金雀异黄酮对冈田酸诱导大鼠血小板Tau蛋白过度磷酸化的保护作用及机制

Protective effects and mechanism of genistein on Tau protein hyperphosphorylation in platelets of rat induced by okadaic acid

目的探讨金雀异黄酮(GS)对冈田酸(OA)诱导大鼠血小板Tau蛋白过度磷酸化的保护作用及其机制。方法通过MTT法观察不同浓度的OA对大鼠血小板存活的影响。应用蛋白免疫印迹方法,检测OA处理后大鼠血小板Tau蛋白磷酸化Ser199和Thr231位点、Tau-1(非磷酸化蛋白)、Tau-5(总Tau蛋白)、糖原合成激酶3β(GSK-3β)和抑制性磷酸化GSK-3βSer9位点的表达情况;并观察GS对OA诱导大鼠血小板上述指标变化的影响。结果 MTT检测结果表明,不同浓度的OA对血小板均有损伤作用,其中80 nmol/L OA对大鼠血小板损伤程度较为合适。Western blot检测结果表明,80 nmol/L OA处理12 h后,Tau蛋白Ser199位点的磷酸化水平增高(P<0.05),Thr231位点的磷酸化水平显著增高(P<0.01);Tau-1的磷酸化水平显著降低(P<0.01);Tau-5无明显变化;GSK-3β的表达无变化而抑制性磷酸化GSK-3βSer9位点的表达减少(P<0.05)。用0.5μmol/L GS预处理后可减轻OA所诱导的Tau蛋白过度磷酸化,同时抑制性磷酸化GSK-3βSer9位点的表达增加(P<0.05)。结论 OA可诱导大鼠血小板Tau蛋白Ser199和Thr231位点的磷酸化水平增高,该作用可能通过激活GSK-3β实现。GS可能通过抑制血小板GSK-3β的活性,最终达到减轻OA所诱导的大鼠血小板Tau蛋白过度磷酸化的作用。

Objective To investigate the protective effects and mechanism of genistein （GS） on Tan protein hyperphospho- rylation in platelets of rat induced by okadaic acid （OA）. Methods The effects of different concentrations of OA on rat platelets survival was measured by MTY assay. Western blot was applied to detect Tan protein phosphorylation in the sites of Ser199 and Thr231, Tau-1 （Non-phosphorylated protein）, Tau-5 （Total Tau protein）, Glycogen synthase kinase-3β （GSK-3β）and inhibitive phosphorylated site of GSK-3β Ser9 and observe the effects of GS on the change of these index in platelets of rat induced by OA. Results MTT assay showed that different concentrations of OA could damage the viability rate of the platelets of rat, and 80 nmol/L OA injury of rat platelets is more appropriate. After 12 h exposure to 80 nmol/L OA, Western blot showed that the increased Tau protein phosphorylation in the site of Ser199 （P〈0.05）, the significant increase of phosphorylation in the site of Thr231 （P〈0.01）, significant decrease of Tan-1 （P〈0.01） and no significant change of the Tan-5 and GSK-3β and decrease of inhibitive phosphorylat- ed site of GSK-β Ser9 （P〈0.05）. Pretreatment with 0.5 μmol/L GS reduced OA inducing Tau protein Hyperphosphorylation, but the expression of inhibitive phosphorylated site of GSK-3β Ser9 was increased （P〈0.05）. Conclusion The increase of Tan phos- phorylation in the sites of Ser199 and Thr231 may be induced by OA in platelets of rat by GSK-β activation. GS may attenuate Tau protein hyperphosphorylation induced by OA in platelets of rat by inhibiting the activation of GSK-β.