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.
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。