米诺环素抑制炎症小体介导的细胞焦亡改善阿尔茨海默病小鼠的认知能力研究

doi:10.12092/j.issn.1009-2501.2020.06.004

摘要/Abstract

摘要： 目的: 研究米诺环素(minocycline, Mino)抑制炎症小体介导的细胞焦亡改善阿尔茨海默病认知能力的作用机制。方法: 脂多糖(lipopolysaccharide, LPS)诱导大鼠神经细胞株PC12构建神经细胞焦亡损伤模型,将细胞分为对照组、LPS组、LPS+Mino组,CCK-8法检测细胞活力,流式细胞术检测细胞凋亡水平,蛋白免疫印迹法检测炎症小体关键蛋白NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor pyrin domain containing 3, NLRP3)、凋亡相关斑点样蛋白(apoptosis-associated speck-like protein containing CARD, ASC)、Caspase-1及pro-Caspase-1的水平以及焦亡执行蛋白Gasdermin-D(GSDMD)、p30-GSDMD的水平,酶联免疫吸附法检测培养基中白介素(IL)-1β、IL-18、肿瘤坏死因子(TNF)-α的表达水平。APP-PS1双转基因小鼠10只,随机分为对照组和实验组,实验组采用50 μg的米诺环素灌胃给药,分别在给药前和给药后3周采用Morris水迷宫实验检测小鼠的认知记忆能力,处死小鼠后取海马CA3区检测组织中NLRP3、ASC、Caspase-1及pro-Caspase-1的蛋白表达,以及炎症因子IL-1β、IL-18、TNF-α、焦亡执行蛋白GSDMD、p30-GSDMD的水平。结果: 米诺环素可以抑制LPS诱导的细胞凋亡,LPS+Mino组细胞活力显著高于LPS组,而细胞凋亡率显著低于LPS组,差异具有统计学意义(P<0.05)。而LPS+Mino组中炎症小体关键蛋白NLRP3、ASC、Caspase-1的表达低于LPS组,同时焦亡执行蛋白p30-GSDMD低于LPS组,GSDMD高于LPS组,培养基中IL-1β、IL-18、TNF-α的水平低于LPS组,差异具有统计学意义(P<0.05)。动物实验中,米诺环素可以显著改善小鼠的认知能力;Morris水迷宫实验中,小鼠潜伏期缩短,相比对照组具有统计学意义(P<0.05)。同时小鼠穿越平台次数明显增多,相比对照组具有统计学意义(P<0.05)。实验组小鼠炎症小体关键蛋白NLRP3、ASC、Caspase-1的表达低于对照组,同时焦亡执行蛋白p30-GSDMD低于对照组,GSDMD高于对照组,炎症因子IL-1β、IL-18、TNF-α的水平低于对照组,差异具有统计学意义(P<0.05)。结论: 米诺环素可以通过抑制炎症小体活化,抑制神经细胞焦亡的发生,同时改善阿尔茨海默病小鼠的认知能力。

关键词: 米诺环素, 阿尔茨海默病, 炎症小体, 细胞焦亡

Abstract: AIM: To study the mechanism of minocycline (Mino) in inhibiting inflammatory corpuscle NLRP3-mediated pyroptosis and improving cognitive ability in Alzheimer's disease. METHODS: Lipopolysaccharide (LPS) was used to induce PC12 to construct a model of neuronal pyroptosis. The cells were divided into control group, LPS group and LPS+Mino group. CCK-8 assay was used to detect cell viability, flow cytometry was used to detect apoptotic level, and Western blot was used to detect the levels of key proteins NLRP3, ASC, Caspase-1 and pro-Caspase-1 in NLRP3 corpuscles, as well as the levels of GSDMD and p30-GSDMD. Enzyme-linked immunosorbent assay was used to detect the expression of interleukin-1β, interleukin-18 and tumor necrosis factor-α in culture medium. APP-PS1 mice were randomly divided into control group and experimental group. The experimental group was given intragastric administration of minocycline 50 μg. The cognitive and memory abilities of mice were tested by Morris test before and 3 weeks after administration. The expressions of NLRP3, ASC, Caspase-1 and pro-Caspase-1 in hippocampal CA3 region were detected after execution, and the expressions of inflammatory factors such as IL-1β, IL-18 and TNF-α were detected. RESULTS: Minocycline could inhibit LPS-induced pyroptosis. Cell viability in LPS+Mino group was significantly higher than that in LPS group, and the apoptotic rate was significantly lower than that in LPS group (P<0.05). The expression of NLRP3, ASC and Caspase-1 in LPS+Mino group was lower than that in LPS group, while the expression of p30-GSDMD was lower than that in LPS group, and GSDMD was higher than that in LPS group. The levels of IL-1β, IL-18 and TNF-α in culture medium were lower than those in LPS group, with significant difference (P<0.05). In animal experiments, minocycline could significantly improve the cognitive ability of mice. In Morris experiment, the latency of mice was shortened, which had statistical significance compared with the control group (P<0.05). At the same time, the number of times of mice crossing the platform increased significantly compared with the control group (P<0.05). The expression of key proteins NLRP3, ASC and Caspase-1 in NLRP3 corpuscle of mice in experimental group was lower than that in control group, while the expression of pyroptosis executive protein p30-GSDMD was lower than that in control group, GSDMD was higher than that in control group, and the levels of inflammatory factors IL-1β, IL-18 and TNF-α were lower than that in control group (P<0.05). CONCLUSION: Minocycline can inhibit the activation of NLRP3 corpuscle and the occurrence of neuronal pyroptosis, and improve the cognitive ability of mice with Alzheimer's disease.

Key words: minocycline, Alzheimer's disease, inflammatory corpuscle, pyroptosis

中图分类号:

R965.2

盛泳佳, 李文燕, 祝迪薇, 王瑾, 顾艳玲. 米诺环素抑制炎症小体介导的细胞焦亡改善阿尔茨海默病小鼠的认知能力研究[J]. 中国临床药理学与治疗学, 2020, 25(6): 625-632.

SHENG Yongjia, LI Wenyan, ZHU Diwei, WANG Jin, GU Yanling. Minocycline inhibits inflammatory corpuscle mediated pyroptosis and improves cognitive ability in Alzheimer's disease mice[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2020, 25(6): 625-632.

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