不同时间氧糖剥夺再灌注对SH-SY5Y细胞线粒体自噬及功能的影响
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摘要
目的:探讨不同时点氧糖剥夺再灌注(OGD/R)后人神经母细胞瘤(SH-SY5Y)细胞线粒体自噬及功能改变。方法:利用无糖DMEM及三气培养箱对SH-SY5Y细胞进行OGD/R,根据OGD/R不同时间将细胞分为control组、3 h/12 h组、3 h/24 h组、6 h/12 h组及6 h/24 h组,OGD/R后采用流式细胞术检测细胞线粒体膜电位及活性氧(ROS)含量,采用Western blot法检测线粒体蛋白Beclin1及P62表达。结果:与control组比较,OGD/R后SH-SY5Y细胞线粒体膜电位发生去极化(P<0.01)、胞内ROS含量升高(P<0.01),3 h/24 h组、6 h/12 h组及6 h/24 h组线粒体Beclin1蛋白表达显著升高(P<0.01),,3 h/12 h、6 h/24 h组线粒体P62蛋白表达升高(P<0.05); OGD/R各组间比较,3 h/24 h组线粒体去极化程度、细胞内ROS含量及线粒体Beclin1蛋白表达水平均较3 h/12 h组显著升高(P<0.01),且6 h/24 h组ROS含量最高(P<0.01)。结论:SH-SY5Y细胞OGD/R后线粒体功能受损且线粒体自噬活性升高,且随着缺血时间及再灌注时间增加线粒体受损加重,线粒体自噬也增加。
Objective: To investigate the mitochondrial function and mitophagy in SH-SY5 Y cells at different time points after oxygen glucose deprivation(OGD)/reperfusion(R). Methods: SH-SY5 Y cells were subjected to OGD by using sugar-free DMEM and three-gas incubator. After deprivation in the corresponding time, reperfusion(R) was performed. The cells were divided into the control group, the 3 h/12 h group, the 3 h/24 h group, the 6 h/12 h group and the 6 h/24 h group according to OGD/R time. The mitochondrial membrane potential changes and reactive oxygen species(ROS) levels were detected by flow cytometry. The expression of mitochondrial proteins Beclin1 and P62 was detected by Western blot. Results: Compared with the controls, the mitochondrial membrane potential of SH-SY5 Y cells was depolarized and the intracellular ROS content increased after OGD/R. The expression of Beclin1 in mitochondrial proteins was observed in 3 h/24 h, 6 h/12 h and 6 h/24 h. The expression of P62 in mitochondrial protein increased in the 3 h/12 h group and 3 h/24 h group. Compared with the OGD/R group, the mitochondrial depolarization increased and the expression of Beclin1 was higher in 3 h/24 h than in 3 h/12 h. The ROS content was the highest in the 6 h/24 h group. Conclusion: After OGD/R in SH-SY5 Y cells, mitochondrial function is impaired and mitochondrial autophagy activity increases, and mitochondrial damage increases with the increase of ischemia time and reperfusion time, and the mitochondrial autophagy also increases.
Objective: To investigate the mitochondrial function and mitophagy in SH-SY5 Y cells at different time points after oxygen glucose deprivation(OGD)/reperfusion(R). Methods: SH-SY5 Y cells were subjected to OGD by using sugar-free DMEM and three-gas incubator. After deprivation in the corresponding time, reperfusion(R) was performed. The cells were divided into the control group, the 3 h/12 h group, the 3 h/24 h group, the 6 h/12 h group and the 6 h/24 h group according to OGD/R time. The mitochondrial membrane potential changes and reactive oxygen species(ROS) levels were detected by flow cytometry. The expression of mitochondrial proteins Beclin1 and P62 was detected by Western blot. Results: Compared with the controls, the mitochondrial membrane potential of SH-SY5 Y cells was depolarized and the intracellular ROS content increased after OGD/R. The expression of Beclin1 in mitochondrial proteins was observed in 3 h/24 h, 6 h/12 h and 6 h/24 h. The expression of P62 in mitochondrial protein increased in the 3 h/12 h group and 3 h/24 h group. Compared with the OGD/R group, the mitochondrial depolarization increased and the expression of Beclin1 was higher in 3 h/24 h than in 3 h/12 h. The ROS content was the highest in the 6 h/24 h group. Conclusion: After OGD/R in SH-SY5 Y cells, mitochondrial function is impaired and mitochondrial autophagy activity increases, and mitochondrial damage increases with the increase of ischemia time and reperfusion time, and the mitochondrial autophagy also increases.
引文
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[15]LI H,LIU Y,LIN L T,et al.Acupuncture reversed hippocampal mitochondrial dysfunction in vascular dementia rats[J].Neurochemistry International,2016,92:35-42.
[16]YU S,ZHENG S,LENG J,et al.Inhibition of mitochondrial calcium uniporter protects neurocytes from ischemia/reperfusion injury via the inhibition of excessive mitophagy[J].Neuroscience Letters,2016,628(15):24-29.
[17]LIN M T,BEAL M F.Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases [J].Nature,2006,443 (7113):787-795.
[18]WALLACE D C.A mitochondrial paradigm of metabolic and degenerative diseases,aging,and cancer:a dawn for evolutionary medicine[J].Annu Rev Genet,2005,39:359-407.
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[2] SUN J,LI Y Z,DING Y H,et al.Neuroprotective effects of gallic acid against hypoxia/reoxygenation-induced mitochondrial dysfunctions in vitro and cerebral ischemia/reperfusion injury in vivo[J].Brain Res,2014,1589:126-139.
[3] LUO Y G,YANG X F,ZHAO S T,et al.Hydrogen sulfide prevents OGD/R-induced apoptosis via improving mitochondrial dysfunction and suppressing an ROS-mediated caspase-3 pathway in cortical neurons[J].Neurochemistry International,2013,63(8):826-831.
[4] LI Y,ZHANG W T,CHEN C,et al.Inotodiol protects PC12 cells against injury induced by oxygen and glucose deprivation/restoration through inhibiting oxidative stress and apoptosis[J].Journal of Applied Biomedicine,2017,16(2):126-132.
[5] YU S,ZHENG S,LENG J,et al.Inhibition of mitochondrial calcium uniporter protects neurocytes from ischemia/reperfusion injury via the inhibition of excessive mitophagy[J].Neuroscience Letters,2016,628:26-29.
[6] YOULE R J,NARENDRA D P.Mechanisms of mitophagy[J].Nat Rev Mol Cell Biol,2011,12(1):9-14.
[7] JOHN T B,ALAN T.Vascular dementia[J].The Lancet,2015,386(10004):1698-1706.
[8] VENKAT P,CHOPP M,CHEN J.Models and mechanisms of vascular dementia[J],Experimental Neurology,2015,272:97-108.
[9] MARUTANI E,KOSUGI S,TOKUDA K,et la.A novel hydrogen sulfide-releasing N-methyl-D-aspartate receptor antagonist prevents ischemic neuronal death[J].J Biol Chem,2012,287(38):32124-32135.
[10]XIONG S Y,XU Y P,MA M M,et al.Neuroprotective effects of a novel peptide,FK18,under oxygen-glucose deprivation in SH-SY5Y cells and retinal ischemia in rats via the Akt pathway[J].Neurochemistry International,2017,108:78-90.
[11]ZHU Q L,LI Y X,ZHOU R,et al.Neuroprotective effects of oxysophocarpine on neonatal rat primary cultured hippocampal neurons injured by oxygen-glucose deprivation and reperfusion[J].Cell Mol Neurobiol,2018,38(2):529-540.
[12]DOYLE K P,SIMON R P,STENZELPOORE M P.Mechanisms of ischemic brain damage.[J].Neuropharmacology,2008,55(3):310-318.
[13]MEHTA S L,MANHAS N,RAGHUBIR R.Molecular targets in cerebral ischemia for developing novel therapeutics[J].Brain Research Reviews,2007,54(1):34-66.
[14]BAKTHAVACHALAM P,SHANMUGAM P S T.Mitochondrial dysfunction-silent killer in cerebral ischemia[J].Journal of the Neurological Sciences,2017,374(15):417-423.
[15]LI H,LIU Y,LIN L T,et al.Acupuncture reversed hippocampal mitochondrial dysfunction in vascular dementia rats[J].Neurochemistry International,2016,92:35-42.
[16]YU S,ZHENG S,LENG J,et al.Inhibition of mitochondrial calcium uniporter protects neurocytes from ischemia/reperfusion injury via the inhibition of excessive mitophagy[J].Neuroscience Letters,2016,628(15):24-29.
[17]LIN M T,BEAL M F.Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases [J].Nature,2006,443 (7113):787-795.
[18]WALLACE D C.A mitochondrial paradigm of metabolic and degenerative diseases,aging,and cancer:a dawn for evolutionary medicine[J].Annu Rev Genet,2005,39:359-407.
[19]秦正红.自噬生物学与疾病[M].2版.北京:科学出版社,2015:409-437.
[20]张坤西,张洁,郭艳苏.P62蛋白的研究[J].脑与神经疾病杂志,2016,24(1):59-61.