ESE1通过影响转录因子NF-κB的活性促进LPS侧脑室注射模型中小胶质细胞活化及神经元凋亡

发布时间：2019-01-23 08:23

【摘要】：目的:研究ESE1在脂多糖(LPS)诱导的大鼠中枢神经系统炎症模型中的表达,并探讨ESE1参与小胶质细胞活化以及神经元凋亡的机制。方法:1.整体水平,建立大鼠侧脑室注射脂多糖(lipopolysaccharide,LPS)的神经炎症模型;取54只Sprague-Dawley(SD)雄性大鼠,随机分为两组:LPS注射组(48只)和假手术组(6只)。通过蛋白质印迹法(Western Blot,WB)和免疫组织化学染色法(immunohistochemistry,IHC)检测LPS注射后ESE1的表达和细胞类型定位变化。2.通过免疫荧光双标记法(double-immunofluorescent staining,IF)检测ESE1在LPS注射后与不同细胞的共定位情况。3.通过WB检测LPS注射后i NOS、cleaved caspase-3、Bax及Bcl-2的表达,并通过IF分别检测i NOS/Iba-1、i NOS/ESE1、Neu N/cleaved-capase-3、ESE1/cleaved-caspase-3的共定位情况,以明确ESE1与小胶质细胞活化及神经元凋亡的关系。4.细胞水平,建立脂多糖诱导的小胶质细胞活化模型,通过WB检测i NOS以及ESE1的表达变化,并通过ELISA检测促炎细胞因子的释放,以及荧光素酶报告基因检测NF-κB的转录活性确定ESE1对小胶质细胞活化的影响。同时,也构建小胶质细胞活化培养基(condition medium,CM)诱导的神经元凋亡模型,通过WB检测cleaved-caspase3以及ESE1的表达变化,以及LDH释放实验确定ESE1对神经元凋亡的影响。结果:1.LPS侧脑室注射后,大脑皮质ESE1表达上调,在1d后达到高峰,随后降低。2.ESE1分别与小胶质细胞以及神经元共定位,并且ESE1与其共定位数目增加。3.LPS注射后,cleaved-caspase3、以及i NOS表达上调,达到峰值后,随后下降;并且i NOS/Iba-1、i NOS/ESE1、Neu N/cleaved-capase-3、ESE1/cleaved-caspase-3定位增加。4.在小胶质细胞中敲低ESE1的表达能明显降低i NOS的表达,以及促炎细胞因子的释放,以及NF-κB的转录活性明显降低。5.在神经元细胞中,敲低ESE1的表达能明显降低CM引起的神经元中cleaved-caspase3以及Bcl-2的水平,同时降低CM引起的LDH释放。结论:ESE1在LPS诱导的中枢神经系统中表达上调,其表达变化主要存在于小胶质细胞以及神经元中,而非星形胶质细胞。在中枢神经系统中,ESE1通过影响NF-κB的转录活性,影响小胶质细胞活化以及神经元的凋亡。
[Abstract]:Aim: to investigate the expression of ESE1 in rat model of central nervous system inflammation induced by lipopolysaccharide (LPS), and to explore the mechanism of ESE1 involved in the activation of microglia and neuronal apoptosis. Methods: 1. 54 male Sprague-Dawley (SD) rats were randomly divided into two groups: LPS injection group (n = 48) and sham operation group (n = 6). Western blotting (Western Blot,WB) and immunohistochemical staining (immunohistochemistry,IHC) were used to detect the expression of ESE1 and cell type localization after LPS injection. 2. Immunofluorescence double labeling (double-immunofluorescent staining,IF) was used to detect the co-localization of ESE1 with different cells after LPS injection. The expression of I NOS,cleaved caspase-3,Bax and Bcl-2 after LPS injection was detected by WB, and the co-localization of I NOS/Iba-1,i NOS/ESE1,Neu Nr / cleaved-capase-3 ESE1 / cleaved-caspase-3 was detected by IF. To clarify the relationship between ESE1 and microglia activation and neuronal apoptosis. 4. At cell level, lipopolysaccharide induced microglial activation model was established. The expression of I NOS and ESE1 was detected by WB, and the release of pro-inflammatory cytokines was detected by ELISA. The transcriptional activity of NF- 魏 B was determined by luciferase reporter gene. The effect of ESE1 on the activation of microglia was determined. At the same time, the neuronal apoptosis model induced by microglial activation medium (condition medium,CM) was also constructed. The expression of cleaved-caspase3 and ESE1 was detected by WB, and the effect of ESE1 on neuronal apoptosis was determined by LDH release assay. Results: after intracerebroventricular injection of 1.LPS, the expression of ESE1 in cerebral cortex increased, reached its peak after 1 day, then decreased. 2.ESE1 co-located with microglia and neurons, and the number of co-localization of ESE1 and ESE1 increased after 3.LPS injection. The expression of cleaved-caspase3, and I NOS were up-regulated, reached the peak and then decreased. And I NOS/Iba-1,i NOS/ESE1,Neu Nr / cleaved-capase-3 ESE1 / cleaved-caspase-3 locus increased by 4. 4%. The expression of knockout ESE1 in microglia decreased the expression of I NOS, the release of proinflammatory cytokines and the transcription activity of NF- 魏 B. In neuronal cells, the low expression of ESE1 could significantly decrease the levels of cleaved-caspase3 and Bcl-2 in neurons induced by CM, and decrease the release of LDH induced by CM. Conclusion: the expression of ESE1 is up-regulated in the central nervous system induced by LPS, and the expression changes are mainly in microglia and neurons, but not astrocytes. In the central nervous system, ESE1 affects the activation of microglia and the apoptosis of neurons by affecting the transcriptional activity of NF- 魏 B.
【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R741

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