自噬抑制剂3-MA上调H_2O_2损伤的PC12细胞氧化应激水平
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摘要
为探究自噬抑制剂6-氨基-3-甲基腺嘌呤(3-methyladenine,3-MA)对损伤细胞氧化应激水平的影响,将3-MA作用于H_2O_2诱导的PC12细胞损伤模型,以自噬增强剂雷帕霉素(rapamycin,Rap)作为对照,探讨自噬与氧化应激的关系。测定线粒体的膜电位和细胞内的活性氧(reactive oxygen species,ROS)与丙二醛(malondialdehyde,MDA)含量,以及超氧化物歧化酶(superoxide dismutase,SOD)和过氧化氢酶(catalase,CAT)活性,评价损伤细胞的氧化应激状态。单丹(磺)酰戊二胺(monodansylcadaverine,MDC)染色,观察损伤细胞的自噬情况。蛋白质印迹分析损伤细胞中的自噬相关蛋白质LC3-II/LC3-I比值变化。实验结果显示:与正常组相比,H_2O_2损伤细胞的ROS水平上升到正常组的141%,MDA含量增加(P<0. 001); CAT与SOD酶活力显著降低(P<0. 001),差异均有统计学意义,证明损伤细胞氧化应激水平增加; MDC染色结果表明,H_2O_2组自噬明显增加。Western印迹结果表明,LC3-II/LC3-I值显著升高(P<0. 05);与损伤组相比,3-MA组MDC染色结果表明,自噬水平降低。Western印迹结果表明,LC3-II/LC3-I值下降;细胞内ROS水平升高,增加到正常组的208%。MDA含量增加(P<0. 001),CAT、SOD酶活力降低(P<0. 001)。综上结果表明,自噬抑制剂可增加H_2O_2诱导的PC12细胞损伤模型的氧化应激水平,增加细胞凋亡。
To investigate the effect of autophagy inhibitor 3-methyladenine(3-MA) on oxidative stress levels in damaged cells,3-MA was applied to the PC12 cell injury model induced by H_2O_2 and autophagy enhancer rapamycin(Rap) was used as a control to explore the relationship between autophagy and oxidative stress. The mitochondrial membrane potential,intracellular reactive oxygen species(ROS) and malondialdehyde(MDA) contents,and activities of superoxide dismutase(SOD) and catalase(CAT)were determined to evaluate the oxidative stress state. The autophagy of the injured cells was observed by the staining of monodansylcadaverine(MDC),and the change of the ratio of autophagy-related protein LC3-II/LC3-I in the damaged cells was analyzed by Western blot experiment. These results showed that compared with the normal group,the ROS level of H_2O_2 injury cells increased to 141%. MDA content increased(P<0. 001),and CAT and SOD activity decreased significantly(P < 0. 001). The difference was statistically significant,which indicated that the oxidative stress level of injured cells increased. The results of MDC staining showed a significant increase in autophagy in H_2O_2 group,and the results of Western blot showed that the value of LC3-II/LC3-I increased significantly(P < 0. 05). MDC staining results in 3-MA group showed that the level of autophagy decreased as compared with the injury group.Western blot results showed that the value of LC3-II/LC3-I decreased. Intracellular ROS level increased to 208% in normal group. MDA content increased(P< 0. 001),whereas the enzyme activity of CAT and SOD decreased(P < 0. 001). Taken together,autophagy inhibitors increase oxidative stress levels and increase apoptosis in H_2O_2-induced PC12 cell injury models.
To investigate the effect of autophagy inhibitor 3-methyladenine(3-MA) on oxidative stress levels in damaged cells,3-MA was applied to the PC12 cell injury model induced by H_2O_2 and autophagy enhancer rapamycin(Rap) was used as a control to explore the relationship between autophagy and oxidative stress. The mitochondrial membrane potential,intracellular reactive oxygen species(ROS) and malondialdehyde(MDA) contents,and activities of superoxide dismutase(SOD) and catalase(CAT)were determined to evaluate the oxidative stress state. The autophagy of the injured cells was observed by the staining of monodansylcadaverine(MDC),and the change of the ratio of autophagy-related protein LC3-II/LC3-I in the damaged cells was analyzed by Western blot experiment. These results showed that compared with the normal group,the ROS level of H_2O_2 injury cells increased to 141%. MDA content increased(P<0. 001),and CAT and SOD activity decreased significantly(P < 0. 001). The difference was statistically significant,which indicated that the oxidative stress level of injured cells increased. The results of MDC staining showed a significant increase in autophagy in H_2O_2 group,and the results of Western blot showed that the value of LC3-II/LC3-I increased significantly(P < 0. 05). MDC staining results in 3-MA group showed that the level of autophagy decreased as compared with the injury group.Western blot results showed that the value of LC3-II/LC3-I decreased. Intracellular ROS level increased to 208% in normal group. MDA content increased(P< 0. 001),whereas the enzyme activity of CAT and SOD decreased(P < 0. 001). Taken together,autophagy inhibitors increase oxidative stress levels and increase apoptosis in H_2O_2-induced PC12 cell injury models.
引文
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[3]Cheung YT,Zhang NQ,Hung CH,et al.Temporal relationship of autophagy and apoptosis in neurons challenged by low molecular weightβ-amyloid peptide[J].J Cell Mol Med,2011,15(2):244-257
[4]Ciechanover A,Kwon YT.Protein quality control by molecular chaperones in neurodegeneration[J].Front Neurosci,2017,11:185
[5]Banerjee R,Beal MF,Thomas B.Autophagy in neurodegenerative disorders:pathogenic roles and therapeutic implications[J].Trends Neurosci,2010,33(12):541-549
[6]Holley AK,Bakthavatchalu V,Velez-Roman JM,et al.Manganese superoxide dismutase:guardian of the powerhouse[J].Int J Mol Sci,2011,12(10):7114-7162
[7]Su B,Wang X,Nunomura A,et al.Oxidative stress signaling in Alzheimer’disease[J].Curr Alzheimer Res,2008,5(6):525-532
[8]Thummasorn S,Shinlapawittayatorn K,Khamseekaew J,et al.Humanin directly protects cardiac mitochondria against dysfunction initiated by oxidative stress by decreasing complex Iactivity[J].Mitochondrion,2018,38:31-40
[9]Vakifahmetoglu-Norberg H,Ouchida AT,Norberg E.The role of mitochondria in metabolism and cell death[J].Biochem Biophys Res Commun,2017,482(3):426-431
[10]He L,Xiao D,Feng J,et al.Induction of apoptosis of liver cancer cells by nanosecond pulsed electric fields(nsPEFs)[J].Med Oncol,2017,34(2):24
[11]Levin B,Kroemer G.Autophagy in the pathogenesis of disease[J].Cell,2008,132(1):27-42
[12]Mizushima N,Levine B,Cuervo AM,et al.Autophagy fights disease through cellular self-degestion[J].Nature,2008,451(7182):1069-1075
[13]Tsagalioti E,Trifonos C,Morari A,et al.Clinical value of nutritional status in neurodegenerative diseases:What is its impact and how it affects disease progression and management?[J].Nutr Neurosci,2018,21(3):162-175
[14]Zhang Y,He X,Wu X,et al.Rapamycin upregulates glutamate transporter and IL-6 expression in astrocytes in a mouse model of Parkinson’s disease[J].Cell Death Dis,2017,8(2):e2611
[15]Liu K,Shi N,Sun Y,et al.Therapeutic effects of rapamycin on MPTP-induced Parkinsonism in mice[J].Neurochem Res,2013,38(1):201-207
[16]Wang H,Ye Y,Zhu Z,et al.MiR-124 regulates apoptosis and autophagy process in MPTP model of Parkinson’s disease by targeting to Bim[J].Brain Pathol,2016,26(2):167-176
[17]Settembre C,Fraldi A,Jahreiss L,et al.A block of autophagy in lysosomal storage disorders[J].Hum Mol Genet,2008,17(1):119-129
[18]Snyder B,Shell B,Cunningham JT,et al.Chronic intermittent hypoxia induces oxidative stress and inflammation in brain regions associated with early-stage neurodegeneration[J].Physiol Rep,2017,5(9).pii:e13258
[19]Salim S.Oxidative stress and the central nervous system[J].JPharmacol Exp Ther,2017,360(1):201-205
[20]Guo H,Chen L,Cui H,et al.Research advances on pathways of Nickel-induced apoptosis[J].Int J Mol Sci,2015,17(1).pii:E10
[21]Wei Y,Zhou F,Zhang D,et al.A graphene oxide based smart drug delivery system for tumor mitochondria-targeting photodynamic therapy[J].Nanoscale,2016,8(6):3530-3538