摘要
目的探讨敲低长链非编码RNA(lncRNA)牛磺酸上调基因1(TUG1)抑制核苷酸结合寡聚结构域样受体蛋白1(NLRP1)炎症小体在缓解阿尔茨海默病进展中的作用。方法选取9~10周龄遗传背景为C57/BL6的野生型小鼠(WT组,10只)或淀粉样前体蛋白(APP)/早老素1(PS1)转基因小鼠(30只)。APP/PS1转基因小鼠随机分为模型(model)组,模型+敲低lncRNA TUG1组[model+lncRNA TUG1短发夹RNA(shRNA)组]和model+shRNA非靶标(NT)组,每组10只。分别采集12周龄第1天(3月龄)和32周龄第1天(8月龄)小鼠外周血和脑皮质组织,并分离皮质中的原代小胶质细胞和原代星形胶质细胞,每个时间点每组5只小鼠。Real-time PCR分别测定3月龄和8月龄上述4个分组小鼠脑皮质组织和原代小胶质细胞中lncRNA TUG1和巨噬细胞移动抑制因子(MIF)mRNA的水平,以及原代星形胶质细胞中补体蛋白C1r和C1s mRNA的水平。ELISA法测定其外周血浆中MIF含量。对3月龄和8月龄小鼠脑皮质原代小胶质细胞和原代星形胶质细胞共培养。CCK-8法测定上述2种细胞的增殖能力。Western blotting分别测定3月龄和8月龄上述4个分组小鼠脑皮质组织中MIF、白细胞介素1β前体(pro-IL-1β)、凋亡相关斑点样蛋白(ASC)、Caspase-1(p20)、Caspase-1(full)、NLRP1及NLRP3蛋白的表达水平。采用免疫荧光染色法测定8月龄各分组小鼠脑皮质组织中β淀粉样蛋白(Aβ)表达。结果3月龄和8月龄时,与WT组小鼠相比,model组小鼠脑皮质组织和原代小胶质细胞中lncRNA TUG1和MIF相对表达水平显著上调,原代小胶质细胞和原代星形胶质细胞增殖能力增强(P<0.05)。与model组相比,model+lncRNA TUG1 shRNA组小鼠脑皮质组织和原代小胶质细胞中lncRNA TUG1和MIF的相对表达水平显著降低,原代小胶质细胞和原代星形胶质细胞增殖能力降低(P<0.05)。与WT组相比,model组小鼠外周血浆中MIF含量显著升高;小鼠脑皮质组织中pro-IL-1β、ASC、Caspase-1(p20)、Caspase-1(full)、NLRP1以及NLRP3的蛋白表达水平显著升高;Aβ免疫荧光强度明显增强(P<0.05)。与model组相比,model+lncRNA TUG1 shRNA组小鼠外周血浆中MIF含量显著降低;小鼠脑皮质组织中pro-IL-1β、ASC、Caspase-1(p20)、Caspase-1(full)和NLRP1的蛋白表达水平显著降低,Aβ免疫荧光强度明显降低(P<0.05),而NLRP3蛋白质的表达水平无明显变化(P>0.05)。与model组相比,model+shRNA NT组小鼠上述所有检测指标差异均无显著性(P>0.05)。结论APP/PS1转基因小鼠脑皮质组织和原代小胶质细胞中lncRNA TUG1和MIF因子表达上调与脑皮质内NLRP1炎症小体激活成正相关,敲低lncRNA TUG1可缓解阿尔茨海默病的进展。
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 Aβimmunofluorescent 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 Aβimmunofluorescent 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.
作者
马婷婷
陈建红
刘爱翠
李海宁
MA Ting-ting;CHEN Jian-hong;LIU Ai-cui;LI Hai-ning(Department of Neurology,General Hospital of Ningxia Medical University,Yinchuan 750004,China)
出处
《解剖学报》
CAS
CSCD
2024年第1期32-42,共11页
Acta Anatomica Sinica
基金
宁夏重点研发项目(2019BEB04008)。
