雷公藤氯内酯醇（T_4）通过激活Wnt/β-catenin信号通路减轻寡聚态Aβ_（1-42）诱导的神经元凋亡
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摘要
目的：观察寡聚态Aβ1-42对原代培养的大鼠皮层神经元凋亡的诱导作用及可能机制,探讨雷公藤氯内酯醇(tripchlorolide, T4)保护大鼠皮层神经元免于发生凋亡的作用及可能机制。
方法：选用孕16-18d的清洁级SD(Sprague-Dawley)大鼠,分离纯化皮层神经元,应用5μmol/l寡聚态Aβ1-42以及Wnt/β-catenin信号通路激动剂Wnt3a和抑制剂Dkk1处理神经元,分别通过MTT法、TUNEL染色和蛋白免疫印迹术观察神经元存活率、细胞凋亡情况以及β-catenin和GSK3β及其磷酸化产物的表达水平,了解Wnt/β-catenin信号通路在寡聚态Aβ1-42诱导的神经元凋亡过程中的作用,并进一步探讨T4保护神经元免于凋亡的可能分子机制。
结果：1、5μmol/l寡聚态Aβ1-42与原代培养的胎鼠皮层神经元共同孵育24h后,MTT检测神经元存活率较正常对照组明显下降,TUNEL染色神经元胞体缩小,细胞突起变短、消失,胞核深染。2、寡聚态Aβ1-42作用于胎鼠皮层神经元24h后,GSK3β、磷酸化GSK3β、磷酸化β-catenin水平升高,非磷酸化β-catenin水平下降,这与Wnt/β-catenin通路抑制剂Dkk1的作用类似。3、用不同浓度T4对胎鼠的皮层神经元进行预处理后再加入寡聚态Aβ1-42共同孵育,发现T4对寡聚态Aβ1-42诱导的神经元损伤有明显的保护作用,表现为T4可显著提高寡聚态Aβ1-42作用后的皮层神经元生存率,减少神经元凋亡；此外T4可选择性降低细胞内β-catenin的磷酸化程度,稳定细胞内β-catenin水平,同时减少GSK3β及其磷酸化产物水平,这与Wnt/β-catenin通路激动剂Wnt3a的作用类似。
结论：1、寡聚态Aβ1-42可诱导胎鼠皮层神经元凋亡；Wnt/β-catenin信号通路是Aβ1-42诱导胎鼠的皮层神经元凋亡的可能途径。2、一定剂量T4预处理可减轻寡聚态Aβ1-42诱导胎鼠的皮层神经元凋亡。T4的神经元保护作用可能是通过活化Wnt/β-catenin信号通路选择性降低细胞内β-catenin的磷酸化程度,稳定细胞内β-catenin水平,同时减少GSK3β及其磷酸化产物水平来实现的。T4在阿尔茨海默病的治疗方面可能具有潜在的应用价值。
Objective:To investigate the effect and the molecular mechanisms of oligomeric Aβ1-42-induced apoptosis in primary cultured rat cortical neurons, and to explore the underlied mechanism of protective action of tripchlorolide (T4) in oligomeric Aβ1-42-treated neurons.
Methods:Primary cultures of cortical neurons were prepared from the embryonic day 16-18d Sprague-Dawley rats. The oligomeric Aβ1-42 (5μM for 24 h) was applied to induce apptotic neuronal death. Prior to treatment with Ap for 24 h, the cultured neurons were pre-incubated with T4 (2.5,10 and 40nM), Wnt3a (Wnt signaling agonists) and Dkkl (inhibitors) for indicated time. Then the cell viability, neuronal apoptosis, and protein levels of Wnt, glycogen synthase kinase 3β(GSK3β),β-catenin and phospho-β-catenin were measured by MTT assay, TUNEL staining and Western blot, respectively.
Results:
1. Oligomeric Aβ1-42 induced apoptotic neuronal cell death in a time-and dose-dependent manner. After treatment with 5μmol/1 oligomeric Aβ1-42 for 24 hours, the cortical neuronal survival was significantly decreased by 20% compared with control group by MTT assay. The neural body shrank, process shortened or dismissed and nucleus was stained black by TUNEL
2. Treatment with oligomeric Aβ1-42 significantly increased the protein levels of phosphorylatedβ-catenin and GSK3β. In contrast, the non-phosphorylatedβ-catenin protein levels were decrease. Wnt signaling inhibitors Dkkl produced the familiar effect on the protein changes of P-catenin and GSK3β.
3. Pretreatment with T4 significantly increased the neuronal cell survival and attenuated apoptotic neuronal death. Moreover, oligomeric Aβ1-42-induced the phosphorylation ofβ-catenin and GSK3βwas markedly inhibited by T4. Additionally, T4 stabilized cytoplasmicβ-catenin to activate P-catenin downsteam signaling. Wnt signaling agonists Wnt3a produced the familiar effect on the protein changes ofβ-catenin and GSK30.
Conclusion:Tripchlorolide protects against the neurotoxicity of Aβby regulating Wnt/β-catenin signaling pathway. This will provide insight into the clinical application of tripchlorolide to Alzheimer's disease.
方法：选用孕16-18d的清洁级SD(Sprague-Dawley)大鼠,分离纯化皮层神经元,应用5μmol/l寡聚态Aβ1-42以及Wnt/β-catenin信号通路激动剂Wnt3a和抑制剂Dkk1处理神经元,分别通过MTT法、TUNEL染色和蛋白免疫印迹术观察神经元存活率、细胞凋亡情况以及β-catenin和GSK3β及其磷酸化产物的表达水平,了解Wnt/β-catenin信号通路在寡聚态Aβ1-42诱导的神经元凋亡过程中的作用,并进一步探讨T4保护神经元免于凋亡的可能分子机制。
结果：1、5μmol/l寡聚态Aβ1-42与原代培养的胎鼠皮层神经元共同孵育24h后,MTT检测神经元存活率较正常对照组明显下降,TUNEL染色神经元胞体缩小,细胞突起变短、消失,胞核深染。2、寡聚态Aβ1-42作用于胎鼠皮层神经元24h后,GSK3β、磷酸化GSK3β、磷酸化β-catenin水平升高,非磷酸化β-catenin水平下降,这与Wnt/β-catenin通路抑制剂Dkk1的作用类似。3、用不同浓度T4对胎鼠的皮层神经元进行预处理后再加入寡聚态Aβ1-42共同孵育,发现T4对寡聚态Aβ1-42诱导的神经元损伤有明显的保护作用,表现为T4可显著提高寡聚态Aβ1-42作用后的皮层神经元生存率,减少神经元凋亡；此外T4可选择性降低细胞内β-catenin的磷酸化程度,稳定细胞内β-catenin水平,同时减少GSK3β及其磷酸化产物水平,这与Wnt/β-catenin通路激动剂Wnt3a的作用类似。
结论：1、寡聚态Aβ1-42可诱导胎鼠皮层神经元凋亡；Wnt/β-catenin信号通路是Aβ1-42诱导胎鼠的皮层神经元凋亡的可能途径。2、一定剂量T4预处理可减轻寡聚态Aβ1-42诱导胎鼠的皮层神经元凋亡。T4的神经元保护作用可能是通过活化Wnt/β-catenin信号通路选择性降低细胞内β-catenin的磷酸化程度,稳定细胞内β-catenin水平,同时减少GSK3β及其磷酸化产物水平来实现的。T4在阿尔茨海默病的治疗方面可能具有潜在的应用价值。
Objective:To investigate the effect and the molecular mechanisms of oligomeric Aβ1-42-induced apoptosis in primary cultured rat cortical neurons, and to explore the underlied mechanism of protective action of tripchlorolide (T4) in oligomeric Aβ1-42-treated neurons.
Methods:Primary cultures of cortical neurons were prepared from the embryonic day 16-18d Sprague-Dawley rats. The oligomeric Aβ1-42 (5μM for 24 h) was applied to induce apptotic neuronal death. Prior to treatment with Ap for 24 h, the cultured neurons were pre-incubated with T4 (2.5,10 and 40nM), Wnt3a (Wnt signaling agonists) and Dkkl (inhibitors) for indicated time. Then the cell viability, neuronal apoptosis, and protein levels of Wnt, glycogen synthase kinase 3β(GSK3β),β-catenin and phospho-β-catenin were measured by MTT assay, TUNEL staining and Western blot, respectively.
Results:
1. Oligomeric Aβ1-42 induced apoptotic neuronal cell death in a time-and dose-dependent manner. After treatment with 5μmol/1 oligomeric Aβ1-42 for 24 hours, the cortical neuronal survival was significantly decreased by 20% compared with control group by MTT assay. The neural body shrank, process shortened or dismissed and nucleus was stained black by TUNEL
2. Treatment with oligomeric Aβ1-42 significantly increased the protein levels of phosphorylatedβ-catenin and GSK3β. In contrast, the non-phosphorylatedβ-catenin protein levels were decrease. Wnt signaling inhibitors Dkkl produced the familiar effect on the protein changes of P-catenin and GSK3β.
3. Pretreatment with T4 significantly increased the neuronal cell survival and attenuated apoptotic neuronal death. Moreover, oligomeric Aβ1-42-induced the phosphorylation ofβ-catenin and GSK3βwas markedly inhibited by T4. Additionally, T4 stabilized cytoplasmicβ-catenin to activate P-catenin downsteam signaling. Wnt signaling agonists Wnt3a produced the familiar effect on the protein changes ofβ-catenin and GSK30.
Conclusion:Tripchlorolide protects against the neurotoxicity of Aβby regulating Wnt/β-catenin signaling pathway. This will provide insight into the clinical application of tripchlorolide to Alzheimer's disease.
引文
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[2]Braak H, Braak E. Frequency of stages of Alzheimer-related lesions in different age categories. Neurobiol Aging.1997,18(4):351-357.
[3]Dong MJ, Peng B, Lin XT, et al. The prevalence of dementia in the People's Republic of China:a systematic analysis of 1980-2004 studies. Age Ageing.2007,36(6):619-624.
[4]Bertoni-Freddari C, Fattoretti P, Casoli T, et al. Neuronal apoptosis in Alzheimer's disease: the role of age-related mitochondrial metabolic competence[J]. Ann N Y Acad Sci, 2009,1171:18-24.
[5]Nikolaev A, McLaughlin T, O'Leary DD, et al. APP binds DR6 to trigger axon pruning and neuron death via distinct caspases[J]. Nature,2009,457(7232):981-989.
[6]Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer's disease:progress and problems on the road to therapeutics. Science.2002,297(5580):353-356.
[7]White JA, Manelli AM, Holmberg KH, et al. Differential effects of oligomeric and fibrillar amyloid-beta 1-42 on astrocyte-mediated inflammation. Neurobiol Dis. 2005,18(3):459-465.
[8]Dahlgren KN, Manelli AM, Stine WB, Jr., et al. Oligomeric and fibrillar species of amyloid-beta peptides differentially affect neuronal viability. J Biol Chem. 2002,277(35):32046-32053.
[9]Lacor PN, Buniel MC, Furlow PW, et al. Abeta oligomer-induced aberrations in synapse composition, shape, and density provide a molecular basis for loss of connectivity in Alzheimer's disease. J Neurosci.2007,27(4):796-807.
[10]Haass C, Selkoe DJ. Soluble protein oligomers in neurodegeneration:lessons from the Alzheimer's amyloid beta-peptide. Nat Rev Mol Cell Biol.2007,8(2):101-112.
[11]Wang Q, Rowan MJ, Anwyl R. Beta-amyloid-mediated inhibition of NMDA receptor-dependent long-term potentiation induction involves activation of microglia and stimulation of inducible nitric oxide synthase and superoxide. J Neurosci. 2004,24(27):6049-6056.
[12]Poling A, Morgan-Paisley K, Panos JJ, et al. Oligomers of the amyloid-beta protein disrupt working memory:Confirmation with two behavioral procedures. Behav Brain Res. 2008.
[13]Knobloch M, Farinelli M, Konietzko U, et al. Abeta oligomer-mediated long-term potentiation impairment involves protein phosphatase 1-dependent mechanisms. J Neurosci.2007,27(29):7648-7653.
[14]Inestrosa NC, Toledo EM. The role of Wnt signaling in neuronal dysfunction in Alzheimer's Disease[J]. Mol Neurodegener,2008;3:9.
[15]Fogarty MP, Kessler JD, Wechsler-Reya RJ. Morphing into cancer:the role of developmental signaling pathways in brain tumor formation[J]. J Neurobiol,2005;64(4):458-475.
[16]Logan CY, Nusse R:The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol 2004,20:781-810.
[17]Nusse R:Wnt signaling and stem cell control. Cell Res 2008,18:523-527.
[18]Steiner JP, Connolly MA, Valentine HL, et al. Neurotrophic actions of nonimmunosuppressive analogues of immunosuppressive drugs FK506, rapamycin and cyclosporin A. Nat Med.1997,3(4):421-428.
[19]Tao X, Cush JJ, Garret M, et al. A phase I study of ethyl acetate extract of the chinese antirheumatic herb Tripterygium wilfordii hook F in rheumatoid arthritis[J]. J Rheumatol, 2001,28(10):2160-2167.
[20]马伟光,张滔,张超,等.有毒药物雷公藤的研究及展望.中华中医药杂志.2006,21(2)：117-120.
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