Abstract：
BACKGROUND: Sepsis-associated encephalopathy (SAE) is a diffuse brain dysfunction activated by microglia. The potential pathological changes of SAE are complex, and the cellular pathophysiological characteristics remains unclear. This study aims to explore the ROS/TXNIP/NLRP3 pathway mediated lipopolysaccharide (LPS)-induced inflammatory response in microglia.
METHODS: BV-2 cells were pre-incubated with 10 μM N-acetyl-L-cysteine (NAC) for 2 h, which were then reacted with 1 μg/mL LPS for 24 h. Western blot assay examined the protein levels of IBA1, CD68, TXNIP, NLRP3, ASC, and Cleaved Caspase-1 in BV-2 cells. The contents of inflammatory factor were detected by ELISA assay. The co-immunoprecipitation assay examined the interaction between TXNIP and NLRP3.
RESULTS: LPS was confirmed to promote the positive expressions of IBA1 and CD68 in BV-2 cells. The further experiments indicated that LPS enhanced ROS production and NLRP3 inflammasome activation in BV-2 cells. Moreover, we also found that NAC partially reversed the facilitation of LPS on the levels of ROS, IL-1β, IL-18, TXNIP, NLRP3, ASC, and Cleaved Caspase-1 in BV-2 cells. NAC treatment also notably alleviated the interaction between TXNIP and NLRP3 in BV-2 cells.
CONCLUSIONS: ROS inhibition mediated NLRP3 signaling inactivation by decreasing TXNIP expression.
METHODS: BV-2 cells were pre-incubated with 10 μM N-acetyl-L-cysteine (NAC) for 2 h, which were then reacted with 1 μg/mL LPS for 24 h. Western blot assay examined the protein levels of IBA1, CD68, TXNIP, NLRP3, ASC, and Cleaved Caspase-1 in BV-2 cells. The contents of inflammatory factor were detected by ELISA assay. The co-immunoprecipitation assay examined the interaction between TXNIP and NLRP3.
RESULTS: LPS was confirmed to promote the positive expressions of IBA1 and CD68 in BV-2 cells. The further experiments indicated that LPS enhanced ROS production and NLRP3 inflammasome activation in BV-2 cells. Moreover, we also found that NAC partially reversed the facilitation of LPS on the levels of ROS, IL-1β, IL-18, TXNIP, NLRP3, ASC, and Cleaved Caspase-1 in BV-2 cells. NAC treatment also notably alleviated the interaction between TXNIP and NLRP3 in BV-2 cells.
CONCLUSIONS: ROS inhibition mediated NLRP3 signaling inactivation by decreasing TXNIP expression.
摘要：
背景：脓毒症相关性脑病（SAE）是由小胶质细胞激活的弥漫性脑功能障碍。SAE的潜在病理变化是复杂的，细胞病理生理特征尚不清楚。本研究旨在探讨ROS/TXNIP/NLRP3通路介导的脂多糖(LPS)诱导的小胶质细胞炎症反应。
方法：将BV-2细胞与10μMN-乙酰-L-半胱氨酸（NAC）预孵育2小时，然后与1μg/mLLPS反应24小时。蛋白质印迹测定法检查了IBA1，CD68，TXNIP，NLRP3,ASC,和BV-2细胞中裂解的Caspase-1。ELISA法检测炎症因子含量。免疫共沉淀测定检查了TXNIP和NLRP3之间的相互作用。
结果：LPS可促进BV-2细胞中IBA1和CD68的阳性表达。进一步的实验表明，LPS增强了BV-2细胞中ROS的产生和NLRP3炎性体的激活。此外，我们还发现NAC部分逆转了LPS对ROS水平的促进作用，IL-1β,IL-18,TXNIP,NLRP3,ASC,和BV-2细胞中裂解的Caspase-1。NAC处理还显著减轻了BV-2细胞中TXNIP和NLRP3之间的相互作用。
结论：ROS抑制通过降低TXNIP表达介导NLRP3信号失活。
方法：将BV-2细胞与10μMN-乙酰-L-半胱氨酸（NAC）预孵育2小时，然后与1μg/mLLPS反应24小时。蛋白质印迹测定法检查了IBA1，CD68，TXNIP，NLRP3,ASC,和BV-2细胞中裂解的Caspase-1。ELISA法检测炎症因子含量。免疫共沉淀测定检查了TXNIP和NLRP3之间的相互作用。
结果：LPS可促进BV-2细胞中IBA1和CD68的阳性表达。进一步的实验表明，LPS增强了BV-2细胞中ROS的产生和NLRP3炎性体的激活。此外，我们还发现NAC部分逆转了LPS对ROS水平的促进作用，IL-1β,IL-18,TXNIP,NLRP3,ASC,和BV-2细胞中裂解的Caspase-1。NAC处理还显著减轻了BV-2细胞中TXNIP和NLRP3之间的相互作用。
结论：ROS抑制通过降低TXNIP表达介导NLRP3信号失活。
