敲除<i>Sirt3</i>后激活自噬对阿尔茨海默病有保护作用

doi:10.3785/j.issn.1008-9292.2020.12.10

浙江大学学报（医学版）

2020, Vol. 49

Issue (6): 750-757 DOI: 10.3785/j.issn.1008-9292.2020.12.10

原著

敲除Sirt3后激活自噬对阿尔茨海默病有保护作用

舒敏¹(

),张文哲¹,金湘博¹,曾玲晖^1,*(

),项迎春^2,*(

)

1. 浙大城市学院医学院, 浙江杭州 310015
2. 浙江医院药剂科, 浙江杭州 310012

Sirt3 gene knockout protects mice from Alzheimer's disease through activating autophagy

SHU Min¹(

),ZHANG Wenzhe¹,JIN Xiangbo¹,ZENG Linghui^1,*(

),XIANG Yingchun^2,*(

)

1. School of Medicine, Zhejiang University City College, Hangzhou 310015, China
2. Department of Pharmacy, Zhejiang Hospital, Hangzhou 310012, China

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摘要：

目的: 探究Sirt3在阿尔茨海默病（AD）发生发展中的作用及可能机制。方法: 体内实验以C57BL/6野生型小鼠和Sirt3基因敲除小鼠为对象，采用腹腔注射D-半乳糖联合脑定位注射β-淀粉样蛋白（Aβ）_1-40建立AD小鼠模型。莫里斯（Morris）水迷宫实验、自发活动开场实验和悬尾实验观察造模前后小鼠学习记忆和焦虑抑郁状态的改变，免疫荧光染色观察脑内海马区域Aβ沉积情况，蛋白质印迹法检测小鼠脑组织中自噬和凋亡相关蛋白表达。体外实验选小鼠皮层原代细胞作为对象，给予Aβ_1-40建立AD体外模型，MTT法检测细胞活性。结果: 体内实验结果显示，野生型小鼠诱发AD后平台潜伏期延长（P < 0.05），穿越平台次数减少、目标象限停留时间缩短（均P < 0.05），提示造模后小鼠的学习记忆能力下降；而Sirt3敲除能够缓解AD所致的学习记忆障碍（均P < 0.05）。相较于野生型小鼠，Sirt3基因敲除小鼠脑内海马区域的Aβ沉积减少（P < 0.05），凋亡蛋白cleaved caspase 3表达减少（P < 0.05）。另外，野生型小鼠诱发AD后LC3-Ⅱ和P62蛋白表达增加（均P < 0.05），提示自噬流受阻；而Sirt3基因敲除小鼠诱发AD后LC3-Ⅱ蛋白表达增加，P62蛋白表达减少（均P < 0.05），提示自噬被激活。小鼠皮层原代细胞AD模型中，Sirt3基因敲除的细胞较野生型细胞死亡减少（P < 0.05）；加用自噬抑制剂氯喹后，Sirt3基因敲除的保护作用消失（P < 0.05）。结论: Sirt3敲除对于D-半乳糖联合Aβ_1-40诱发的AD有保护作用，这种保护作用可能与自噬流活化有关。

关键词： 阿尔茨海默病; Sirt3; 自噬; β-淀粉样蛋白; 学习; 记忆; 小鼠

Abstract:

Objective: To investigate the protective effect of Sirt3 gene knockout on Alzheimer's disease (AD) in mice. Methods: The animal model of AD was established by intraperitoneal injection of D-galactose and brain-localized injection of amyloid β-protein (Aβ)_1-40 in wild type C57BL/6 mice and Sirt3 gene knockout mice. Morris water maze, Y maze and tail suspension test were used to assess the cognitive function and anxiety-like behaviors in mice. Aβ deposition in the hippocampus was detected by immunofluorescent staining. Western blotting analysis was conducted to detect the expression of related proteins in the brain. Mouse cortical primary neurons were cultured and AD cell model was established. MTT assay was used to detect cell viability after modeling. Results: Behavioral results showed that cognitive deficits were found in wide type mice after induction of AD as its prolonged escape latency (P < 0.05) and decreased crossing number of platform and target zone duration (all P < 0.05); while the knockout of Sirt3 alleviated cognitive deficit induced by AD (all P < 0.05). Aβ immunofluorescence staining showed that the deposition of Aβ in the hippocampal region and expression of cleaved caspase 3 in the brain in Sirt3 knockout mice was reduced compared with that of wild type mice (all P < 0.05). The expression of LC3-Ⅱ and P62 increased after AD was induced in wild type mice, while the autophagy in Sirt3 knockout mice was activated as the increase expression of LC3-Ⅱ and decrease expression of P62 (all P < 0.05). In the AD cell model, the results of MTT assay were consistent with the animal experiments, and the protective effect of Sirt3 knockdown was eliminated after the treatment of the autophagy inhibitor chloroquine (all P < 0.05). Conclusion: The knockdown of Sirt3 shows a protective effect on AD induced by D-galactose and Aβ_1-40 in mice, which may be related to its function of activating autophagy.

Key words: Alzheimer's disease Sirt3 Autophagy Amyloid β-protein Learning Memory Mice

收稿日期: 2020-09-20 出版日期: 2021-01-14

CLC:

R741.02

基金资助: 浙江省科技计划项目(2018C37131);2018年度高层次留学回国人员（团队）在杭创新创业项目

通讯作者: 曾玲晖,项迎春 E-mail: shumin123@zju.edu.cn;zenglh@zucc.edu.cn;xych999@126.com

作者简介: 舒敏(1996-), 女, 硕士研究生, 主要从事神经系统疾病研究; E-mail:shumin123@zju.edu.cn; https://orcid.org/0000-0001-9119-5103

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引用本文:

舒敏,张文哲,金湘博,曾玲晖,项迎春. 敲除Sirt3后激活自噬对阿尔茨海默病有保护作用[J]. 浙江大学学报（医学版）, 2020, 49(6): 750-757.

SHU Min,ZHANG Wenzhe,JIN Xiangbo,ZENG Linghui,XIANG Yingchun. Sirt3 gene knockout protects mice from Alzheimer's disease through activating autophagy. J Zhejiang Univ (Med Sci), 2020, 49(6): 750-757.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2020.12.10 或 http://www.zjujournals.com/med/CN/Y2020/V49/I6/750

表 1 野生型小鼠和Sirt3基因敲除小鼠诱发阿尔茨海默病前后莫里斯水迷宫实验结果

表 2 野生型小鼠和Sirt3基因敲除小鼠诱发阿尔茨海默病前后自发活动开场实验和悬尾实验结果

图 1 野生型小鼠和Sirt3基因敲除小鼠诱发阿尔茨海默病后海马组织β-淀粉样蛋白沉积情况

图 2 野生型小鼠和Sirt3基因敲除小鼠诱发阿尔茨海默病后凋亡和自噬相关蛋白电泳图

表 3 野生型小鼠和Sirt3基因敲除小鼠诱发阿尔茨海默病前后凋亡和自噬相关蛋白表达量比较

图 3 不同处理后小鼠阿尔茨海默病模型细胞活性变化(n=3)

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