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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 the protective effect and mechanism of Sarsasapogenin (SAR) on cortical neurons induced by glutamate. Methods: The neurons of the rat neonatal rat cerebral cortex were cultured for 7 days and then used in the experiment. The morphological changes of apoptosis were observed by Hoechst33258 nuclear staining. The expressions of SYP, p-PDK1, p-Akt473 and p-mTOR protein in neurons were detected by Western blotting The expression level. Results: Morphological observation showed that glutamate (150μmol / L) could significantly inhibit the dendritic growth of neurons. Glutamate 150μmol / L + SAR 30μmol / L group can significantly antagonize the inhibition of glutamate. Glutamate 150μmol / L + SAR30μmol / L + blocking agent can block the effect of SAR. Glutamate reduces neuronal cell viability and increases the percentage of apoptotic neuronal cells. Glutamate 150μmol / L + SAR 30μmol / L group could significantly inhibit glutamate-induced neuronal cell viability and increase the percentage of apoptosis. The glutamate 150μmol / L + SAR 30μmol / L + blockade could block the protective effect of SAR. Western blot results showed that glutamate can significantly reduce the expression of SYP, p-PDK1, p-Akt473, p-mTOR protein. Glutamate 150μmol / L + SAR 30μmol / L group could significantly increase the expression of SYP, p-PDK1, p-Akt473 and p-mTOR protein. Glutamate 150μmol / L + SAR 30μmol / L + could significantly decrease the protein expression of SYP, p-PDK1, p-Akt473 and p-mTOR in neurons. CONCLUSIONS: SAR attenuates glutamate-induced injury of cultured cortical neurons in vitro and this effect may be related to the PI3K / Akt / mTOR signaling pathway.