松果菊苷调控抗增殖蛋白表达抑制MPP~+诱导的SH-SY5Y细胞凋亡

Echinacoside regulates prohibitin expression and inhibits MPP+-induced apoptosis in SH-SY5Y cells

目的探讨松果菊苷(echinacoside,ECH)调控抗增殖蛋白(prohibitin,PHB)表达,及对MPP^(+)诱导的SH-SY5Y细胞凋亡的效应及机制。方法选取SH-SY5Y细胞分为control组、MPP^(+)组、MPP^(+)+ECH组、NC+MPP^(+)组、NC+MPP^(+)+ECH组、PHB-RNAi+MPP^(+)+ECH组。CCK-8检测细胞存活率,倒置相差显微镜观察细胞形态,Hoechst33342荧光染色法观察凋亡细胞,流式细胞仪检测细胞凋亡率、活性氧含量及线粒体膜电位,Western blot检测PHB、Akt、p-Akt、Bcl-2、Bax、cleaved caspase-3相对蛋白表达。结果与control组比较,MPP^(+)组细胞存活率显著下降;细胞生长状态明显变差;细胞内活性氧含量增高、线粒体膜电位降低、凋亡相关蛋白表达增加、凋亡率升高。与MPP^(+)组比较,MPP^(+)+ECH组细胞存活率显著上升;细胞生长状态明显好转;细胞内活性氧含量降低、线粒体膜电位升高、凋亡相关蛋白表达降低、凋亡率明显下降;PHB和p-Akt表达量明显上升。与NC+MPP^(+)+ECH组比较,PHB-RNAi+MPP^(+)+ECH组p-Akt水平降低,细胞凋亡率上升。结论松果菊苷能减轻MPP^(+)诱导的SH-SY5Y细胞凋亡,此效应可能是通过上调PHB表达,使Akt磷酸化,从而保护细胞线粒体功能来实现的。

Aim To investigate the effect of Echinacoside(ECH)regulating the expression of prohibitin(PHB)on MPP-induced apoptosis of SH-SY5 Y cells and the underlying mechanism.Methods SH-SY5 Y cells were selected and divided into control group,MPPgroup,MPP^(+)ECH group,NC+MPPgroup,NC+MPP^(+)ECH group,PHB-RNAi+MPP^(+)ECH group.Cell survival rate was determined by CCK-8 assay.Cell morphology was observed using an inverted phase contrast microscope;the apoptotic cells were observed by Hoechst33342 fluorescence staining,whereas apoptotic rate,reactive oxygen species content,and mitochondrial membrane potential were analyzed by flow cytometry.The relative protein expressions of PHB,Akt,p-Akt,Bcl-2,Bax,and cleaved-caspase3 were determined by Western blot.Results Compared with control group,the cell survival rate of MPPgroup significantly decreased.The growth state of the cells became significantly worse.Intracellular ROS content increased,mitochondrial membrane potential decreased,apoptosis-related protein expression increased and the apoptotic rate increased.Compared with MPPgroup,MPP^(+)ECH group significantly increased cell viability.The growth status of cells was significantly improved.Intracellular ROS content decreased,mitochondrial membrane potential increased,apoptosis-related protein expression decreased,and the apoptotic rate decreased significantly.The expression levels of PHB and p-Akt significantly increased.Compared with NC+MPP^(+)ECH group,p-Akt level decreased and the cell apoptotic rate increased in PHB-RNAi+MPP^(+)ECH group.Conclusions Echinoside can reduce MPP-induced apoptosis of SH-SY5 Y cells,which may be realized by upregulating PHB expression and phosphorylation of Akt to protect mitochondrial function.