**2. Results**

#### *2.1. E*ff*ects of Juglone on Cell Viability*

To determine the juglone concentration range that is non-toxic to J774.1 cells, cells were treated with increasing concentrations of juglone (3.1–50 μM), followed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. When juglone was used at 10 μM or less, no significant cytotoxic e ffects were observed (*p* > 0.05; Figure 1). Therefore, juglone was used at 2.5, 5, or 10 μM in subsequent experiments.

**Figure 1.** Assessment of the cytotoxic e ffects of juglone in J774.1 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (**a**) Chemical structure of juglone. (**b**) J774.1 cells were treated with increasing concentrations of juglone (3.1–50 μM) for 24 h, followed by quantification of cell viability using the MTT assay. Data are presented as mean ± standard deviation (SD) of three independent experiments. \* *p* < 0.05; \*\* *p* < 0.01; vs. control.

#### *2.2. E*ff*ects of Juglone on Reactive Oxygen Species (ROS) Production*

ROS have a strong oxidative capacity and are essential mediators of NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome activation. To determine the ability of juglone to inhibit lipopolysaccharide (LPS)-induced ROS production in J774.1 cells, we used the cell-permeable ROS-sensitive dye DCFH-DA, which is non-fluorescent in a reduced state and emits fluorescence upon oxidation by ROS. We found that treatment with LPS enhanced ROS production in J774.1 cells. However, when cells were treated with 10 μM juglone, ROS production was significantly impaired (Figure 2a). The fluorescence intensity in LPS-treated cells was approximately 120%, while the fluorescence intensity in juglone-treated (10 μM) cells after LPS stimulation was only 84.5%. Thus, juglone may exert anti-inflammatory effects by suppressing ROS generation by macrophages during inflammation.

**Figure 2.** Effects of juglone on the production of inflammatory mediators. (**a**) reactive oxygen species (ROS) production in J774.1 cells treated with lipopolysaccharide (LPS). Cells were treated with different concentrations of juglone (2.5, 5, and 10 μM) for 2 h, followed by treatment with 1 μg/mL LPS for an additional 24 h. (**b**) NO production in J774.1 cells treated with LPS. Cells were treated with increasing concentrations of juglone (3.1–50 μM or 0 μM control) for 2 h, followed by treatment with 1 μg/mL LPS for an additional 24 h. The levels of NO in the cell culture media were measured using Griess reagent. (**<sup>c</sup>**,**d**) Effects of juglone on the LPS/ATP-induced secretion of interleukin-1β (IL-1β) and IL-18 in J774.1 cells. J774.1 macrophages were treated with different concentrations of juglone for 2 h, followed by treatment with 1 μg/mL LPS for 6 h and treatment with 5 mM ATP for 1 h. The levels of IL-1β and IL-18 secreted in the culture medium were analyzed by enzyme-linked immunosorbent assay (ELISA). Data are presented as mean ± SD of three independent experiments. \* *p* < 0.05; \*\* *p* < 0.01 vs. LPS or LPS + ATP cells, respectively.

#### *2.3. E*ff*ects of Juglone on Nitric Oxide (NO) Production*

Inflammatory mediators, such as reactive nitrogen species and ROS, are essential mediators of inflammatory responses, especially in the initial stages of inflammation. Therefore, we assessed the effects of juglone in LPS-induced NO production. We found that even though LPS treatment resulted in a significant increase in NO production in J774.1 cells, juglone treatment suppressed LPS-induced NO production in a dose-dependent manner (*p* < 0.05; Figure 2b).

#### *2.4. E*ff*ects of Juglone on the Secretion of the Pro-Inflammatory Cytokines IL-1*β *and IL-18*

Inflammatory cytokines are important mediators of immune responses and orchestrate the initial stages of inflammation. The pro-inflammatory cytokines IL-1β and IL-18 are known to mediate the first steps of an inflammatory immune response [22]. Therefore, targeting these upstream mediators of inflammation has emerged as a promising approach to treat inflammation-related diseases. Thus, we sought to investigate whether juglone could affect the levels of mRNA, protein, or extracellular secretion of IL-18 and IL-1β. We found that treatment with juglone suppressed the induction of IL-18 and IL-1β at the mRNA and protein level in response to LPS and ATP in J774.1 cells (Figure 3 and Figure S1). The secretory inhibition of pro-inflammatory cytokines (IL-1β and IL-18) of juglone on murine macrophage cells was studied and the results are shown Figure 2c,d. Treatment of macrophage cells with juglone caused a concentration-dependent reduction in their IL-1β (5 μM, \* *p* < 0.05) and IL-18 (10 μM, \*\* *p* < 0.01). Juglone-treated cell was reduced 25.9% of IL-1β secretion (5 μM, \* *p* < 0.05) and 22.6% of IL-18 secretion (10 μM, \*\* *p* < 0.01) compared to the LPS plus STP-primed control group. These results suggested that juglone could inhibit the initial steps of the inflammatory immune response by suppressing the expression and secretion of the pro-inflammatory cytokines IL-1β and IL-18 in macrophages.

**Figure 3.** Effects of juglone on IL-1β and IL-18 expression in LPS-treated J774.1 cells. J774.1 macrophages were treated with different concentrations of juglone (2.5, 5, and 10 μM) for 2 h, followed by treatment with 1 μg/mL LPS for 6 h and 5 mM ATP for 1 h. (**a**) The mRNA levels of IL-1β and IL-18 in J774.1 cells were determined with RT-qPCR. Glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) was used as a reference gene. (**b**) The protein levels of IL-1β and IL-18 in J774.1 cells were assessed by Western blotting and are presented as relative to β-actin intensity. Data are presented as mean ± SD. \* *p* < 0.05; \*\* *p* < 0.01 vs. LPS + ATP treated cells.

#### *2.5. E*ff*ects of Juglone on NLRP3 and Caspase-1 Expression*

We then assessed the e ffects of juglone treatment on the mRNA and protein levels of NLRP3 and caspase-1. We found that even though NLRP3 was induced in J774.1 cells following stimulation with LPS and ATP, treatment with juglone suppressed the LPS/ATP-mediated NLRP3 induction at the mRNA level in a dose-dependent manner. The mRNA levels of caspase-1 were not significantly a ffected by juglone treatment (Figure 4a). Importantly, juglone treatment suppressed the LPS/ATP-mediated NLRP3 ATPase upregulation at the protein level in J774.1 macrophages (Figure 4b and Figure S2). Juglone also reduced the levels of cleaved caspase-1 in a concentration-dependent manner (Figure 4b). These results suggested that juglone could inhibit the interaction of NLRP3 and apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC) and subsequent NLRP3 inflammasome formation by downregulating the expression of NLRP3. Moreover, the decreased NLRP3 expression might impair ASC speck formation, leading to reduced cleavage and subsequent activation of the downstream signaling mediator, caspase-1.

**Figure 4.** J774.1 cells were treated with various concentrations of juglone for 2 h and then treated with 1 μg/mL LPS for 24 h. (**a**) The mRNA levels of NACHT, LRR and PYD domains-containing protein 3 (NLRP3) and caspase-1 in J774.1 cells were determined by RT-qPCR. GAPDH was used as a reference gene. (**b**) The relative protein levels of NLRP3, procaspase-1, and cleaved caspase-1 were assessed by Western blotting and are presented as relative to β-actin intensity. Data are presented as the mean ± SD.\* *p* < 0.05; \*\* *p* < 0.01 vs. LPS + ATP treated cells.

#### *2.6. E*ff*ects of Juglone on the ATPase Activity of NLRP3*

The pyrin ATP-binding domain of NLRP3 exhibits ATPase activity, which is essential for NLRP3 inflammasome oligomerization. Therefore, we investigated the effects of juglone treatment on the ATPase activity of NLRP3 in J774.1 cells. Treatment with LPS and ATP in J774.1 macrophages increased the ATPase activity of NLRP3 (Figure 5). However, treatment with 10 μM of juglone significantly reduced the ATPase activity of NLRP3 (*p* < 0.05).

**Figure 5.** Effects of juglone on the ATPase activity of NLRP3 in LPS/ATP-treated J774.1 cells. J774.1 macrophages were treated with different concentrations of juglone for 2 h, followed by treatment with 1 μg/mL LPS for 6 h and 5 mM ATP for an additional hour. Analysis of the ATPase activity of NLRP3 was performed using a reaction mixture containing 40 mM Tris, 80 mM NaCl, 8 mM MgAc2, 1 mM EDTA, and 4 mM ATP, pH 7.5. Data are presented as mean ± SD of three independent experiments. \* *p* < 0.05 vs. LPS/ATP treated cells.
