圣草酚对过氧化氢所致SH-SY5Y细胞线粒体动力学失衡及凋亡的影响

Effect of eriodictyol on the imbalance of mitochondrial dynamics and apoptosis in SH-SY5Y cells induced by hydrogen peroxide

背景:线粒体动力学异常与神经退行性疾病中氧化应激现象密切相关。课题组前期研究发现圣草酚可以减轻阿尔茨海默病神经损伤,但其是否对线粒体动力学有调控作用尚未明确。目的:探讨圣草酚抑制过氧化氢诱导的SH-SY5Y细胞凋亡的具体机制。方法:将SH-SY5Y细胞分为如下3组:PBS对照组、过氧化氢(250μmol/L)模型组、过氧化氢(250μmol/L)+圣草酚治疗组(10μmol/L)。干预24 h,应用试剂盒检测丙二醛水平和超氧化物歧化酶活性,显微镜下观察细胞形态,TUNEL染色观察细胞凋亡情况,JC-1染色观察线粒体膜电位,免疫荧光染色法和Western blot检测细胞凋亡蛋白以及线粒体融合和线粒体分裂蛋白的表达。结果与结论:①与过氧化氢组相比,圣草酚治疗组丙二醛水平显著减少,超氧化物歧化酶活性显著增加;②与PBS对照组相比,过氧化氢组细胞密度下降,胞体肥大,细胞凋亡率增加,线粒体模电位下降,Bcl-2表达减少,Bax表达增加;与过氧化氢组相比,圣草酚治疗组上述指标得到明显改善;③与PBS对照组相比,过氧化氢组细胞线粒体分裂相关蛋白p-DRP1和FIS1的表达量升高,而线粒体融合相关蛋白OPA1和Mfn2的表达量降低;与过氧化氢组相比,圣草酚治疗可以降低p-DRP1和FIS1的表达,增加OPA1和Mfn2的表达;④结果说明,圣草酚可通过调控线粒体动力学抑制过氧化氢诱导的SH-SY5Y细胞凋亡。

BACKGROUND:Abnormal mitochondrial dynamics is closely related to oxidative stress after affecting neurodegenerative diseases.Our previous studies have shown that eriodictyol can reduce nerve injury in Alzheimer’s disease,but whether it has a regulatory effect on mitochondrial dynamics is not clear.OBJECTIVE:To explore the mechanism of eriodictyol on SH-SY5Y cell apoptosis induced by hydrogen peroxide(H_(2)O_(2)).METHODS:SH-SY5Y cells were divided into three groups:PBS control group,H_(2)O_(2)(250μmol/L)group,and H_(2)O_(2)(250μmol/L)plus eriodictyol(10μmol/L)treatment group(eriodictyol group).The intervention was performed for 24 hours.The kits were utilized to observe the level of malondialdehyde and the activity of superoxide dismutase.Cell morphology was observed under the microscope.TUNEL staining was used to observe the apoptosis.JC-1 staining was used to observe mitochondrial membrane potential.The expression levels of apoptotic proteins and mitochondrial fusion and fission protein-related proteins were detected by immunofluorescence staining and western blot assay.RESULTS AND CONCLUSION:(1)A significant decrease of malondialdehyde levels and a significant increase of superoxide dismutase activity were found in eriodictyol treated group,when compared with the H_(2)O_(2) group.(2)Compared with the PBS control group,the H_(2)O_(2) group showed decreased cell density,cytosolic hypertrophy,increased apoptosis,decreased mitochondrial membrane potential,decreased Bcl-2 expression,and increased Bax expression.Compared with the H_(2)O_(2) group,above indexes were significantly improved in the eriodictyol group.(3)Compared with PBS control group,the expression levels of mitochondrial fission-related proteins dynamin-related protein 1(p-DRP1)and mitochondrial fission protein 1(FIS1)were elevated and the expression levels of mitochondrial fusion-related proteins optic atrophic protein 1(OPA1)and mitofusin 2(Mfn2)were decreased in the H_(2)O_(2) group.Compared with the H_(2)O_(2) group,eriodictyol treatment decreased the expression levels of p-DRP1 and FIS1 and increased the expression of OPA1 and Mfn2.(4)The results indicate that eriodictyol can inhibit H_(2)O_(2)-induced apoptosis in SH-SY5Y cells by regulating mitochondrial dynamics.