Compound 4′-O-methylbroussochalcone B attenuates LPS/ATP-induced macrophage pyroptosis in vitro through modulation of the NLRP3 inflammasome and MAPK/NF-κB pathways

Background The compound 4′-O-methylbroussochalcone B (4′-O-M) is a chalcone-type flavonoid isolated from Psoralea corylifolia , a traditional Asian medicinal herb. While studies have reported its antioxidant and anti-inflammatory activities, its effects on macrophage inflammatory signaling and NLRP3 inflammasome activation remain unclear. Therefore, in this in vitro study, we aimed to clarify the anti-inflammatory actions and underlying molecular mechanisms of 4′-O-M in lipopolysaccharide (LPS)/adenosine triphosphate (ATP)-stimulated J774A.1 macrophages, with a particular emphasis on mitochondrial reactive oxygen species (mtROS)-dependent NLRP3 inflammasome activation and MAPK/NF-κB signaling pathways. Methods J774A.1 macrophages were pretreated with graded concentrations of 4′-O-M, followed by LPS priming and ATP stimulation to activate the inflammasome. Levels of nitric oxide (NO), prostaglandin E 2 (PGE 2 ), and cytokines (interleukin [IL]-6, tumor necrosis factor-α [TNF-α], IL-1β, IL-18) in J774A.1 macrophages were quantified by ELISA. MAPK/NF-κB phosphorylation and inflammasome-related proteins (NLRP3, ASC, caspase-1, IL-1β, gasdermin D) were assessed by immunoblotting. mtROS production was evaluated using the MitoSOX™ Red assay. Results 4′-O-M markedly reduced LPS-induced inflammatory mediators, suppressing NO and PGE 2 production and IL-6 and TNF-α secretion by inhibiting MAPK and NF-κB phosphorylation. As the response progressed to the inflammasome activation stage, 4′-O-M further reduced IL-1β and IL-18 release and limited caspase-1 activation. Additionally, 4′-O-M decreased the production of mtROS, an upstream signal associated with NLRP3 activation, suggesting that this reduction may contribute to its inhibitory effects on the inflammasome pathway. Conclusion 4′-O-M mitigates macrophage inflammatory responses in vitro via the dual suppression of MAPK/NF-κB-mediated priming and mtROS-dependent NLRP3 inflammasome activation. Thus, 4′-O-M is a promising natural scaffold for developing therapeutics targeting inflammasome-driven inflammatory diseases.