**1. Introduction**

Innate immune cells, including macrophages and dendritic cells, play crucial roles in the initiation of inflammatory immune responses upon activation of inflammasome complexes that induce the maturation of inflammatory cytokines [1]. Pattern-recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), initiating signaling cascades that lead to inflammation and release of inflammatory cytokines [2,3]. Toll-like receptors (TLRs) and NOD-like receptors (NLRs) called PRR are membrane-bound and cytoplasmic receptors, respectively, which recognize PAMPs and DAMPs [4]. The immune responses initiated by the interaction between TLRs and PAMPs are mediated through MyD88-dependent and TRIF-dependent pathways [2,3,5]. When TLRs recognize and bind lipopolysaccharides (LPS) or other extracellular PAMPs, the transcription factor nuclear factor (NF)-κB is activated and induces the expression of several pro-inflammatory cytokines, including interleukin (IL)-1β and IL-18 [6]. On the other hand, NLRs, PRRs found in the cytoplasm, are activated by DAMPs, including adenosine triphosphate (ATP), monosodium urate crystals, β-amyloid, and nigericin [7].

P2X7, a membrane-bound PPR that recognizes DAMP, is a membrane-bound receptor that binds extracellular ATP, promoting the release of K<sup>+</sup> [8,9], and subsequently, inducing the formation of inflammasomes after binding of NLRP3 (NACHT, LRR and PYD domains-containing protein 3) to ASC (apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain) and pro-caspase-1. The formation of NLRP3 inflammasomes activates pro-caspase-1, which induces pyroptosis by cleaving pro-IL-1β and pro-IL-18 into their active forms, which initiate the innate immune response [7]. In addition, intracellular danger signals, such as mitochondrial damage and reactive oxygen species (ROS) production, can promote assembly and activation of NLRP3 inflammasomes [10–12]. Inflammatory factors and cytokines can also prime NLRP3 inflammasomes, and ROS inhibitors have been reported to suppress NLRP3 priming [13]. Therefore, the treatment of macrophages with potent antioxidative agents has emerged as a promising strategy to block NLRP3 inflammasome priming.

Juglone (5-hydroxy-1,4-naphthoquinone) is a natural phenolic compound isolated from the roots, stems, and leaves of walnut trees [14]. Juglone is produced during allelopathy and inhibits the germination or growth of surrounding plants by inhibiting specific enzymes necessary for metabolic function, mitochondrial respiration, and photosynthesis [15]. Recent studies have reported that juglone has a variety of pharmacological e ffects, including anti-inflammatory, antioxidative, anticancer, and antimicrobial e ffects, by inhibiting ROS-producing enzymes and preventing oxidative stress [14,16–21]. However, the e ffect of juglone on NLRP3 inflammasome formation and activation remains elusive. The aim of this study was to investigate the potential of juglone to inhibit the NLRP3 inflammasome-mediated inflammatory e ffects of LPS and ATP.
