中药复方金思维含药血清调控PI3K/AKT/GSK-3β信号通路对拟阿尔茨海默病细胞模型tau蛋白磷酸化的影响

Effects of traditional Chinese medicine compound GAPT serum on regulation of PI3K/AKT/GSK-3βsignaling pathway through Ptau in Alzheimer disease cell models

目的探讨中药复方金思维含药血清对拟阿尔茨海默病(Alzheimer disease,AD)细胞模型tau蛋白磷酸化的影响及作用机制。方法采用Aβ25-35诱导人神经母细胞瘤细胞(SH-SY5Y)建立拟AD细胞模型,CCK-8法筛选金思维含药血清、PI3K/AKT/GSK-3β通路抑制剂LY294002最佳作用浓度。将SH-SY5Y细胞分为正常组、模型组、金思维低剂量组、金思维中剂量组、金思维高剂量组以及LY294002组。蛋白免疫印迹法检测金思维含药血清对SH-SY5Y细胞tau蛋白磷酸化以及PI3K/AKT/GSK-3β通路相关蛋白的影响。结果20μmol/L Aβ25-35作用于SH-SY5Y细胞24 h可建立最佳拟AD细胞模型。相对于正常组,模型组P-AKT显著降低(P<0.05),P-tau显著升高(P<0.01)。相对于模型组,金思维低剂量含药血清组PI3K的表达量显著升高(P<0.05),高剂量含药血清组GSK-3β的表达量显著降低(P<0.05),中剂量和高剂量含药血清组P-tau的表达量显著降低(P<0.01、P<0.001)。加入通路抑制剂LY294002后,P-PI3K、PI3K、P-AKT、AKT、GSK-3β、P-tau蛋白的表达量较模型组差异无统计学意义(P>0.05)。结论金思维含药血清可降低拟AD细胞模型tau蛋白磷酸化水平,其机制可能与PI3K/AKT/GSK-3β通路的激活有关。

Objective To investigate the effect and mechanism of traditional Chinese medicine compound GAPT serum on regulation of P-tau in Alzheimer disease(AD)cell models.Methods Aβ25-35 induced human neuroblastoma cells(SH-SY5Y)were used to establish an AD cell model,and the optimal concentration of PI3K/AKT/GSK-3βsignaling pathway inhibitor LY294002 and GAPT serum were screened by CCK-8.SH-SY5Y cells were divided into normal group,model group,GAPT low-dose group,GAPT medium-dose group,GAPT high-dose group and LY294002 group.The effect of GAPT serum on tau protein phosphorylation and PI3K/AKT/GSK-3βpathway related proteins in SH-SY5Y cells was detected by western blot.Results SH-SY5Y cells were treated with 20μmol/L Aβ25-35 for 24 h to establish the best AD cell model.Compared with the normal group,P-AKT was significantly reduced(P<0.05)and P-tau was significantly increased(P<0.01).Compared with the model group,the expression of PI3K(P<0.05)was significantly increased in the GAPT low-dose group,the expression of GSK-3β(P<0.05)in the GAPT high-dose group,and the expression of P-tau(P<0.01)in the GAPT medium-dose and high-dose group(P<0.01)(P<0.001).After the addition of pathway inhibitor LY294002,the expression of P-PI3K,PI3K,P-AKT,AKT,GSK-3βand P-tau was not significantly different from that in the model group(P>0.05).Conclusions GAPT serum can reduce the expression of P-tau in AD cell models,and the mechanism may be related to the activation of PI3K/AKT/GSK-3βsignaling pathway.