关键词: 阿尔茨海默病, 长链非编码RNA, 牛磺酸上调基因1, 巨噬细胞移动抑制因子, 核苷酸结合寡聚结构域样受体蛋白1, 免疫印迹法, 小鼠 

Abstract:

Objective  To investigate the relieving effects of knockdown of long non-coding RNA（lncRNA）taurine up-regulated gene 1 (TUG1) on inhibiting nucleotide binding oligomerization domain like receptor protein 1(NLRP1) inflammasome and the progression of Alzheimer’s disease.    Methods  Wild-type (WT group,10 mice) or amyloid precursor protein (APP)/presenilin-1 (PS1) transgenic mice (30 mice) with a genetic background of C57/BL6 aged 9-10 weeks were used in this study. APP/PS1 transgenic mice were randomly divided into model group, model+lncRNA TUG1 short hairpin RNA (shRNA) group and model+shRNA non target（NT）group (n=10). Blood samples, cerebral cortex tissues, primary microglial cells and primary astrocytes were collected from mice 12 weeks of age on day 1 (3-month-old) and 32 weeks of age on day 1 (8-month-old), with 5 mice per group at each time point. Real-time PCR analysis was used to detect the expression levels of lncRNA TUG1 and macrophage migration inhibitory factor (MIF) mRNA in cerebral cortex tissues and primary microglial cells, and C1r and C1s mRNA levels in primary astrocytes of 3-month-old and 8-month-old mice in the above 4 groups, respectively. ELISA was used to determine the MIF in plasma samples of the above 4 groups of mice. Primary microglia and astrocytes from the cerebral cortex of 3-month-old and 8-month-old mice were co-cultured. CCK-8 method  was used to determine the proliferation ability of the above cells. Western blotting was used to determine the expression levels of MIF, pro interleukin-1β(pro-IL-1β), apoptosis associated speck-like protein containing a caspase recrult domain(ASC), Caspase-1 (p20), Caspase-1 (full), NLRP1 and NLRP3 in cerebral cortex tissues of 3-month-old and 8-month-old mice. Immunofluorescent staining was used to determine amyloid beta（Aβ）in cerebral cortex of 8-month-old mice.    Results  At the age of 3-month-old and 8-month-old, compared with the WT group, the relative expression level of lncRNA TUG1 and MIF in cerebral cortex tissues and primary microglia of model group mice was significantly up-regulated, with primary microglial cells and astrocytes proliferation ability enhanced (P<0.05). Compared with the model group, the relative expression level of lncRNA TUG1 and MIF cerebral cortex tissues and primary microglia of model+lncRNA TUG1 shRNA group were significantly down-regulated, with primary microglial cells and astrocytes proliferation ability decreased (P<0.05). Compared with the WT group, MIF factor in the peripheral plasma of model group increased significantly, with pro-IL-1β，ASC，Caspase-1 (p20)，Caspase-1 (full), NLRP1 and NLRP3 expression level up-regulated in the model group mice cerebral cortex tissues, with increased Aβ immunofluorescent indensity (P<0.05). Compared with the model group, MIF factor in the peripheral plasma, and pro-IL-1β，ASC，Caspase-1 (p20)，Caspase-1 (full) and NLRP1 expression in the model+lncRNA TUG1 shRNA group mice cerebral cortex tissues were down-regulated, and Aβ immunofluorescent indensity decreased (P<0.05), while NLRP3 expression level were not changed (P>0.05). There was no significant difference between the model group and the model+shRNA NT group mice of all the above factors (P>0.05).   Conclusion  In APP/PS1 transgenic mice, up-regulation of lncRNA TUG1 and MIF are positively associated with the activation of NLRP1 inflammasome in mice cerebral cortex tissues and primary microglia. Knock-down of lncRNA TUG1 can ameliorate the progression of Alzheimer’s disease.

Key words:

Alzheimer’s disease, Long non-coding RNA, Taurine up-regulated gene 1, Macrophage migration inhibitory factor, Nucleotide binding oligomerization domain like receptor protein 1, Western blotting, Mouse