白英生物碱对叔丁基过氧化氢诱导的人神经瘤母细胞系SH-SY5Y细胞氧化应激和凋亡的作用机制研究

Study on the mechanisms of solanum lyratum thunberg alkaloid against tert-butyl hydroperoxide-induced oxidative stress and apoptosis in human neuroma metrocyte cell line SH-SY5Y cells

目的探讨白英生物碱对叔丁基过氧化氢(tBHP)诱导的人神经瘤母细胞系SH-SY5Y细胞氧化应激和细胞凋亡的作用。方法将人神经瘤母细胞系SH-SY5Y细胞分为对照组、tBHP模型组、白英生物碱(10、20、40 mg/L)组和维生素E(100μmol/L)组。采用甲基噻唑蓝法检测细胞活力,倒置显微镜观察细胞形态学变化,TUNEL法检测细胞凋亡,流式分析法检测线粒体膜电位(罗丹明123荧光强度)和活性氧水平[二氯荧光素(DCF)荧光强度],蛋白质印迹法检测细胞凋亡相关蛋白Bcl-2和Bax的表达。结果 tBHP模型组细胞存活率和罗丹明123荧光强度明显低于对照组,细胞凋亡率和DCF荧光强度明显高于对照组;10、20、40 mg/L白英生物碱组和100μmol/L维生素E组细胞存活率和罗丹明123荧光强度明显高于tBHP模型组[(48±18)%、(61±3)%、(74±19)%、(86±35)%比(43±3)%;(60. 84±0. 11)%、(71.32±0.24)%、(79.68±0.15)%、(70.47±0. 32)%比(55.43±0. 23)%],细胞凋亡率和DCF荧光强度明显低于tBHP模型组[(31.11±0.09)%、(18.34±0.21)%、(13. 58±0. 51)%、(22. 82±0. 39)%比(37. 16±0.27)%;(62.34±0.53)%、(41.22±0.44)%、(38.57±0.65)%、(49.18±0.52)%比(75.42±0.46)%],差异均有统计学意义(均P<0.05)。20mg/L白英生物碱和100μmol/L维生素E能增加tBHP诱导的SH-SY5Y细胞中Bcl-2蛋白表达,同时抑制Bax蛋白表达。结论白英生物碱能够抑制SH-SY5Y细胞中活性氧产生,降低tBHP诱导的SH-SY5Y细胞的氧化应激,减少细胞凋亡。

Objective To investigate the mechanisms of solanum lyratum thunberg alkaloid（STA） against tert-butyl hydroperoxide（tBHP）-induced oxidative stress and apoptosis in human neuroma metrocyte cell line SH-SY5Y cells in vitro. Methods Human neuroma metrocyte cell line SH-SY5Y cells were divided into control group, tBHP model group, STA（10, 20, 40 mg/L） group and vitamin E（100 mol/L） group. Cell viability was measured by MTT assay. Cell morphology was observed by light microscope. Cell apoptosis was detected by TUNEL assay. Mitochondrial membrane potential[fluorescence intensity of rhodamine-123（Rh123）] and reactive oxygen species（ROS）[fluorescence intensity of dichlorofluorescein（DCF）] were detected by flow cytometric assay. Expressions of Bax and Bcl-2 protein were tested by western blotting. Results Cell survival rate and fluorescence intensity of Rh123 in tBHP model group were significantly lower than those in control group; apoptosis rate and fluorescence intensity of DCF in tBHP model group were significantly higher than those in control group; cell survival rate and fluorescence intensity of Rh123 in 10, 20, 40 mg/L STA groups and vitamin E group were significantly higher than those in tBHP model group[（48±18）%,（61±3）%,（74±19）%,（86±35）% vs （43±3）%; （60.84±0.11）%,（71.32±0.24）%,（79.68±0.15）%,（70.47±0.32）% vs （55.43±0.23）%]; apoptosis rate and fluorescence intensity of DCF in 10, 20, 40 mg/L STA groups and vitamin E group were significantly lower than those in tBHP model group[（31.11±0.09）%,（18.34±0.21）%,（13.58±0.51）%,（22.82±0.39）% vs （37.16±0.27）%; （62.34±0.53）%,（41.22±0.44）%,（38.57±0.65）%,（49.18±0.52）% vs （75.42±0.46）%]（all P〈0.05）. Western blotting results indicated that 20 mg/L STA and 100 μmol/L vitamin E could increase the expression of BCL-2 protein and inhibit the expression of BAX protein in tBHP-induced SH-SY5Y cells. Conclusion STA can significantly inhibit tBHP-induced ROS production, oxidative stress and apoptosis in SH-SY5Y cells.