One of the mechanisms by which NLRP3 inflammasome can be activated is ROS generation (Tschopp and Schroder, 2010), and mROS formation seems particularly critical for NLRP3 activation (Zhou et al., 2011). We therefore explored the potential role of ethanol-induced mROS in NLRP3 inflammasome activation. To assess superoxide production by mitochondria in cultured astrocytes, we used the MitoSOXTM Red reagent in live cells, a fluorogenic dye that specifically targets mitochondria in live cells, and a flow cytometry analysis. For these experiments, astrocytes were treated with ethanol (10 and 50 mM), LPS and ATP for 24 h in the presence or absence of Mito-TEMPO [a mitochondria (m) ROS scavenger] or a Z-YVAD-FMK (specific caspase-1 inhibitor) or z-VAD-FMK (inhibitor of caspase proteases) or a NLRP3 bp. Then mROS production was measured. The results indicate that treating WT astrocytes with ATP or LPS or ethanol (10 mM or 50 mM) markedly induced mROS production (Figure 4A). Notably, the mROS production induced by ethanol (10 and 50 mM) or LPS or ATP was significantly abrogated by Mito-TEMPO, z-YVAD-FMK, or Z-VAD-FMK by the presence of NLRP3
