知母皂苷元通过上调PI3K/Akt信号通路减轻淀粉样β蛋白诱导的乳大鼠海马神经元突触损伤

Sarsasapogenin inhibits amyloid beta-protein induced decrease of synaptophysin in hippocampal neurons of neonatal rats via up-regulating PI3K/Akt/GSK3 pathway

目的探讨知母皂苷元(Sar)减轻淀粉样β蛋白(Aβ)诱导的海马神经元突触损伤的信号转导机制。方法取出生0～24 h SD乳大鼠海马神经元,体外培养7 d。海马神经元分别加入磷脂酰肌醇-3-激酶(PI3K)特异性阻断剂LY294002 30μmo.lL-1或蛋白激酶B(Akt)特异性阻断剂曲西立滨1μmol.L-11 h后,加Sar 30和100μmo.l L-1作用1 h,再加Aβ1-4250 nmo.l L-1作用24 h。应用突触囊泡蛋白(SYP)免疫荧光染色观察突触的改变。Western蛋白质印迹法检测海马神经元SYP、磷酸化Akt(p-Akt)和磷酸化糖原合成酶3β(p-GSK3β)表达水平的改变。结果与正常对照组相比,Aβ1-42组培养的海马神经元SYP,p-Akt和p-GSK3β表达水平明显减低(P<0.01)。与Aβ1-42组相比,Aβ1-42+Sar 30和100μmo.l L-1组海马神经元SYP,p-Akt和p-GSK3β表达水平明显增加(P<0.01)。给予LY294002作用后,SYP和p-Akt表达水平明显降低(P<0.05)。给予曲西立滨作用后,SYP和p-GSK3β表达水平明显降低(P<0.05)。单独给予LY294002,海马神经元SYP表达无变化,p-Akt表达水平明显降低(P<0.01)。单独给予曲西立滨,海马神经元SYP和p-GSK3β蛋白表达水平明显降低(P<0.01)。结论 Sar通过上调PI3K/Akt/GSK3β信号通路对抗Aβ1-42诱导的海马神经元突触损伤。

OBJECTIVE To investigate the signaling mechanisms responsible for the effect of sarsasapogenin（Sar） against the amyloid beta-protein fragment 1-42（Aβ1-42） induced decrease of synaptophysin（SYP） in hippocampal neurons.METHODS The primary neurons from the hippocampus of neonatal rats were cultured for 7 d,then exposed to Aβ1-42 50 nmol·L-1 for 24 h in the presence or absence of Sar 30 and 100 μmol·L-1.LY294002 30 μmol·L-1 and triciribine 1 μmol·L-1 were added to the cultures 1 h before Sar treatment.The protein expression of SYP was determined by immunofluorescence and Western blotting.Phosphorylated Akt（p-Akt） and glycogen synthase kinase 3（p-GSK3β） in the cell extracts were detected by Western blotting.RESULTS Compared with normal control group,Aβ1-42 decreased the protein expression of SYP,p-Akt and p-GSK3β（P0.01）,which was significantly prevented by Sar 30 and 100 μmol·L-1（P0.01）.LY294002 and triciribine abrogated the effect of Sar on SYP expression.Sar prevented the Aβ1-42-induced decrease in p-Akt and p-GSK3β,and this effect of Sar was abrogated by LY294002 and triciribine.CONCLUSION Activation of the PI3K/Akt/GSK3β pathway is responsible for the protective effect of Sar against Aβ1-42 neurotoxicity in cultured hippocampal neurons.