星形胶质细胞调控下红花黄色素对Aβ_(1-42)介导的海马神经元突触损伤的保护作用研究
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摘要
目的探讨星形胶质细胞存在下红花黄色素对Aβ_(1-42)诱导的神经元突触损伤的保护作用。方法采用Aβ_(1-42)损伤海马神经元模拟体外AD,不同浓度(0.01、0.1、1 g/L)红花黄色素进行干预,MTT法测定细胞存活率选择最佳浓度。建立纯化海马神经元培养体系(NE-S)和星形胶质细胞和神经元共混合培养体系(MIX-S),两种培养体系分别设立正常对照组、Aβ_(1-42)(15μmol/L)模型组、红花黄色素给药组。倒置显微镜下观察细胞形态;Western Blot检测神经元内SAP102蛋白表达水平。结果 (1)红花黄色素(0.01、0.1、1 g/L)能够显著提高神经元的存活率,且具有剂量依赖性;(2)神经元形态结果显示在MIX-S的模型组中神经元突触萎缩状态较NE-S的模型组稍微有所改善。红花黄色素(1 g/L)能明显改善NE-S和MIX-S中的神经元突触萎缩状态,但NE-S和MIX-S中的红花黄色素1 g/L给药组神经元形态没有差异性;(3) Western Blot结果显示在MIX-S的模型组中SAP102蛋白表达较NE-S的模型组有所增加。在NE-S和MIX-S中的红花黄色素1 g/L给药组SAP102蛋白表达都明显上调,但NE-S和MIX-S中的红花黄色素1 g/L给药组SAP102蛋白含量没有差异性。结论星形胶质细胞的存在可增加Aβ_(1-42)损伤的神经元突触蛋白的表达,但并不提高SY对神经元突触损伤的保护作用。
Objective To investigate the protective effects of safflower yellow on synaptic damage in hippocampal neurons induced by Aβ_(1-42) in the presence of astrocytes. Methods Neurons were cultured in vitro,with contained Aβ_(1-42) for preparing AD models and different concentrations of safflower yellow(0.01、0.1、1 g/L) were used for intervention,then cell viability was assayed by tetramethyl triazole salt(MTT) to choose the best concentrations.We established the pure hippocampal neuronal cultures system(NE-S) and neuron-astrocytes co-cultures system(MIX-S),then the two culture systems were set as normal control group,Aβ_(1-42)(15 μmol/L) model group,and safflower yellow group respectively.Morphological picture of cultured neurons were observed by inverted microscope.The SAP102 protein levels were detected by Western blot. Results Safflower yellow significantly increased the cell survival rate in a dose-dependent manner.We founded that the neuronal synaptic atrophy in the model group in MIX-S was slightly improved compared with the model group in NE-S.In NE-S and MIX-S,the neuronal synaptic atrophy in the safflower yellow 1 g/L group was significantly improved,but the neuronal morphology in the safflower yellow 1 g/L group in MIX-S was as near as make no difference compared with the safflower yellow 1 g/L group in NE-S.The results of Western blot showed that the protein expression of SAP102 was increased in the model group in MIX-S compared with the model group in NE-S.In NE-S and MIX-S,the levels of SAP102 protein in safflower yellow 1 g/L group were both significantly up-regulated,but the level of SAP102 protein seems to be unaltered in 1 g/L safflower yellow group between NE-S and MIX-S. Conclusion The presence of astrocytes can increase the expression of synaptic proteins in neurons injured by Aβ_(1-42),but can't enhance the protective effect of SY on damage of synaptic in cultured neurons.
Objective To investigate the protective effects of safflower yellow on synaptic damage in hippocampal neurons induced by Aβ_(1-42) in the presence of astrocytes. Methods Neurons were cultured in vitro,with contained Aβ_(1-42) for preparing AD models and different concentrations of safflower yellow(0.01、0.1、1 g/L) were used for intervention,then cell viability was assayed by tetramethyl triazole salt(MTT) to choose the best concentrations.We established the pure hippocampal neuronal cultures system(NE-S) and neuron-astrocytes co-cultures system(MIX-S),then the two culture systems were set as normal control group,Aβ_(1-42)(15 μmol/L) model group,and safflower yellow group respectively.Morphological picture of cultured neurons were observed by inverted microscope.The SAP102 protein levels were detected by Western blot. Results Safflower yellow significantly increased the cell survival rate in a dose-dependent manner.We founded that the neuronal synaptic atrophy in the model group in MIX-S was slightly improved compared with the model group in NE-S.In NE-S and MIX-S,the neuronal synaptic atrophy in the safflower yellow 1 g/L group was significantly improved,but the neuronal morphology in the safflower yellow 1 g/L group in MIX-S was as near as make no difference compared with the safflower yellow 1 g/L group in NE-S.The results of Western blot showed that the protein expression of SAP102 was increased in the model group in MIX-S compared with the model group in NE-S.In NE-S and MIX-S,the levels of SAP102 protein in safflower yellow 1 g/L group were both significantly up-regulated,but the level of SAP102 protein seems to be unaltered in 1 g/L safflower yellow group between NE-S and MIX-S. Conclusion The presence of astrocytes can increase the expression of synaptic proteins in neurons injured by Aβ_(1-42),but can't enhance the protective effect of SY on damage of synaptic in cultured neurons.
引文
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[14]Chen Y,Vartiainen N E,Ying W,et al.Astrocytes protect neurons from nitric oxide toxicity by a glutathione dependent mechanism[J].J Neurochem,2001,77:1601-1610.
[15]Fellin T,Carmignoto G.Neuron-to-astrocyte signaling in the brain represent a distinct multifunctional unit[J].J Physiol,2004,559:3-15.
[16]M Alcangiom.Brain-derived neurotrophic factor induced NMDA receptor submit one phosphory lation via ERKand PKC in the rat spina l cord[J].Eur JNeurosci,2004,20(7):1769-1778.
[17]Shaked I,Ben Dror I,Vardimon L.Glutamine synthetase enhances the clearance of extracellular glutamate by the neural retina[J].J Neurochem,2002,83:574-580.
[18]Esposito Z,Belli L,Toniolo S,et al.Amyloidβ,glutamate,excitotoxicity in Alzheimer's disease:are we on the right track[J].CNS Neurosci Therapeut,2013,19:549-555.
[19]莫玉言,侯加卫,周张玖智,胡艳丽.红花黄色素对Aβ_(1-42)诱导的SH-SY5Y细胞损伤的保护作用研究[J].中药药理与临床,2018,34(4):40-44.Mo Y Y,Hou J W,Zhou Z J Z.Jiuzhi,Hu Yanli.Study on the protective effects of safflower yellow on injury of cultured SH-SY5Y cells induced by Aβ1-42[J].Chinese medicine pharmacology and clinical,2018,34(4):40-44.
[2]Sakono M,Zako T.Amyloid oligomers:formation and toxicity of Abeta oligomers[J].FEBS J,2010,227:1348-1358.
[3]苏东宁,常丽荣,武艳.突触相关蛋白102的结构、功能及相关研究进展[J].解剖学报,2017,48(5):628-632.Su Dongning,Chang Lirong,Wu Yan.Structure,functions and relative diseases of synapse-associated protein 102[J].Axta Anatomica Sinica,2017,48(5):628-632.
[4]Igor A,Mireille B,Pierre J M.Astrocyte-neuron metabolic relation-ships:for better and for worse[J].Trends Neurosci,2011,34:76-87.
[5]Garwood C J,Pooler A M,Atherton J,et al.Astrocytes are important mediators of Aβ-induced neurotoxicity and tau phosphorylation in primary culture[J].Cell Death Dis,2011,167:1-9.
[6]Qin H,Benveniste E N.Elisa methodology to quantify astrocyte production of cytokines/chemokines in vitro[J].Methods Mol Biol,2012,814:235-249.
[7]杨晓媛,任玉芳.红花黄色素药理作用研究进展[J].热带医学杂志,2015,15(3):421-424.Yang Xiaoyuan,Ren Yufang.Advances in pharmacological studies of safflower yellow[J].Trop Med,2015,15(3)421-424.
[8]张璐,翟薇,庞杰,等.红花黄色素对Aβ_(1-42)诱导的痴呆大鼠脑组织炎症因子释放的影响[J].中药药理与临床,2017,33(1):85-89.Zhang L,Zhai W,Pang J,et al.Research of inhibition mechanism of wogonin on lung cancer tumor cell growth and metastasis suppressor[J].Chinese medicine pharmacology and clinical,2017,33(1):85-89.
[9]Shi X M,Zhang H,Zhou Z J,et al.Effects of safflower yellow on beta-amyloid deposition and activation of astrocytes in the brain of APP/PS1 transgenic mice[J].Biomedicine&Pharmacotherapy,2017,98:553-565.
[10]Falco A S,Silva R F,Vaz A R,et al.Cross-talk between neurons and astrocytes in response to bilirubin:early beneficial effects[J].Neurochemical Research,2013,38(3):644-659.
[11]Findeis M A.The role of amyloid beta peptide 42 in Alzheimer's disease[J].Pharmacol Ther,2007,116(2):266-286.
[12]Lisa B H,Lydia L,Donald D N.Mitochondria:pharmacological manipulatian of cell death[J].J Clin Invest,2008,23(6):627-630.
[13]Tu S,Okamoto S I,Lipton S A,et al.Oligomeric Aβ-induced synaptic dysfunction in Alzheimer’s disease[J].Molecular Neurodegeneration,2014,9(1):48.
[14]Chen Y,Vartiainen N E,Ying W,et al.Astrocytes protect neurons from nitric oxide toxicity by a glutathione dependent mechanism[J].J Neurochem,2001,77:1601-1610.
[15]Fellin T,Carmignoto G.Neuron-to-astrocyte signaling in the brain represent a distinct multifunctional unit[J].J Physiol,2004,559:3-15.
[16]M Alcangiom.Brain-derived neurotrophic factor induced NMDA receptor submit one phosphory lation via ERKand PKC in the rat spina l cord[J].Eur JNeurosci,2004,20(7):1769-1778.
[17]Shaked I,Ben Dror I,Vardimon L.Glutamine synthetase enhances the clearance of extracellular glutamate by the neural retina[J].J Neurochem,2002,83:574-580.
[18]Esposito Z,Belli L,Toniolo S,et al.Amyloidβ,glutamate,excitotoxicity in Alzheimer's disease:are we on the right track[J].CNS Neurosci Therapeut,2013,19:549-555.
[19]莫玉言,侯加卫,周张玖智,胡艳丽.红花黄色素对Aβ_(1-42)诱导的SH-SY5Y细胞损伤的保护作用研究[J].中药药理与临床,2018,34(4):40-44.Mo Y Y,Hou J W,Zhou Z J Z.Jiuzhi,Hu Yanli.Study on the protective effects of safflower yellow on injury of cultured SH-SY5Y cells induced by Aβ1-42[J].Chinese medicine pharmacology and clinical,2018,34(4):40-44.