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目的探讨人参皂苷Rg1是否通过调节GSK-3β/PP2A活性而减轻凝聚态Aβ25～35诱导的胎鼠皮层神经元Tau蛋白过度磷酸化。方法选用孕期(18±2)d的SD大鼠,分离纯化胎鼠皮层神经元。实验分为阴性对照组、模型组、LiCl处理组、Rg1预处理组。阴性对照组不加任何处理因素;模型组用20μmol/LAβ25～35作用于皮层神经元12h;LiCl处理组用10mmol/LLiCl和20μmol/LAβ25～35共同作用于皮层神经元12h;Rg1预处理组分别用5、10、20、40、80μmol/LRg1预处理皮层神经元24h,再加入20μmol/LAβ25～35作用12h。通过免疫印迹法和免疫细胞化学染色法检测皮层神经元Tau蛋白磷酸化水平、总Tau蛋白水平和糖原合成酶3β(GSK-3β)表达水平,通过非放射性免疫法检测皮层神经元蛋白磷酸酯酶2A(PP2A)活性。结果模型组Tau蛋白在Ser396、Ser199/202和Thr231位点的磷酸化水平、总Tau蛋白水平和GSK-3β的蛋白表达水平均增加,但PP2A的活性不受影响;LiCl处理组和Rg1预处理组,Tau蛋白在Ser396、Ser199/202和Thr231位点的磷酸化水平、总Tau蛋白水平和GSK-3β的蛋白表达水平均降低(P<0.05)。在Rg1预处理组中,以20μmol/LRg1预处理后Tau蛋白磷酸化水平、总Tau蛋白水平和GSK-3β的表达水平下降最为明显,而且PP2A的活性明显增强(P<0.01)。结论人参皂苷Rg1可通过上调PP2A活性和下调GSK-3β活性从而减轻凝聚态Aβ25～35所诱导的皮层神经元Tau蛋白过度磷酸化。
Objective To investigate whether ginsenoside Rg1 can reduce Tau protein hyperphosphorylation induced by condensed Aβ25-35 in fetal rat cortical neurons by regulating GSK-3β/PP2A activity. Methods Fetal rat cortical neurons were isolated and purified from Sprague-Dawley rats (18 ± 2 days) during pregnancy. The experiments were divided into negative control group, model group, LiCl treatment group, and Rg1 pretreatment group. The negative control group did not add any treatment factors; the model group was treated with 20μmol/LAβ25-35 for 12h in the cortical neurons; the LiCl treatment group was treated with 10mmol/L LiCl and 20μmol/LAβ25-35 for 12h in the cortical neurons; the Rg1 pretreatment group was Cortical neurons were pretreated with 5, 10, 20, 40, and 80 μmol/LRg1 for 24 hours, and 20 μmol/LAβ25-35 was added for 12 hours. The levels of Tau phosphorylation, total tau protein and glycogen synthase 3β (GSK-3β) expression in cortical neurons were detected by immunoblotting and immunocytochemical staining. The cortical neurons were detected by non-radioactive immunoassay. Enzyme 2A (PP2A) activity. Results The phosphorylation level, total tau protein level, and GSK-3β protein level of Tau protein in Ser396, Ser199/202 and Thr231 were increased in the model group, but the activity of PP2A was not affected; LiCl treatment group and Rg1 pretreatment In the group, the phosphorylation levels, total tau levels, and GSK-3β protein levels of Tau at Ser396, Ser199/202, and Thr231 decreased (P<0.05). In Rg1 pretreatment group, the level of Tau phosphorylation, total tau protein and GSK-3β expression were significantly decreased after pretreatment with 20 μmol/LRg1, and the activity of PP2A was significantly increased (P<0.01). Conclusion Ginsenoside Rg1 can reduce the hyperphosphorylation of Tau protein in cortical neurons induced by condensed Aβ25-35 by up-regulating PP2A activity and down-regulating GSK-3β activity.