知母皂苷元对谷氨酸引起的皮层神经元损伤的保护作用研究

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。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发育情况;用MTT法测定细胞活力;Ho-echst33258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、caspase-3、钙蛋白酶Ⅰ蛋白表达水平。结果形态学观察结果显示谷氨酸可明显抑制神经元树突的生长发育,表现为神经元树突总长度明显降低、一级树突数目明显减少、最大分支级数明显减少及胞体面积缩小。SAR(10、30、100μmol.L-1)可明显抑制谷氨酸对神经元树突生长发育的抑制作用,并呈明显浓度依赖。MTT和Ho-echst33258核染色结果显示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。SAR(10、30、100μmol.L-1)能明显对抗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。Western印迹结果显示谷氨酸可明显降低SYP蛋白表达水平及增加活性caspase-3、钙蛋白酶Ⅰ蛋白表达水平。SAR(10、30、100μmol.L-1)可明显对抗谷氨酸引起SYP蛋白表达降低及活性caspase-3、钙蛋白酶Ⅰ蛋白表达增加。结论知母皂苷元能够明显对抗谷氨酸引起的皮层神经元损伤作用。

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

Aim To investigate whether Sarsasapogenin can protect cortical neurons from glutamate-induced neurotoxicity.Methods Cortical neurons cultures were prepared from postnatal Sprague-Dawley(SD) rats in 24 h.After cultured 7 d,cortical neurons were used for experiment with inverted phase-contrast microscopy.The total dendrite branch length(TDBL),number of primary-order dendrite(PDN),maximum branch order(MBO),and soma size were analyzed.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 blot was used for SYP,caspase-3 and Calpain Ⅰ protein expression.Results Incubation of cortical neurons with Glu(150 μmol·L-1) for 48 h decreased TDBL,PDN,MBO,and soma size.Compared with the normal controls,the decrease of cell viability and the increase of apoptotic cells in cultured cortical neurons were observed after 24 h's incubation with Glu.Glutamate induced the decreases of SYP and the increases of active caspase-3 and μ-calpain protein expression.Sarsasapogenin inhibited glutamate-induced decrease in TDBL,PDN,MBO,soma size,and cell viability and increase of apoptotic cells,decreases of synaptophysin protein expression,increases of active caspase-3 and μ-calpain protein expression.Conclusion Sarsasapogenin can protect cultured cortical neutons from glutamate-induced damage.