解剖学报

解剖学报 ›› 2016, Vol. 47 ›› Issue (6): 731-737.doi: 10.16098/j.issn.0529-1356.2016.06.002

• 神经生物学 • 上一篇    下一篇

当归多糖延缓D-半乳糖所致巢蛋白-绿色荧光蛋白小鼠脑衰老作用及其机制

姚辉1,2 陈粼波1 陈雄斌1 刘颖1 夏婕妤1 汪子铃1 向玥1,2 熊丽溶1 王璐1 王顺和1,2* 王亚平1*   

  1. 1. 重庆医科大学干细胞与组织工程研究室，组织学与胚胎学教研室; 2.重庆医科大学病理学教研室， 重庆 400016
  • 收稿日期:2016-06-23 修回日期:2016-08-17 出版日期:2016-12-06 发布日期:2016-12-06
  • 通讯作者: 王顺和;王亚平 E-mail:ypwangcq@aliyun.com
  • 基金资助:

    当归多糖调控血液造血干细胞衰老的机制;人参皂苷Rg1延缓神经干细胞衰老调控机理

Anti-aging effects of angelica sinensis polysaccharides on brain aging induced by D-galactose in Nestin-green fluorescent protein transgenic mice and its mechanism

YAO Hui 1,2 CHEN Lin-bo1 CHEN Xiong-bin1 LIU Ying1 XIA Jie-yu1 WANG Zi-ling1 XIANG Yue 1,2 XIONG Li-rong1 WANG Lu1 WANG Shun-he 1,2* WANG Ya-ping 1*   

  1. 1. Laboratory of Stem Cell and Tissue Engineering，Department of Histology and Embryology，Chongqing Medical University; 2. Department of Pathology，Chongqing Medical University，Chongqing 400016，China
  • Received:2016-06-23 Revised:2016-08-17 Online:2016-12-06 Published:2016-12-06
  • Contact: WANG Shun-he ;WANG Ya-ping  E-mail:ypwangcq@aliyun.com

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摘要：

目的 探讨当归多糖(ASP)延缓 D-半乳糖(D-Gal)所致巢蛋白（Nestin）-绿色荧光蛋白(GFP)小鼠脑衰老作用及可能机制。 方法 6～8周龄雄性Nestin-GFP转基因小鼠40只，随机分为正常组(n=10)、ASP正常组(n=10)、脑衰老模型组(n=10)、ASP脑衰老模型组(n=10)。脑衰老模型组：小鼠皮下注射D-gal 200 mg/kg qd×42 d;ASP脑衰老模型组：从脑衰老模型建立的16d 起腹腔注射ASP 140 mg/kg qd×27 d；ASP正常组：皮下注射等量生理盐水，第16天 起腹腔注射ASP (剂量与时间同上)；正常组：小鼠皮下注射等量生理盐水42d。模型建立完成第2天水迷宫实验检测各组小鼠的空间记忆能力；取海马制备冷冻切片，观察海马区神经干细胞荧光强度；衰老相关β-半乳糖苷酶(SA-β-Gal) 染色观察海马组织中染色阳性细胞百分率；酶标比色法检测海马区超氧化物歧化酶 (SOD)、总抗氧化能力（T-AOC）和丙二醛（MDA）含量；酶联免疫吸附测定(ELISA)检测海马区白细胞介素(IL)-1β，IL-6和肿瘤坏死因子(TNF)-α炎症因子变化。 结果 脑衰老模型组小鼠空间记忆能力减退，海马齿状回(DG区)荧光强度降低，SA-β-Gal 染色阳性颗粒增加，海马组织 SOD 活性和T-AOC降低，MDA 含量升高，IL-1β，IL-6 和TNF-α含量增多。与脑衰老模型组比较ASP脑衰老模型组小鼠空间记忆能力有明显改善，海马DG区荧光强度增强，SA-β-Gal 染色阳性颗粒减少，海马组织SOD 活性和T-AOC升高，MDA 含量降低，IL-1β，IL-6 和TNF-α含量减少。 结论 ASP能延缓D-Gal所致小鼠脑衰老，其机制可能与抑制氧化应激损伤、下调炎症因子水平、维持海马区神经干细胞数量有关。

关键词: 当归多糖, 脑衰老模型, 神经干细胞, 酶联免疫吸附测定, 巢蛋白-绿色荧光蛋白转基因小鼠

Abstract:

Objective To explore the anti-aging effects of angelica sinensis polysaccharides(ASP) on brain aging induced by D-galactose in Nestin-green fluorescent protein (GFP )transgenic mice and its mechanism. Methods Male Nestin-GFP transgenic mice (n=40) aging from 6 to 8 weeks old were randomly divided into the normal, ASP normal, brain aging model and ASP brain aging model groups. The brain aging model group was subcutaneously injected with D-galactose (200 mg/kg), qd×42 days. The ASP brain aging model group was intraperitoneally injected with ASP (140 mg/kg) since the 16th day of the replication in the brain aging model, qd×27 days. The ASP normal group was subcutaneously injected with the same amount of saline, qd×15 days, and following intraperitoneally injected with ASP (140 mg/kg) qd×27 days. The normal group was subcutaneously injected with an equal volume of saline within the same time. The related experiment was performed on the second day after finishing copying the model. Learning and memory abilities were measured by Morris water maze. Frozen sections were made to observe the hippocampus fluorescence intensity. The number of senescent cells were detected by senescence-associated β-galactosidase (SA-β-Gal) staining. The activities of superoxide dismutase (SOD), total antioxidant capacity (T-AOC) and contents of malonaldehyde (MDA) in hippocampus were quantified by chromatometry. The level of interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α proinflammatory cytokines in hippocampus were detected by methods enzyme-linked immunosorbent assay (ELISA). Results In the brain aging model group, the spatial learning and memory capacities were weaken, the fluorescence intensity decreased in the dentate gyrus (DG) of hippocampus, SA-β-Gal positive granules increased in section of brain tissue，and the activity of SOD, T-AOC decreased in hippocampus while the contents of MDA increased in hippocampus. The level of IL-1β, IL-6 and TNF-α increased in hippocampus. Compared with the brain aging model group, the spatial learning and memory capacities in the ASP brain aging group, were enhanced, the fluorescence intensity increased in the DG area of hippocampus, SA-β-Gal positive granules decreased in section of brain tissue, and the activity of SOD, T-AOC increased in hippocampus while the contents of MDA decreased in hippocampus. The level of IL-1β, IL-6 and TNF-α in hippocampus decreased. Conclusion ASP can antagonize brain aging induced by D-galactose in mice. In addition, improvement of antioxidant ability, down regulation of the level of proinflammatory cytokines and maintaining the number of neural stem cells in hippocampus may be the underlying anti-aging mechanism of ASP.

Key words: Angelica sinensis polysaccharide, Brain aging model, Neural stem cell, Enzyme-linked immunosorbeit assay, Nestin-green fluorescent protein transgenic mouse