**3. Discussion**

The NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome can be activated by a broad range of stimuli that belong either to pathogen-associated molecular patterns (PAMPs) released during viral, bacterial, fungal, or protozoa infection [23] or to danger-associated molecular patterns (DAMPs) of endogenous or exogenous origin, like extracellular ATP and reactive oxygen species (ROS) [24]. The present study demonstrates that juglone inhibits IL-1b and IL-18 secretion in activated macrophages by suppressing various pro-inflammatory signaling molecules and pathways.

Juglone has been reported to have potent antioxidative effects and prevent various oxidative stress-related diseases by inhibiting ROS-producing enzymes [25,26]. Additionally, juglone has been suggested to have anticancer effects, which are least partly mediated by inhibition of NF-κB signaling and NF-κB-mediated expression of inflammatory cytokines [27,28]. However, the effects of juglone NLRP3 inflammasome formation in macrophages are understudied.

IL-1β, IL-18, and NO have been highlighted by numerous studies as essential mediators of inflammation and have been implicated in autoimmune diseases and other inflammatory conditions [29]. LPS, a major component of Gram-negative bacteria, can induce the expression of inflammatory cytokines and NO. Herein, we reported that juglone treatment inhibited the production of NO in LPS-stimulated J774.1 cells in a concentration-dependent manner. ROS are also involved in inflammatory immune responses and have been shown to induce formation and activation of the NLRP3 inflammasome in response to various exogenous stimuli [30]. In addition, excessive intracellular ROS levels can lead to apoptosis or necrosis [31]. To investigate the effects of juglone on ROS production by macrophages, a DCFH-DA assay was performed in J774.1 cells treated with LPS. Our results suggested that LPS stimulation induced ROS production and that juglone could suppress LPS-induced ROS generation in a dose-dependent manner. Therefore, by reducing ROS production in macrophages, juglone could potentially inhibit activation of the NLRP3 inflammasome.

ATP-induced P2X7R activation promotes the rapid production of large amounts of ROS, which, in turn, stimulates activation of the NLRP3 inflammasome [32]. It has been previously reported that inhibition of the ATPase activity of NLRP3 could decrease the self-oligomerization of NLRP3, as well as its interaction with ASC, which is critical for inflammasome activation [31].

In this study, we found that juglone treatment suppresses not only the expression of NLRP3 but also the ATPase activity of NLRP3, and these were followed by the inhibition of active caspase-1 (Figure 6). These results sugges<sup>t</sup> that juglone could potentially impair the formation of NLRP3 inflammasome, and, thus, the inhibition of pro-caspase-1 activation in macrophages. We also found that treatment with juglone could suppress the LPS/ATP-mediated induction of IL-1β and IL-18 in J774.1 cells, both at the mRNA and protein levels. These correspond to the previous research results that juglone inhibits pro-inflammatory cytokines (TNFα, IL-1 β, and IL-6) and adhesion molecules (VCAM-1 and ICAM-1) expression through the inhibition of <sup>I</sup>κB-phosphorylation-mediated NF-κB activation [17,33].

**Figure 6.** Proposed mechanism underlying the anti-inflammatory e ffects of juglone in macrophages. Pre-treatment of macrophages with juglone suppressed ATP-induced IL-1β, IL-18 and NLRP3 secretion in LPS-primed J774.1 mouse macrophages. Juglone also caused downregulation of ATPase activity and ROS and NO production. Juglone also reduced the mRNA expression and activation of IL-1β, IL-18, NLRP3 and caspase-1. The results show that juglone inhibits IL-1β and IL-18 secretion and NLRP3 formation in activated macrophages.

Furthermore, our extended study results showed that the secretion levels of IL-1β and IL-18 in macrophages were inhibited by juglone treatment in a dose dependent manner. Of note, pro-IL-1β and pro-IL-18 are cleaved to their active forms by caspase-1 upon activation by NLRP3 inflammasome. The expression of IL-18 showed relatively small change rather than that of IL-1β. It may be due to distinct regulation for Il-1β and IL-18. It is reported that there are distinct licensing requirements for processing of IL-1β and IL-18 activation by NLRP3 inflammasome in mice [34]. Considering that juglone is natural product, it is valuable that juglone shows anti-inflammatory e ffects in 10 μM of concentration. Because juglone might be categorized as a potential anticancer compound based on the criteria established by the National Cancer Institute [35] that any compound with IC50 value of ≤4 μg/mL has the potential to be an anticancer compound. In view of these present findings and the existing reports of its use in traditional folk medicine as an anti-inflammatory agent, juglone deserves further studies to justify its potential as an anti-inflammatory agent, using a spectrum of preclinical models. Hence, our results indicate that juglone treatment could reduce the production of mature inflammatory cytokines IL-1β and IL-18 by suppressing their transcription and translation levels as well as caspase-1-mediated cleavage to their active forms. Therefore, our findings sugges<sup>t</sup> that juglone could be used as a potential therapeutic compound for treating inflammatory diseases in the future.
