抑制PI3K/AKT/GSK-3β信号通路对冈田酸诱导原代星形胶质细胞衰老情况的影响

Effect of inhibiting PI3K/AKT/GSK-3β signaling pathway on the senescence of primary astrocytes induced by okadaic acid

目的研究冈田酸(OA)诱导的原代星形胶质细胞中通过抑制磷酯酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)/糖原合成激酶(GSK)-3β信号通路对p21、p53衰老蛋白表达水平和衰老阳性细胞表达的影响,探讨OA诱导星形胶质细胞衰老情况及相关机制。方法免疫荧光鉴定原代星形胶质细胞,CCK8法筛选OA药物浓度,然后用LY294002、OA分别处理或联合处理细胞,Western印迹测定细胞中p-PI3K、p-AKT、p-GSK-3β、p21、p53蛋白表达水平的变化及β-半乳糖苷酶染色检测衰老阳性细胞。结果免疫荧光鉴定原代星形胶质细胞结果显示其纯度达到95%以上,CCK8法筛选出OA药物处理浓度为30 nmol/L,OA组中p-PI3K、p-AKT、p-GSK-3β、p21、p53蛋白表达较正常对照组显著升高(P<0.05),衰老阳性细胞数明显增加,但LY294002+OA组p-PI3K、p-AKT、p-GSK-3β、p21、p53蛋白表达水平较OA组明显降低,并且衰老阳性细胞数也明显减少(P<0.05)。结论通过OA处理能使星形胶质细胞发生衰老,LY294002处理能通过抑制PI3K/AKT/GSK-3β信号通路缓解细胞的衰老情况,提示高磷酸化Tau诱导星形胶质细胞衰老可能与PI3K/AKT/G3K-3β信号通路密切相关。

Objective To study the effects of the inhibition of phosphatidylinositol 3-hydroxykinase(PI3K)/protein kinase B(AKT)/glycogen synthetic kinase(GSK)-3βsignaling pathway in the primary astrocytes induced by okadaic acid(OA)on the expression levels of p21 and p53 senescence proteins and the expression of senescence-positive cells,to explore the senescence of astrocytes induced by OA and related mechanisms.Methods Immunofluorescence was used to identify primary astrocytes,the concentration of OA drugs was screened by CCK8 method,and then the cells were treated separately or combined with LY294002 and OA,the expression levels of p-PI3K,p-AKT,p-GSK-3β,p21 and p53 protein were determined by Western blot and senescence-positive cells were determined byβ-galactosidase staining.Results The results of immunofluorescence identification of primary astrocytes showed that the purity of the primary astrocytes was more than 95%.The concentration of OA drugs screened by CCK8 was 30 nmol/L.Compared with the normal control group,the expression levels of p-PI3K,p-AKT,p-GSK-3β,p21 and p53 protein were significantly increased,and the number of senescence-positive cells was significantly increased in OA group(P<0.05).Compared with OA group,the expression levels of p-PI3K,p-AKT,p-GSK-3β,p21 and p53 protein were significantly decreased,and the number of senescence-positive cells was significantly decreased in LY294002+OA group(P<0.05).Conclusions OA treatment could make astrocytes senescence,LY294002 treatment could alleviate cell senescence by inhibiting PI3K/AKT/GSK-3βsignaling pathway,which suggest that hyperphosphorylated Tau inducing astrocyte senescence might closely relate to PI3K/AKT/G3K-3βsignaling pathways.