Fig 2: Activation of NLRP3 with nigericin and overexpression of NLRP3 reversed the antipyroptotic effects of CAL in vitro. (a) and (b) Representative immunofluorescent staining of NLRP3 and caspase-1. (c) NLRP3 activation by nigericin abolished CAL anti-pyroptotic effects in vitro. Representative Western blot images of NLRP3, ASC, Caspase-1, IL-1ß, IL-18, and GSDMD in H9c2 cells. (d) Forced overexpression of NLRP3 reversed CAL antipyroptotic effects in vitro. Protein expressions of NLRP3, ASC, caspase-1, IL-1ß, IL-18, and GSDMD in H9c2 cells were examined by Western blotting.

Fig 3: 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. (c,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.

Fig 4: Proposed mechanism underlying the anti-inflammatory effects of juglone in macrophages. Pre-treatment of macrophages with juglone suppressed ATP-induced IL-1ß, 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-1ß, IL-18, NLRP3 and caspase-1. The results show that juglone inhibits IL-1ß and IL-18 secretion and NLRP3 formation in activated macrophages.

Fig 5: Uric acid-induced NLRP3 inflammasome activation and pyroptosis are regulated by CTSB. (A) Western blot was conducted to evaluate the protein level of CTSB, NLRP3, caspase-1, GSDMD and GAPDH in HK-2 cell lysates. (B–E) Scatter plots showing the densitometry analysis of CTSB, NLRP3, caspase-1 and GSDMD normalized by GAPDH. (F) Mechanisms of uric acid-induced autophagy promotes HN. Chronic exposure to uric acid induces dysregulation of autophagy, which is mediated by p53 pathway. The activation of autophagy further activated NLRP3 inflammasome and pyroptosis. The release of CTSB from autolysosome plays a crucial role in the activation of NLRP3 inflammasome and pyroptosis, which leads to the release of IL-1ß and IL-18. Blockade of autophagy inhibits the aforementioned responses so that prevents the occur of hyperuricemic nephropathy. Data are expressed as mean ± SEM. ***P<0.001; ****P<0.0001.