枸杞多糖调节线粒体动力学改善过氧化氢诱导SH-SY5Y细胞的凋亡

Lycium barbarum polysaccharide regulates mitochondrial dynamics to improve H2O2-induced apoptosis of SH-SY5Y cells

背景:大量研究结果证明,神经退行性疾病与氧化应激损伤和线粒体动力学失衡密切相关。课题组前期研究表明枸杞多糖具有神经保护作用,但其能否通过调控线粒体动力学异常来改善氧化应激损伤引发的细胞凋亡尚不明确。目的:研究枸杞多糖对过氧化氢诱导人源神经母瘤细胞SH-SY5Y凋亡的影响。方法:将SH-SY5Y细胞分3组培养:对照组常规培养24 h,过氧化氢组加入过氧化氢处理24 h,枸杞多糖组加入枸杞多糖处理2 h后再加入过氧化氢处理24 h。处理结束后,采用试剂盒检测细胞沉淀中丙二醛、谷胱甘肽和超氧化物歧化酶水平,JC-1试剂盒检测线粒体膜电位,MTT法检测细胞活力,TUNEL法检测细胞凋亡情况,免疫荧光染色与Western Blot检测线粒体动力学相关蛋白(磷酸化启动蛋白1、线粒体分裂蛋白1、线粒体融合蛋白1、线粒体融合蛋白2、视神经萎缩症蛋白1)与凋亡蛋白(Bax、Bcl-2、Caspase-3)表达。结果与结论:①与对照组相比,过氧化氢组丙二醛水平升高(P<0.05),超氧化物歧化酶和谷胱甘肽水平降低(P<0.05);与过氧化氢组相比,枸杞多糖组丙二醛水平降低(P<0.05),超氧化物歧化酶和谷胱甘肽水平升高(P<0.05);②过氧化氢组线粒体膜电位低于对照组(P<0.05),枸杞多糖组线粒体膜电位高于过氧化氢组(P<0.05);③与对照组相比,过氧化氢组细胞凋亡率与Bax、Caspase-3蛋白表达升高(P<0.05),细胞活力与Bcl-2蛋白表达降低(P<0.05);与过氧化氢组相比,枸杞多糖组细胞凋亡率与Bax、Caspase-3蛋白表达降低(P<0.05),细胞活力与Bcl-2蛋白表达升高(P<0.05);④与对照组相比,过氧化氢组磷酸化启动蛋白1、线粒体分裂蛋白1的蛋白表达升高(P<0.05),线粒体融合蛋白1、线粒体融合蛋白2、视神经萎缩症蛋白1的蛋白表达降低(P<0.05);与过氧化氢组相比,枸杞多糖组磷酸化启动蛋白1、线粒体分裂蛋白1的蛋白表达降低(P<0.05),线粒体融合蛋白1、线粒体融合蛋白2、视神经萎缩症蛋白1的蛋白表达升高(P<0.05);⑤结果表明,枸杞多糖可通过调节线粒体动力学来改善氧化应激损伤诱导的SH-SY5Y细胞凋亡。

BACKGROUND:A large number of studies have shown that neurodegenerative diseases are closely related to oxidative stress injury and the imbalance of mitochondrial dynamics.Lycium barbarum polysaccharides have a neuroprotective effect.However,it is not clear whether lycium barbarum polysaccharides can ameliorate apoptosis induced by oxidative stress injury by regulating abnormal mitochondrial dynamics.OBJECTIVE:To study the effect of lycium barbarum polysaccharides on apoptosis induced by H2O2 in SH-SY5Y human neuroblastoma cells.METHODS:SH-SY5Y cells were cultured in three groups.The control group was cultured for 24 hours.The hydrogen peroxide group was treated with H2O2 for 24 hours,and the lycium barbarum polysaccharide group was treated with lycium barbarum polysaccharide for 2 hours and then treated with H2O2 for 24 hours.After treatment,the levels of malondialdehyde,glutathione,and superoxide dismutase in the precipitation of the cells were detected by kit.Mitochondrial membrane potential was detected by JC-1 kit.Cell viability was detected by MTT assay.Apoptosis was detected by TUNEL.The expression levels of mitochondrial dynamicsrelated proteins(phosphorylated promoter protein 1,mitochondrial fission protein 1,mitochondrial fusion protein 1,mitochondrial fusion protein 2,and optic atrophy protein 1)and apoptotic proteins(Bax,Bcl-2,and Caspase-3)were detected by immunofluorescence staining and western blot assay.RESULTS AND CONCLUSION:(1)Compared with the control group,the levels of malondialdehyde were increased(P<0.05),and the levels of superoxide dismutase and glutathione were decreased(P<0.05)in the H2O2 group.Compared with the H2O2 group,the malondialdehyde level was decreased(P<0.05),and the superoxide dismutase and glutathione levels were increased(P<0.05)in the lycium barbarum polysaccharide group.(2)The mitochondrial membrane potential in the H2O2 group was lower than that in the control group(P<0.05),and that of lycium barbarum polysaccharide group was higher than that of the H2O2 group(P<0.05).(3)Compared with the control group,the apoptosis rate and the expression of Bax and Caspase-3 protein were increased(P<0.05),while the cell viability and the expression of Bcl-2 protein were decreased(P<0.05)in the H2O2 group.Compared with the H2O2 group,the apoptosis rate and the expression of Bax and Caspase-3 protein were decreased(P<0.05),while the cell viability and the expression of Bcl-2 protein were increased(P<0.05)in the lycium barbarum polysaccharide group.(4)Compared with the control group,the protein expression levels of phosphorylated promoter protein 1 and mitochondrial fission protein 1 were increased(P<0.05),and the protein expression levels of mitochondrial fusion protein 1,mitochondrial fusion protein 2,and optic atrophy protein 1 were decreased(P<0.05)in the H2O2 group.Compared with the H2O2 group,the protein expression levels of phosphorylated promoter protein 1 and mitochondrial fission protein 1 were decreased(P<0.05),and the protein expression levels of mitochondrial fusion protein 1,mitochondrial fusion protein 2,and optic atrophy protein 1 were increased(P<0.05)in the lycium barbarum polysaccharide group.(5)These results indicate that lycium barbarum polysaccharide can improve SH-SY5Y cell apoptosis caused by oxidative stress damage by regulating mitochondrial dynamics.