摘要目的 观察雌二醇(E2)对糖原合成激酶-3β(GSK-3β)活性的影响并其机制.方法 E2作用于大鼠原代皮层神经元,噻唑蓝(MTT)法检测其对细胞活力的影响;放射免疫法测定其对GSK-3活性的影响,Western blot检测其对GSK-3β表达的影响;检测雌激素受体抑制剂ICI-182780和各种信号转导激酶抑制剂对E2诱导的GSK-3β磷酸化和对GSK-3活性的影响.结果 在1、10、100、200、500 nmol/L浓度范围内,相对于对照组,E2对大鼠皮层神经元的细胞活力分别为98.4％、107.5％、105.8％、95.6％和82.3％;1、10、100 nmol/L的E2能够剂量依赖性的降低GSK-3的活性,分别为对照组的85.6％ 、78.5％和65.4％;Western blot显示E2剂量依赖性的增加了GSK-3β磷酸化(9Ser),而对非磷酸化的GSK-3β无显著影响;雌激素受体抑制剂ICI-182780拮抗了E2对GSK-3β的磷酸化作用;E2诱导的GSK-3β的磷酸化作用能被丝裂原活化蛋白激酶(MAPK)的抑制剂PD98059、蛋白激酶C(PKC)抑制剂GF109203X所部分抑制,而Akt的抑制剂Akt-I对其无显著影响;E2降低GSK-3活性至对照组的65.4％ (P =0.000),加入ICI、PD98059、GF109203X和Akt-I后,E2诱导的GSK-3的活性分别为对照组的98.7％、89.6％、94.9％和67.3％,这与GSK磷酸化表达检测相一致.结论 E2能够降低GSK-3活性,表现为使GSK-3β磷酸化(9Ser)增加;E2对GSK-3β作用是雌激素受体依赖性的,其对GSK-3β的磷酸化作用可能是通过MAPK和PKC信号通路所介导的.

abstractsObjective To investigate the effects of 17β-estradiol (E2) on glycogen synthase kinase-3β (GSK-3β) activity and the possible related mechanisms.Methods E2 was added to rat primary cortical neurons and its effect on cell viability was measured by methyl thiazol tetrazolium (MTF) assays;its effect on GSK-3 activity was detected directedly.The phosphorylated (9Ser) and non-phosphorylated GSK-3β expression after drug treatment was detected by Western blotting.The effects of estrogen receptor blocker and various kinase inhibitors on GSK-3 phosphorylation and GSK-3 activity were also measured.Results As conpared with control group,in a concentration range of 1,10,100,200 and 500 nmol/L,E2 didn't significantly decrease cell viability in rat primary cortical neurons (the corresponding cell viability was 98.4％,107.5％,105.8％,95.6％ and 82.3％ respectively).At concentrations of 1,10 and 100 nmol/L,E2 dose-dependently decreased GSK-3 activity (to 85.6％,78.5％ and 65.4％ of control group respectively).Western blotting indicated that E2 also dose-dependently increased GSK-3β (9Ser) phosphorylation without significantly affecting non-phosphorylated GSK-3β expression.The effect of E2 on GSK-33 phosphorylation could be partially blocked by estrogen receptor antagonist ICI-182780.The GSK-3β phosphorylation induced by E2 could be partially reversed by mitogen-activated protein kinase (MAPK) inhibitor PD98059 and protein kinase C (PKC) inhibitor GF109203X,but not by an Akt inhibitor Akt-I.The GSK-3 activity was decreased to 65.4％ (P =0.000) of control group after E2 administration,and this effect could be partially reversed by ICI,PD98059,GF109203X administration,but not by Akt-I.These data were in accordance with those of Western blotting.Conclusion E2 can decrease GSK-3 activity as indicated by increased GSK-3β (9Ser) phosphorylation in an estrogen receptor-dependent manner,and the effect of E2 on GSK-3 phosphorylation may be mediated through MAPK and PKC signaling pathways.