白藜芦醇对内质网应激介导的原代神经元细胞作用机制

Mechanism of Action of Resveratrol in Attenuating Endoplasmic Reticulum Stress Induced by Tunicamycin in Primary Neurons

目的:探讨白藜芦醇(resveratrol,Res)对衣霉素(tunicamycin,TM)诱导内质网应激(endoplasmic reticulum stress,ERS)模型的神经保护作用及其作用机制。方法:取怀孕18 d孕鼠分离的胎鼠海马神经元进行原代培养,设置对照组(不作处理)、TM组(质量浓度2.5μg/mL)、Res组(5μmol/L)和Res+TM组(5μmol/L Res预处理+质量浓度2.5μg/mL TM进一步共处理),采用细胞计数试剂盒(cell counting kit-8,CCK8)检测海马神经元存活率并筛选TM和Res最佳作用浓度和时间;利用流式细胞仪分别检测原代神经元凋亡水平和周期状态,通过Western blot分析细胞ERS、自噬、凋亡和周期的相关蛋白变化。结果:5μmol/L Res预处理10 h后能减轻TM对原代神经元细胞存活率的抑制作用,使神经元细胞周期重激活,促使其由G1/G0期向S期进行。与TM组相比,Res预处理10 h组(R10T组)的葡萄糖调节蛋白78(glucose regulated protein 78,GRP78)、Caspase 3相对表达量极显著或显著降低(P<0.01、P<0.05),而自噬相关蛋白Beclin-1、微管结合蛋白1轻链3(microtubule-associated protein1 light chain 3,LC3)B相对表达量显著升高(P<0.05),磷酸化糖原合成酶激酶3β(phosphorylated glycogen synthase kinase 3β,p-GSK3β)、凋亡相关蛋白B淋巴细胞瘤-2(B-cell lymphoma 2,Bcl2)蛋白相对表达量高度显著或极显著升高(P<0.001、P<0.01),抑制TM引起的细胞凋亡。结论:Res可以减轻TM引起的原代神经元ERS,对原代神经元起到保护作用,其作用机制与Res对细胞存活率、周期分布和自噬、凋亡相关蛋白表达调节有关。

Objective:To investigate the neuroprotective effect of resveratrol(Res)on tunicamycin(TM)-induced endoplasmic reticulum stress(ERS)and its mechanism.Methods:Fetal hippocampal neurons from female mice at 18 days of pregnancy were used for primary culture.The experiment was divided into a control group(without drug treatment),a TM group(2.5μg/mL),a Res group(5μmol/L)and a Res+TM group(5μmol/L Res+2.5μg/mL TM).Cell Counting Kit-8(CCK8)was used to detect the survival rate of hippocampal neurons and the optimal concentration and action time of TM and Res were determined.Flow cytometry was used to detect the cell apoptosis and cycle of primary neurons,and Western blot was used to analyze the expression of proteins related to endoplasmic reticulum stress,autophagy,apoptosis and cell cycle.Results:Preconditioning with Res(5μmol/L)for 10 h could reduce the inhibitory effect of TM on the survival rate of primary neurons and reactivate the cell cycle of neurons,promoting its transition from G1/G0 to S phase.Compared with the TM group,preconditioning with Res for 10 h significantly decreased the relative expression levels of glucose-regulated protein 78(GRP78)(P<0.01),caspase 3(P<0.05),but significantly increased the protein expression levels of autophagyrelated protein Beclin-1(P<0.05),microtubule-associated protein 1 light chain 3B(LC3B)(P<0.05),phosphorylated glycogen synthase kinase 3β(p-GSK3β)(P<0.001)and apoptosis-related protein B-cell lymphoma 2(Bcl2)(P<0.01),as well as inhibited cell apoptosis induced by TM.Conclusion:Res can attenuate TM-induced ERS in primary neurons and protect primary neurons.The mechanism is related to the regulation of Res on cell survival rate,cell cycle distribution and the expression of autophagy-and apoptosis-related proteins.