丁香脂素对谷氨酸钠诱导的SH-SY5Y细胞兴奋性损伤的保护作用
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摘要
目的:基于谷氨酸钠诱导人神经母细胞瘤细胞(SH-SY5Y)细胞损伤构建的模型,观察从枫香槲寄生中提取得到丁香脂素的保护,并探讨其作用机制。方法:采用谷氨酸钠构建SH-SY5Y细胞损伤模型,实验分为正常组,损伤模型组(谷氨酸钠50 mmol·L~(-1),谷氨酸钠50 mmol·L~(-1)+DMSO),丁香脂素组(6. 25,12. 5,25μmol·L~(-1)),通过细胞计数法,细胞形态学观察,Annexin V-FITC/碘化丙啶(PI)细胞凋亡检测、活性氧(ROS)检测、线粒体膜电位以及蛋白免疫印迹法(Western blot)等方法,评价丁香脂素对谷氨酸钠诱导的神经兴奋性损伤的神经保护活性。结果:与正常组比较,模型组的细胞存活率明显降低(P <0. 01),ROS显著升高(P <0. 01),线粒体膜电位显著降低(P <0. 01),聚腺苷二磷酸-核糖聚合酶(PARP),PARP1蛋白表达显著降低(P <0. 01),细胞的凋亡率显著增大(P <0. 01);与模型组比较,丁香脂素组(6. 25,12. 5,25μmol·L~(-1))呈浓度依赖性增加细胞的存活率(P <0. 01),减少ROS的积累(P <0. 01),显著恢复线粒体膜电位的变化(P <0. 01),显著上调PARP,PARP1蛋白(P <0. 01),显著降低细胞凋亡率(P <0. 01)。结论:提示丁香脂素对谷氨酸钠诱导的SH-SY5Y神经细胞的兴奋性损伤具有显著的保护活性,其作用机制可能是通过抗氧化应激,修复线粒体功能及DNA损伤等通路来显著降低谷氨酸钠诱导的神经细胞凋亡。
Objective: To establish a model for the injury of human neuroblastoma cell( SH-SY5 Y induced by sodium glutamate,and to observe the protective effect of syringaresinol on cell damage from Viscum liquidambaricolum hayataon,and to explore its mechanism. Method: Construction of SH-SY5 Y cell injury model using sodium glutamate. The experiment was divided into normal cell group, injury model group( sodium glutamate 50 mmol·L~(-1),sodium glutamate 50 mmol·L~(-1)+ DMSO),syringaresinol experimental group( 6. 25,12. 5,25 μmol·L~(-1)),by cell counting,cell morphology observation,Annexin V-FITC/PI apoptosis detection,ROS reactive oxygen species detection, mitochondrial membrane potential, and Western blot, evaluation of syringaresinol on glutamate-induced neuronal excitability injury neuroprotective activity. Result: Compared with normal group,the cell survival rate of the model group was significantly decreased( P < 0. 01),ROS accumulation was significant( P < 0. 01),mitochondrial membrane potential was significantly decreased( P < 0. 01),and the expression of poly-ADP-ribose polymerase( PARP) and PARP1 protein was significantly decreased( P < 0. 01),the apoptotic rate of cells also increased significantly( P < 0. 01). Compared with the model group, the syringaresinol group( 6. 25,12. 5,25 μmol·L~(-1)) showed a concentration-dependent increase in cells. Survival rate( P < 0. 01),decreased ROS accumulation( P < 0. 01),restored mitochondrial membrane potential( P <0. 01),up-regulated PARP,PARP1 protein( P < 0. 01),decreased apoptosis rate( P < 0. 01). Conclusion:Syringaresinol has significant protective activity against excitatory damage induced by sodium glutamate in SH-SY5 Y neurons,the mechanism may be through anti-oxidative stress,repairing mitochondrial function and DNA damage to significantly reduce sodium glutamate-induced neuronal apoptosis.
Objective: To establish a model for the injury of human neuroblastoma cell( SH-SY5 Y induced by sodium glutamate,and to observe the protective effect of syringaresinol on cell damage from Viscum liquidambaricolum hayataon,and to explore its mechanism. Method: Construction of SH-SY5 Y cell injury model using sodium glutamate. The experiment was divided into normal cell group, injury model group( sodium glutamate 50 mmol·L~(-1),sodium glutamate 50 mmol·L~(-1)+ DMSO),syringaresinol experimental group( 6. 25,12. 5,25 μmol·L~(-1)),by cell counting,cell morphology observation,Annexin V-FITC/PI apoptosis detection,ROS reactive oxygen species detection, mitochondrial membrane potential, and Western blot, evaluation of syringaresinol on glutamate-induced neuronal excitability injury neuroprotective activity. Result: Compared with normal group,the cell survival rate of the model group was significantly decreased( P < 0. 01),ROS accumulation was significant( P < 0. 01),mitochondrial membrane potential was significantly decreased( P < 0. 01),and the expression of poly-ADP-ribose polymerase( PARP) and PARP1 protein was significantly decreased( P < 0. 01),the apoptotic rate of cells also increased significantly( P < 0. 01). Compared with the model group, the syringaresinol group( 6. 25,12. 5,25 μmol·L~(-1)) showed a concentration-dependent increase in cells. Survival rate( P < 0. 01),decreased ROS accumulation( P < 0. 01),restored mitochondrial membrane potential( P <0. 01),up-regulated PARP,PARP1 protein( P < 0. 01),decreased apoptosis rate( P < 0. 01). Conclusion:Syringaresinol has significant protective activity against excitatory damage induced by sodium glutamate in SH-SY5 Y neurons,the mechanism may be through anti-oxidative stress,repairing mitochondrial function and DNA damage to significantly reduce sodium glutamate-induced neuronal apoptosis.
引文
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[15] Buchanan R J,Gjini K,Darrow D,et al. Glutamate and GABA concentration changes in the globus pallidus internus of Parkinson's patients during performance of implicit and declarative memory tasks:a report of two subjects[J]. Neurosci Lett,2015,589(3):73-78.
[16] Yong S,Seung J B,Hyun-O P. Involvement of heme oxygenase-1 expression in neuroprotection by piceatannol,a natural analog and a metabolite of resveratrol,against glutamate-mediated oxidative injury in HT22 neuronal cells[J]. Amino Acids,2013,45(2):393-401.
[17] Shukla S,Sharma A,Pandey V K,et al. Concurrent acetylation of Fox O1/3a and p53 due to sirtuins inhibition elicit Bim/PUMA mediated mitochondrial dysfunction and apoptosis in berberine-treated Hep G2cells[J]. Regul Toxicol Pharmacol,2015,291:70-83.
[18] Hyun S K,Yeo J K,Young R S. An overview of carcinogenic heavy metal:molecular toxicity mechanism and prevention[J]. J Cancer Prev,2015,20(4):232-400.
[2] LIU Z W,ZHOU T Y,Ziegler A C,et al. Oxidative stress in neurodegenerative diseases:from molecular mechanisms to clinical applications[J]. Oxid Med Cell Longev,2017,2017:1-11.
[3] Yoko N,Kensaku T,Kouji W,et al. Antioxidative compounds isolated from kokuto,non-centrifugal cane sugar[J]. Biosci Biotechnol Biochem,1996,60(10):1714-1716.
[4] El-Hassan A,El-Sayed M,Hamed A I,et al. Bioactive constituents of leptadenia arborea[J]. Fitoterapia,2003,74(1/2):184-187.
[5] Sunil R,FANG X P,Anderson J E,et al. Bioactive constituents from the twigs of asimina parviflora[J]. J Nat Prod,1992,55(10):1462-1467.
[6] Kenzo C,Matsumi Y,Emiko U,et al. Neuritogenesis of Herbal(+)-and(-)-Syringaresinols separated by chiral HPLC in PC12h and neuro2a cells[J]. Biol Pharm Bull,2002,25(6):791-793.
[7] Nishizaw Y. Glutamate release and neuronal damage in ischemia[J]. Life Sci,2001,69(4):369-381.
[8]冯波,王蓉,盛树力.神经退行性疾病研究中拟神经细胞模型:人神经母细胞瘤株SH-SY5Y的来源特性及应用[J].中国临床康复,2006,10(6):121-123.
[9]韩凤昭,彭涛,王林,等.红景天苷衍生物S01对谷氨酸及缺氧缺糖所致SH-SY5Y细胞损伤的保护作用[J].中国药理学与毒理学杂志,2008,22(3):180-185.
[10] Park H W,Cho S Y,Kim H H,et al. Enantioselective induction of SIRT1 gene by syringaresinol from Panax ginseng berry and acanthopanax senticosus harms stem[J]. Bioorg Med Chem Lett,2015,25(2):307-309.
[11] Beal M F. Aging, energy, and oxidative stress in neurodegenerative diseases[J]. Ann Neurol,1995,38(3):357-366.
[12] Yunes R,Estrella C R,García S,et al. Postnatal administration of allopregnanolone modifies glutamate release but not BDNF content in striatum samples of rats prenatally exposed to ethanol[J]. Biomed Res Int,2015,27(10):6.
[13] Girbovan C,Plamondon H. Resveratrol downregulates glutamate-1transporter expression and microglia activation in the hippocampus following cerebral ischemia reperfusion in rats[J]. Brain Res,2015,1608:203-214.
[14] Guerriero R M,Giza C C,Rotenberg A. Glutamate and GABA imbalance following traumatic brain injury[J].Curr Neurol Neurosci,2015,15(5):1-11.
[15] Buchanan R J,Gjini K,Darrow D,et al. Glutamate and GABA concentration changes in the globus pallidus internus of Parkinson's patients during performance of implicit and declarative memory tasks:a report of two subjects[J]. Neurosci Lett,2015,589(3):73-78.
[16] Yong S,Seung J B,Hyun-O P. Involvement of heme oxygenase-1 expression in neuroprotection by piceatannol,a natural analog and a metabolite of resveratrol,against glutamate-mediated oxidative injury in HT22 neuronal cells[J]. Amino Acids,2013,45(2):393-401.
[17] Shukla S,Sharma A,Pandey V K,et al. Concurrent acetylation of Fox O1/3a and p53 due to sirtuins inhibition elicit Bim/PUMA mediated mitochondrial dysfunction and apoptosis in berberine-treated Hep G2cells[J]. Regul Toxicol Pharmacol,2015,291:70-83.
[18] Hyun S K,Yeo J K,Young R S. An overview of carcinogenic heavy metal:molecular toxicity mechanism and prevention[J]. J Cancer Prev,2015,20(4):232-400.