知母皂苷元对谷氨酸诱导的皮层神经元损伤的保护作用及其机制

Protective effects of Sarsasapogenin against glutamate-induced neurotoxicity in the cultured cortical neurons in rats and the mechanisms

目的:观察知母皂苷元(Sarsasapogenin,SAR)对谷氨酸引起的皮层神经元损伤的保护作用及其机制研究。方法:大鼠乳鼠大脑皮层神经元,培养7 d后用于实验。倒置相差显微镜观察神经元树突生长情况;用MTT法测定细胞活力;Hoechst33258核染色观察细胞凋亡的形态学改变;Western印迹法检测神经元SYP、p-PDK1、p-Akt473、p-mTOR蛋白表达水平。结果:形态学观察结果显示谷氨酸(150μmol/L)可明显抑制神经元树突的生长发育。谷氨酸150μmol/L+SAR 30μmol/L组可明显对抗谷氨酸的抑制作用。谷氨酸150μmol/L+SAR30μmol/L+各阻断剂组能够阻断SAR的作用。谷氨酸可降低神经元细胞活力及增加神经元细胞凋亡百分比。谷氨酸150μmol/L+SAR 30μmol/L组能明显抑制谷氨酸引起的神经元细胞活力降低及细胞凋亡百分比增加。谷氨酸150μmol/L+SAR 30μmol/L+各阻断剂组能够阻断SAR的保护作用。Western印迹结果显示谷氨酸可明显降低SYP、p-PDK1、p-Akt473、p-mTOR蛋白表达水平。谷氨酸150μmol/L+SAR 30μmol/L组可明显增加SYP、p-PDK1、p-Akt473及p-mTOR蛋白表达水平。谷氨酸150μmol/L+SAR 30μmol/L+各阻断剂组能够明显降低神经元SYP、p-PDK1、p-Akt473及p-mTOR的蛋白表达水平。结论:SAR对谷氨酸引起的体外培养皮层神经元的损伤有保护作用,这种作用可能与PI3K/Akt/mTOR信号转导通路有关。

Objective：To investigate whether Sarsasapogenin can protect cortical neurons from glutamate-induced neurotoxicity and the mechanisms of signal transduction.Methods：Cortical neurons cultures were prepared from postnatal Sprague-Dawley（SD） rats in 24 h.After 7 d cultured,cortical neurons were used for experiment,using inverted phase-contrast microscopy,we analyzed the total dendrite branch length（TDBL）,number of primary-order dendrite（PDN）,maximum branch order（MBO）,and soma size.The cell viability was assessed by the 3（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT） assay.The neuronal apoptosis was quantified by scoring the percentage of cells with apoptotic nuclear morphology after Hoechst 33258 staining.Western blotting was used for SYP,p-PDK1,p-Akt473,p-mTOR protein expression.Results：Incubation of cortical neurons with Glu（150 μmol/L） for 48 h decreases in TDBL,PDN,MBO,and soma size.The decrease of cell viability and the increase of apoptotic cells in cultured cortical neurons were observed incubated with Glu for 24 h compared with the normal controls.Glutamate（150 μmol/L） group induced the decreases of synaptophysin,p-PDK1,p-Akt473,p-mTOR protein expression.Sarsasapogenin inhibited glutamate-induced decrease in TDBL,PDN,MBO,soma size,and cell viability and increase of apoptotic cells,decreases of synaptophysin,p-PDK1,p-Akt473,p-mTOR protein expression.Glu ＋ SAR30 ＋ LY294002 group,Glu ＋ SAR30 ＋ TCBN group,Glu ＋ SAR30 ＋ Rapa group decreases in TDBL,PDN,MBO,soma size,and cell viability,increases in apoptotic cells.Glu ＋ SAR30 ＋ LY294002 group induced the decrease in synaptophysin,p-PDK1,p-Akt473 and p-mTOR protein expression.Glu ＋ SAR30 ＋ TCBN group decreased synaptophysin,p-Akt473,and p-mTOR protein expression.Glu ＋ SAR30 ＋ Rapa group decreased synaptophysin,p-mTOR protein expression.Conclusion：We demonstrate that Sarsasapogenin can protect cultured cortical neutons from glutamate-induced damage,which mainly via activated PI3K/Akt/mTOR signal pathway.