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目的:探讨银杏内酯B对谷氨酸诱导的人神经母细胞瘤(SH-SY5Y)细胞凋亡的保护作用及其机制。方法:用2mmol/L谷氨酸诱导SH-SY5Y细胞凋亡建立模型,以不同浓度的银杏内酯B(10、50和250μmol/L)进行保护,MTT比色法检测细胞存活率;Hoechst 33258染色法观察药物作用后细胞凋亡的形态学变化;流式细胞仪检测细胞凋亡率;Fura-2/AM双波长荧光法测定细胞内钙离子浓度;Western blot检测线粒体和胞浆中Cyt C蛋白的表达。结果:2 mmol/L谷氨酸作用SH-SY5Y细胞24 h后细胞存活率为0.721±0.013,与正常组相比明显降低;细胞核固缩裂解,呈凋亡特征性改变;细胞凋亡率为(13.27±0.08)%、细胞内钙离子浓度为359±11 nmol/L,均明显升高。与凋亡组相比,银杏内酯B(10、50、250)μmol/L保护组细胞存活率分别为0.768±0.014、0.819±0.013、0.897±0.010,均明显升高;细胞核核固缩裂解减少,形态有所改善;细胞凋亡率分别为11.33±0.57、7.02±0.47、5.85±0.24;细胞内钙离子浓度为302±9 nmol/L、243±9 nmol/L、186±8 nmol/L,均明显降低;Western blot结果显示,与凋亡组相比银杏内酯B保护组可以显著减少细胞线粒体以及胞浆中Cyt C蛋白的含量。结论:银杏内酯B对谷氨酸诱导的SH-SY5Y细胞凋亡具有保护作用,其机制可能与银杏内酯B降低了细胞内钙离子的浓度及阻断了Cyt C从线粒体释放入胞浆中有关。
Objective: To investigate the protective effect and mechanism of ginkgolide B on apoptosis of human neuroblastoma (SH-SY5Y) cells induced by glutamate. Methods: Apoptosis of SH-SY5Y cells was induced by 2 mmol / L glutamic acid and protected by different concentrations of ginkgolide B (10, 50 and 250 μmol / L). Cell viability was measured by MTT colorimetric assay. Hoechst 33258 The morphological changes of apoptotic cells were observed by the staining method. The apoptosis rate was detected by flow cytometry. The intracellular calcium concentration was determined by Fura-2 / AM dual-wavelength fluorescence method. The Cyt C Protein expression. Results: The cell survival rate of SH-SY5Y cells treated with 2 mmol / L glutamate for 24 h was 0.721 ± 0.013, which was significantly lower than that of the normal group. The cell nucleus pyknosis was characterized by apoptotic changes. The apoptosis rate was (13.27 ± 0.08)%, the intracellular calcium concentration was 359 ± 11 nmol / L, both significantly increased. Compared with the apoptotic group, the survival rates of Ginkgolide B (10,50,250) μmol / L group were 0.768 ± 0.014, 0.819 ± 0.013 and 0.897 ± 0.010, respectively, which were significantly increased. The nuclear pyknosis The cell apoptosis rates were 11.33 ± 0.57, 7.02 ± 0.47 and 5.85 ± 0.24, respectively. The intracellular calcium concentrations were 302 ± 9 nmol / L, 243 ± 9 nmol / L, 186 ± 8 nmol / L, all decreased significantly. Compared with the apoptosis group, Ginkgolide B protective group could significantly reduce the content of Cyt C protein in mitochondria and cytoplasm. CONCLUSION: Ginkgolide B has a protective effect on glutamate-induced apoptosis in SH-SY5Y cells, which may be related to the fact that Ginkgolide B reduces the intracellular calcium concentration and blocks the release of Cyt C from mitochondria into the cytoplasm In the relevant.