肝脏Kupffer细胞缺失的APP/PS1小鼠模型建立及表型分析

Establishment and phenotypic analysis of an APP/PS1 mouse model with hepatic Kupffer cell depletion

摘要

目的 建立肝脏Kupffer细胞缺失的APP/PS1小鼠模型, 系统分析该模型在阿尔茨海默病(AD)认知功能、脑内AD病理特征、肝脏功能及炎症等方面的表型变化, 为探究Kupffer细胞与AD发病机制的关系提供实验依据。方法 利用APP/PS1双转基因AD小鼠, 通过腹腔注射氯膦酸盐构建Kupffer细胞缺失的APP/PS1小鼠模型; 通过Morris水迷宫实验评估认知功能; 硫磺素S染色、免疫组织化学法(6E10抗体标记)检测脑内β淀粉样蛋白(Aβ)沉积; 免疫组织化学法检测脑内胶质纤维酸性蛋白(星形胶质细胞活化标志物)和离子钙结合适配分子1(小胶质细胞活化标志物)的表达; 肝组织免疫荧光及血清检测分析外周Aβ代谢; Western blotting分析肝脏中低密度脂蛋白受体相关蛋白1(LRP-1)的表达水平; ELISA检测血清中肝脏功能指标及肝脏中炎症因子水平。结果 Kupffer细胞缺失加剧APP/PS1小鼠空间记忆障碍(P<0.05), 增加小鼠皮层和海马体中Aβ沉积(P<0.05)、星形胶质细胞与小胶质细胞活化(P<0.05); 肝组织Aβ沉积增加(P<0.01); 血清Aβ40及Aβ42水平显著升高(P<0.01), 肝脏清除Aβ主要受体LRP-1的表达上调(P<0.01); Kupffer细胞缺失对APP/PS1小鼠肝脏功能无显著影响(P>0.05), 但可抑制肝脏炎症(P<0.05)。结论 成功建立了肝脏Kupffer细胞缺失的APP/PS1小鼠模型, 该模型呈现更严重的AD病理特征和认知功能障碍, 揭示了肝脏Kupffer细胞可通过调控外周Aβ清除参与AD的病理进程。

Abstract

Objective To establish an APP/PS1 mouse model with hepatic Kupffer cell depletion, systematically analyze the phenotypic changes of the model in terms of Alzheimer's disease (AD) cognitive function, brain AD pathological characteristics, liver function and inflammation, and provide an experimental basis for exploring the association between Kupffer cells and the pathogenesis of AD. Methods APP/PS1 double-transgenic AD mice were used to construct an APP/PS1 mouse model with Kupffer cell depletion by intraperitoneal injection of clodronate; cognitive function was evaluated by Morris water maze; thioflavin S staining and immunohistochemistry (labeled with 6E10 antibody) were used to detect amyloid-beta (Aβ) deposition in the brain; the expression of glial fibrillary acidic protein (astrocyte activation marker) and ionized calcium-binding adapter molecule 1 (microglial activation marker) in the brain were detected by means of immunohistochemistry; hepatic tissue immunofluorescence and serum detection were used to analyze peripheral Aβ metabolism; Western blotting was used to analyze the expression level of low-density lipoprotein receptor-related protein 1 (LRP-1) in hepatic tissue; ELISA was used to detect the levels of liver function indicators in serum and inflammatory cytokines in liver tissue. Results Kupffer cell depletion exacerbated spatial memory impairment and increased Aβ deposition (P<0.05), as well as astrocyte and microglial activation in the cortex and hippocampus of APP/PS1 mice (P<0.05); increased Aβ deposition in liver tissue (P<0.01), significantly elevated serum Aβ40 and Aβ42 levels (P<0.01), and upregulated expression level of LRP-1-the primary receptor for hepatic clearance of Aβ (P<0.01). Kupffer cell depletion had no significant effect on liver function of APP/PS1 mice (P>0.05), but could inhibit liver inflammation (P<0.05). Conclusion We successfully established an APP/PS1 mouse model with hepatic Kupffer cell depletion. This model exhibited more severe AD pathological features and cognitive impairment, revealing that hepatic Kupffer cells can be involved in the pathological process of AD by regulating peripheral Aβ clearance.

关键词

阿尔茨海默病 / 肝脏 / Kupffer细胞 / APP/PS1小鼠 / β淀粉样蛋白 / 模型建立 / 表型分析 / 低密度脂蛋白受体相关蛋白1

Key words

Alzheimer's disease / liver / Kupffer cells / APP/PS1 mice / amyloid-beta / model establishment / phenotypic analysis / low-density lipoprotein receptor-related protein 1

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基金资助

国家自然科学基金青年科学基金(82405017)

陕西基础科学(化学、生物学)研究院基础科学研究计划青年项目(23JHQ038)

咸阳市重点研发计划项目(L2025-ZDYF-JBFZ-027)