乌头碱抑制GSK-3β对抗β淀粉样蛋白诱导的神经细胞损伤

Aconitine attenuates neuronal damage induced byβ-amyloid via inhibition of GSK-3β

本研究旨在探讨乌头碱抑制糖原合成酶激酶3β(GSK-3β)对抗β淀粉样蛋白片段1-40(Aβ_(1-40))诱导的神经细胞损伤的药理作用。基于细胞安全性测试设置5 nmol/L为后续实验的乌头碱适宜浓度。以20μmol/L的Aβ1-40孵育24 h建立SH-SY5Y神经细胞损伤细胞模型。设置3个实验分组:正常对照组、Aβ_(1-40)细胞损伤模型对照组、乌头碱干预组,后者以5 nmol/L的乌头碱预孵育12 h。ELISA检测细胞培养上清液乳酸脱氢酶(LDH),流式细胞术分析细胞凋亡与坏死,Western blotting检测细胞GSK-3β磷酸化水平。结果显示,乌头碱干预组细胞培养上清液LDH水平、细胞凋亡率与坏死率以及细胞的GSK-3β磷酸化水平均显著低于模型对照组,提示乌头碱可显著减轻Aβ1-40导致的细胞损伤,此作用可能与其抑制GSK-3β过磷酸化有关。

This research was designed to investigate the pharmacological effects of aconitine on attenuating neuronal damage induced by amyloidβprotein fragment 1-40(Aβ_(1-40))via inhibition of glycogen synthase kinase-3β(GSK-3β).Based on cell safety tests,5 nmol/L was set as the appropriate concentration of aconitine used in following experiments.A cell-damage model was established by incubating SH-SY5Y cells with Aβ_(1-40)(20μmol/L)for 24 hours.3 experimental groups were set up:the normal control,Aβ1-40-damaged cell model control and aconitine-treated group,while the last group was pre-treated with aconitine(5 nmol/L)for 12 hours.The lactate dehydrogenase(LDH)in cell culture supernatant was measured using ELISA.The apoptosis and necrosis were analyzed by flow cytometry.The cell GSK-3βprotein phosphorylation was detected by Western blotting.Compared with the Model,the significantly lower LDH in cell culture supernatant,cell apoptosis or necrosis rate and phosphorylated GSK-3βlevels were detected in the aconitine-treated group.These data suggested that aconitine can substantially alleviate the neuronal damage caused by Aβ1-40,and the benefit may be related to the inhibition of GSK-3βhyper-phosphorylation by aconitine.