Fig. 1: Nimbolide inhibits NLRP3 inflammasome activation and pyroptosis in macrophages.

A Heatmap representing the screening of 126 natural products (10 μM) for IL-1β inhibition in culture supernatant of BMDMs. The arrow indicates NIM. The heatmap represents the mean values from these independent experiments. B Structure of NIM. C Cell viability of BMDMs treated with increasing concentrations of NIM for 24 h, assessed by CCK-8 assay. D Immunoblot analysis of cleaved Caspase-1 (p20) and IL-1β in culture supernatants (SN) and proteins in whole-cell lysates (Lys) of BMDMs. Cells were primed with LPS (500 ng/mL) for 3 h, treated with NIM (1, 2, or 5 μM) or the inhibitor MCC950 (5 μM) for 30 min, and stimulated with ATP (2.5 mM) for 30 min. E, F Levels of IL-1β (E) and IL-6 (F) in culture supernatants from BMDMs treated as in D, measured by ELISA. G Immunoblot analysis of full-length gasdermin D (GSDMD, p53) and the cleaved N-terminal fragment (p32) in lysates of BMDMs treated as in D. H Representative fluorescence images of BMDMs stained with Propidium Iodide (PI, red) and Hoechst (blue). Scale bar: 100 μm. The cells were stimulated with LPS (500 ng/mL) for 3 h, treated with NIM (5 μM) or MCC950 (5 μM) as previously described, then were challenged with ATP (2.5 mM) for 1 h. I Quantification of the percentage of PI-positive cells from (H). J Lactate dehydrogenase (LDH) release in supernatants of BMDMs. The cells were stimulated with LPS (500 ng/mL) for 3 h, treated with NIM (1, 2, and 5 μM) or MCC950 (5 μM) as previously described, then were challenged with ATP (2.5 mM) for 1 h. Data are representative of three biologically independent experiments and presented as the mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test (E, F, I, J). *P < 0.05, **P < 0.01, ***P < 0.001, significantly different; ns, not significant.