26.76)%,差异有统计学意义(P<0.05);相对于硫酸亚铁处理组,硒酸钠预处理显著降低了tau在Thr231、Ser396和Ser404等位点的过度磷酸化,分别降低了(51.08±13.37)%、(42.59±13.65)%和(38.42±13.80)%,差异有统计学意义(P<0.05)。与对照组比较,硫酸亚铁处理的细胞中活性氧(ROS)水平显著增加至(155.4±3.79)%;与硫酸亚铁处理组比较,硒酸钠预处理的细胞中ROS水平减少了(38.70±4.52)%,差异均有统计学意义(P<0.05);与对照组比较,用硒酸钠处理的细胞的ROS水平无显著变化。Western blot结果显示硒酸钠和硫酸亚铁处理未改变细胞中PP2A的表达水平,但是与对照组比较,硫酸亚铁处理的细胞中p-PP2A/PP2A的比例显著增加至(186.1±20.57)%,而与硫酸亚铁处理组比较,硒酸钠预处理组细胞中p-PP2A/PP2A的变化降低(42.89±12.05)%,差异均有统计学意义(P<0.05)。另外,与对照组比较,硫酸亚铁处理组细胞中p-GSK3β/GSK3β显著降低至(62.05±3.99)%,而与硫酸亚铁处理组比较,硒酸钠预处理组的细胞中pGSK3β/GSK3β水平增加了(23.73±9.48)%,差异均有统计学意义(P<0.05)。结论经过硒酸钠预处理的细胞在一定程度上抑制过量铁带来的细胞氧化应激水平、PP2A和GSK3β活性的变化,从而减少过量铁引起的tau过度磷酸化。

Objective To explore the function and mechanism of sodium selenate on affecting tau phosphorylation induced by iron. Methods Four different treatment groups were designed： （1） control group, SH- SY5Y cells were treated with ascorbie acid ; （2） cells were treated with 50 μmol/L ferrous sulfate for 24 h; （3） cells were pretreated with 0.1 μmol/L sodium selenate for 12 h, then treated with 50 μmol/L ferrous sulfate for 24 h; （4） cells were pretreated with 0.1 μmol/L sodium selenate for 12 h, then treated with ascorbic acid solution for 24 h. The oxidative stress was detected by Reactive Oxygen Species Assay. The activities of PP2A and GSK313 and tau hyperphosphorylation were determined by western blot. Results The results showed that the expression levels of total tau （ tau5 ） were not altered significantly among these four groups. However, the levels of phosphorylated tau at Thr231, Ser396 and Ser404 were markly increased by （77.40＋26.62）% , （116.80＋30.90）% and （78.80±26.76）%, respectively in SH-SY5Y cells treated with iron （P〈0.05）. It was worth notingthat sodium selenate treatment significantly reduced the levels of phosphorylated tau by （51.08 ± 13.37）% at Thr231, （42.59±13.65）% at Ser396 and （38.42±13.80）% at Ser404, compared with iron treatment alone （P〈0.05）. There was a significant increase to （155.40±3.79）% in the levels of ROS in ferrous iron-treated cells compared with that of the control （P〈0.01）. However, cells pre-treated with sodium selenate exhibited a （38.70±4.52）% decrease in ROS production compared with that of ferrous iron treatment （P〈0.05）. No significant changes were observed in the ceils treated with sodium selenate alone compared with the control. Western blot results showed that treatment with sodium selenate and ferrous sulfate did not alter the PP2A expression level in the cell However, the ratio of p-PP2A/PP2A was increased significantly to （186.10±20.57）% in iron-treated cells compared with the control. Sodium selenate treatment significantly attenuated the increase by （42.89± 12.05） （P〈0.05） in the ratio of p-PP2A/PP2A caused by iron treatment. Furthermore, the ratio of p- GSK3 β/GSK3 β was dramatically reduced to （62.05± 3.99）% （P〈0.05） in cultures treated with iron compared with the control, whereas in cultures pretreated with sodium selenate and then treated with iron the p-GSK3β/GSK3β level was increased by （23.73±9.48）% （P〈0.05） compared with iron-treated ceils. Conclusion Pretreatment of cells with sodium selenite could inhibit the levels of cellular oxidative stress induced by excess iron, the activation of PP2A and GSK3β, and thus reduced excessive iron-induced tau hyperphosphorylation.