神经干细胞衰老与核因子E2相关因子2/抗氧化反应元件信号通路的关系

吴奇; 王顺和; 程霄; 向玥; 陈粼波; 汪子铃; 肖含先之; 姜蓉; 王璐; 王亚平

doi:10.16098/j.issn.0529.1356-2020.06.002

PDF(2373 KB)

解剖学报 ›› 2020, Vol. 51 ›› Issue (6) : 815-820. DOI: 10.16098/j.issn.0529.1356-2020.06.002

神经生物学

神经干细胞衰老与核因子E2相关因子2/抗氧化反应元件信号通路的关系

吴奇^1,2王顺和^1,2 程霄^1,2向玥^1,2陈粼波¹汪子玲¹ 肖含先之¹ 姜蓉¹王璐¹ 王亚平^1*

作者信息 +

Relationship between neural stem cells aging and nuclear factor erythroid 2 related factor 2/antioxidant response element signaling pathway

WU Qi^1,2WANG Shun-he^1,2 CHENG Xiao^1,2 XIANG Yue^1,2 CHEN Lin-bo¹ WANG Zi-ling¹ XIAO Ha-xian-zhi¹ JIANG Rong¹ WANG Lu¹ WANG Ya-ping^1*

Author information +

文章历史 +

摘要

目的建立D-半乳糖（D-gal）诱导小鼠神经干细胞（NSCs）体外衰老模型，探讨NSCs衰老与细胞核因子E2相关因子2（Nrf2）/抗氧化反应元件（ARE）信号通路的关系。方法取新生C57BL/6J小鼠全脑，分离、鉴定NSCs，培养至第3代，随机分为对照组和衰老组。对照组细胞在NSCs培养基中常规培养48 h，衰老组细胞在对照组基础上加入D-gal（终浓度为10 g/L）。细胞计数试剂盒8（CCK-8）法检测NSCs增殖活力；衰老相关β-半乳糖苷酶（SA-β-gal）染色检测衰老阳性神经球百分率；锥虫蓝染色检测细胞存活率；酶标比色法检测细胞培养上清液中超氧化物歧化酶（SOD）与过氧化氢酶（CAT）活性和丙二醛（MDA）含量；Western boltting检测NSCs中Nrf2和血红素加氧酶 1（HO-1）蛋白表达水平；Real-time PCR检测GCLC和GCLM基因表达水平。结果与对照组相比，衰老组NSCs增殖活力明显下降，锥虫蓝染色显示，NSCs存活率下降明显，S-β-gal染色阳性神经球百分率显著上升，SOD活性与CAT活性均显著降低，MDA含量显著升高，NSCs中Nrf2/ARE信号通路相关蛋白Nrf2和HO-1表达水平显著下调，通路相关GCLC和GCLM基因表达下调。结论采用D-gal可以构建NSCs体外衰老模型，其氧化损伤机制可能与抑制Nrf2/ARE抗氧化信号通路有关。

Abstract

Objective To establish aging model of mouse neural stem cells (NSCs) in vitro and study the relationship between D-galactose (D-gal)-induced NSCs aging and nuclear factor erythroid 2 related factor 2 (Nrf2)/antioxidant response elemen (ARE) signaling pathway. Methods The whole brain of newborn C57BL/6J mouse was obtained. NSCs were isolated and identified, and cultured the cells to the third generation. The NSCs were randomly divided into control group and aging group. The NSCs of control group were cultured in NSCs medium for 48 hours, and the NSCs of aging group were added with D-galactose (D-gal,final concentration of 10 g/L) on the basis of the control group. Cell counting kit-8(CCK-8) was used to detect the activity of NSCs; Senescence-associated β-galactosidase (SA-β-gal) staining was used to detect the percentage of senescence-positive neurospheres; Trypan blue staining was used to detect cell viability; Enzyme-labeled colorimetry assay was used for detection of the activity of superoxide dismutases (SOD), catalase (CAT) and malondialdehyde (MDA) in the cell culture supernatants; Western blotting was used to detect the expression of Nrf2, heme-oxygenase-1 (HO-1) protein; Real-time PCR was applied to detect the expression level of GCLC, GCLM genes. Results Compared with the control group, the proliferative activity of NSCs in the aging group decreased significantly, the percentage of SA-β-gal staining positive neurons increased obviously, the activity of trypan blue stained NSCs decreased explicitly, SOD activity and CAT activity decreased markedly, and MDA content increased significantly. The expression levels of Nrf2/ARE signaling pathway-related proteins Nrf2 and HO-1 were significantly down-regulated in cells, the expression of pathway-associated GCLC and GCLM genes was down-regulated. Conclusion The addition of D-gal to the culture system can construct an in vitro aging model of NSCs. The possible mechanism is closely related to the inhibition of the antioxidant activity of Nrf2/ARE signaling pathway.

导出引用

吴奇王顺和程霄向玥陈粼波汪子玲肖含先之姜蓉王璐王亚平. 神经干细胞衰老与核因子E2相关因子2/抗氧化反应元件信号通路的关系[J]. 解剖学报. 2020, 51(6): 815-820 https://doi.org/10.16098/j.issn.0529.1356-2020.06.002

WU Qi WANG Shun-he CHENG Xiao XIANG Yue CHEN Lin-bo WANG Zi-ling XIAO Ha-xian-zhi JIANG Rong WANG Lu WANG Ya-ping. Relationship between neural stem cells aging and nuclear factor erythroid 2 related factor 2/antioxidant response element signaling pathway[J]. Acta Anatomica Sinica. 2020, 51(6): 815-820 https://doi.org/10.16098/j.issn.0529.1356-2020.06.002

中图分类号： R338.1

参考文献

［1］ Ni J, Fang Y, Hu Y, et al. Effect of Tenuigenin on low density lipoprotein receptor-related protein 1 level changes of Alzheimer’s disease rat in hippocampus［J］. Acta Anatomica Sinica,2016,47(6):744-749.（in Chinese）

倪杰,房宇,胡燕,等.细叶远志皂苷对阿尔茨海默病大鼠海马低密度脂蛋白受体相关蛋白1水平的影响［J］.解剖学报,2016,47(6):744-749.

［2］ Wang X, Wang C, Pei G. α-Secretase ADAM10 physically interacts with β-secretase BACE1 in neurons and regulates CHL1 proteolysis［J］. J Mol Cell Biol, 2018, 10(5):411-422.

［3］ Dong XH, Gao WJ, Kong WN, et al. Neuroprotective effect of the active components of three Chinese herbs on brain iron load in a mouse model of Alzheimer’s disease［J］. Exp Ther Med, 2015, 9(4):1319-1327.

［4］ Zhang Y, He ML. Deferoxamine enhances alternative activation of microglia and inhibits amyloid beta deposits in APP/PS1 mice［J］. Brain Res, 2017, 1677(1):86-92.

［5］ Bilkeigorzo A. Genetic mouse models of brain ageing and Alzheimer’s disease ［J］. Pharmacol Ther, 2014, 142(2): 244-257.

［6］ Xu J, Wang J, Wimo A, et al. The economic burden of dementia in china, 1990-2030: Implications for health policy［J］. Bull World Health Organ, 2017, 95(1):18-26.

［7］ Wang ZhL, Zhang Y, Wang J. Role of Nogo-A and Nogo-A receptors in Alzheimer’s disease［J］. Acta Anatomica Sinica, 2018,49(4):549-555.（in Chinese）

王兆伦,张艳,王君. Nogo-A及其受体在阿尔茨海默病中的作用［J］.解剖学报,2018,49(4):549-555.

［8］ Dong XH, Bai JT, Kong WN, et al. Effective components of Chinese herbs reduce central nervous system function decline induced by iron overload［J］. Neural Regen Rese, 2015, 10(5): 778-785.

［9］ Dong XH, Gao WJ, Kong WN, et al. Neuroprotective effect of the active components of three Chinese herbs on brain iron load in a mouse model of Alzheimer’s disease［J］. Exp Ther Med, 2015, 9(4):1319-1327.

［10］ Ballard C, Gauthier S, Corbett A, et al. Alzheimer’s disease［J］. Lancet, 2011, 377(9770):1019-1031.

［11］ Wortmann M. World Alzheimer Report 2014:dementia and risk reduction［J］. J Alzheimer’s Assoc, 2015, 11(7): 837.

［12］ Barage SH, Sonawane KD. Amyloid cascade hypothesis: pathogenesis and therapeutic strategies in Alzheimer’s disease［J］. Neuropeptides, 2015, 52:1-18.

［13］ Endres K, Anders A, Kojro E, et al. Tumor necrosis factorD-α converting enzyme is processed by proprotein-convertases to its mature form which is degraded upon phorbol ester stimulation［J］. Eur J Biochem, 2010, 270(11):2386-2393.

［14］ Devi L, Ohno M. Effects of BACE1 haploinsufficiency on APP processing and Aβ concentrations in male and female 5XFAD Alzheimer mice at different disease stages［J］. Neuroscience, 2015, 307:128-137.

［15］ Manzine PR, Ettcheto M, Cano A, et al. ADAM10 in Alzheimer’s disease: pharmacological modulation by natural compounds and its role as a peripheral marker.［J］. Biomed Pharmacother,2019,113:108661.

［16］ Tackenberg C, Nitsch RM. The secreted APP ectodomain sAPPα, but not sAPPβ, protects neurons against Aβ oligomer-induced dendritic spine loss and increased tau phosphorylation ［J］. Mol Brain, 2019, 12(1):27.

［17］ Peterslibeu C, Campagna J, Mitsumori M, et al. sAβPPα is a potent endogenous inhibitor of BACE1［J］. J Alzheimers Dis, 2015, 47(3):545-555.

［18］ Barger SW, Harmon AD. Microglial activation by Alzheimer amyloid precursor protein and modulation by apolipoprotein E ［J］. Nature, 1998, 388(6645):878-881.

［19］ Su X, Zhang Zh, Yang LJ, et al. Granulocyte colony stimulating factor improving the earning and memory function and its anti-inflammatory mechanism of Alzheimer’s disease model rats［J］. Acta Anatomica Sinica, 2019,50(1):29-34.（in Chinese）

苏翔,张臻,杨霖璟,等.粒细胞集落刺激因子改善阿尔茨海默病模型大鼠学习记忆功能及抗炎作用机制［J］.解剖学报,2019,50(1):29-34.

［20］ Joseph JA, Carey A, Brewer GJ, et al. Dopamine and a beta-induced stress signaling and decrements in Ca2+ buffering in primary neonatal hippocampal cells are antagonized by blueberry extract［J］. J Alzheimers Dis, 2007, 11(4):433-446.

［21］ Luo XJ, Lin M, Wang ChM. Role and research progress of inflammation in the mechanism of Alzheimer’s disease［J］.Journal of Tropical Medicine, 2016(1):119-122.（in Chinese）

罗小金, 林茂, 王春梅. 炎症反应在阿尔茨海默病发病机制中的研究进展［J］. 热带医学杂志, 2016(1):119-122.