促红细胞生成素对Aβ25-35诱导的细胞tau蛋白磷酸化的抑制作用

Effect of erythropoietin on β-amyloid peptide-induced tau phosphorylation in SH-SY5Y cells

目的探讨促红细胞生成素（Epo）对凝聚态β淀粉样蛋白25—35片断（Aβ25-35）诱导的SH—SY5Y细胞tau蛋白磷酸化的影响。方法MTT法观察不同浓度的Epo（0、5、10、20、50U1单独作用24h对SH—SY5Y细胞存活率的影响；20μmol／L的凝聚态Aβ25-35作用于SH—SY5Y细胞不同时间点（0min、30min、1h、3h、6h、12h、24h1后，Western blot法检测tau蛋白磷酸化（Ser199、Ser396及taul）水平的变化；观察不同浓度的Epo（5、10、20u）预处理细胞3h后对Aβ25-35作用的影响以及P13K／Akt抑制剂LY294002（50μmol／L）预处理细胞1h后EpO抑制作用受到的影响。结果不同浓度的Epo作用24h后，MTT示细胞存活率无明显变化。20μmol／L的Aβ25-35作用不同时间点后，tau蛋白Ser396、Ser199位点的磷酸化水平3h时开始增加，6h达到最高峰，12h后又逐渐下降，24h时仍维持较高水平，与0min、30min时比较，差异均有统计学意义（P〈0．05）；而总的tau蛋白没有任何变化。5、10、20U Epo预处理均可有效地抑制Aβ25-35引起的Ser396、Ser199位点的磷酸化，与正常对照组比较，差异均有统计学意义（氏0．05）。LY294002预处理细胞后，Epo的作用受到抑制。结论Epo可通过P13K／Akt途径对Aβ25-35诱导的tau蛋白磷酸化发挥抑制作用，本研究为研究AD的发病机制及探索新的有效治疗药物提供了重要的理论基础。

Objective To investigate the effect of erythropoietin （Epo） on tau hyperphosphorylation induced by β-amyloid peptide 25-35 （Aβ25-35） in SH-SY5Y cells. Methods MTT assay was employed to identify the changes in the viability of SH-SY5Y cells following Epo treatment at 0, 5, 10, 20, and 50 U. Western blot was used to detect the levels oftau phosphorylation at Ser396, Ser199 and Taul at different time points after treatment with 20 μmol/L Aβ25-35. The effect of a 3-hour Epo pretreatment at 5, 10, and 20 U on the actions of Aβ25-35 was evaluated. The inhibitory effect of Epo on Aβ25-35-induced neurotoxicity after pretreatment with the PI3K/Akt inhibitor LY294002 was assessed to explore the possible mechanism of Epo. Results The viability of SH-SY5Y cells showed no obvious changes in response to Epo exposure at different doses. Western blot showed that Aβ25-35 induced increased phosphorylation at Ser396 and Ser199 3 h after the treatment. The phosphorylation reached the peak level at 6 h after Aβ25-35 treatment and gradually decreased after 12 h, but stilled maintained a significantly higher level at 24 h,compared with 0 min, 30 min （P〈0.05）. The total tau underwent no significant changes in response to Aβ25-35 treatment （P〉0.05）. Epo pretreatment at 5, 10, and 20 U efficiently inhibited Aβ25-35-induced tau phosphorylation in comparison with that in the control cells（P〈0.05）, and application of LY294002 resulted in obvious inhibition of the effect of Epo. Conclusion Epo can inhibit Aβ25-35-induced tau phosphorylation via PI3K/Akt signaling pathway, and this finding provides an important theoretical basis for studying the pathogenesis and management of AD.