Shu Min, Zhang Wenzhe, Jin Xiangbo, Zeng Linghui, Xiang Yingchun
School of Medicine, Zhejiang University City College, Hangzhou 310015, China.
Department of Pharmacy, Zhejiang Hospital, Hangzhou 310012, China.
Zhejiang Da Xue Xue Bao Yi Xue Ban. 2020 Dec 25;49(6):750-757. doi: 10.3785/j.issn.1008-9292.2020.12.10.
To investigate the protective effect of gene knockout on Alzheimer's disease (AD) in mice.
The animal model of AD was established by intraperitoneal injection of D-galactose and brain-localized injection of amyloid β-protein (Aβ) in wild type C57BL/6 mice and 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.
Behavioral results showed that cognitive deficits were found in wide type mice after induction of AD as its prolonged escape latency (<0.05) and decreased crossing number of platform and target zone duration (all <0.05); while the knockout of alleviated cognitive deficit induced by AD (all <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 knockout mice was reduced compared with that of wild type mice (all <0.05). The expression of LC3-Ⅱ and P62 increased after AD was induced in wild type mice, while the autophagy in knockout mice was activated as the increase expression of LC3-Ⅱ and decrease expression of P62 (all <0.05). In the AD cell model, the results of MTT assay were consistent with the animal experiments, and the protective effect of knockdown was eliminated after the treatment of the autophagy inhibitor chloroquine (all <0.05).
The knockdown of shows a protective effect on AD induced by D-galactose and Aβ in mice, which may be related to its function of activating autophagy.
研究基因敲除对小鼠阿尔茨海默病（AD）的保护作用。
通过向野生型C57BL/6小鼠和基因敲除小鼠腹腔注射D-半乳糖及脑内注射淀粉样β蛋白（Aβ）建立AD动物模型。采用Morris水迷宫、Y迷宫和悬尾试验评估小鼠的认知功能和焦虑样行为。通过免疫荧光染色检测海马中Aβ沉积。进行蛋白质免疫印迹分析以检测脑中相关蛋白的表达。培养小鼠皮质原代神经元并建立AD细胞模型。建模后用MTT法检测细胞活力。
行为学结果显示，诱导AD后野生型小鼠出现认知缺陷，逃避潜伏期延长（<0.05），穿越平台次数和目标区域停留时间减少（均<0.05）；而基因敲除可减轻AD诱导的认知缺陷（均<0.05）。Aβ免疫荧光染色显示，与野生型小鼠相比，基因敲除小鼠海马区Aβ沉积及脑中裂解的半胱天冬酶3表达减少（均<0.05）。野生型小鼠诱导AD后LC3-Ⅱ和P62表达增加，而基因敲除小鼠自噬被激活，表现为LC3-Ⅱ表达增加和P62表达减少（均<0.05）。在AD细胞模型中，MTT法检测结果与动物实验一致，自噬抑制剂氯喹处理后基因敲低的保护作用消失（均<0.05）。
基因敲低对D-半乳糖和Aβ诱导的小鼠AD具有保护作用，这可能与其激活自噬的功能有关。
