Fig. 1

Virtual screening and validation of potential NLRP3 inflammasome inhibitors in BMDMs. (A) The schematic model of screening NLRP3 inflammasome inhibitors from a nature compound library. (B) Five potential NLRP3 inflammasome inhibitors from 5,088 natural products were selected based on docking scores. (C) LPS (100 ng/mL) primed-BMDMs were treated with candidates and then stimulated with ATP (5 mmol/L). The levels of IL-1β in the supernatant were measured by ELISA, n = 6. MCC950 as a positive drug for inhibiting NLRP3 inflammasome activation. (D) The binding affinity between candidate compounds and purified human NLRP3 protein was examined by SPR assay. (E) Levels of IL-1β and caspase-1 in SN (Supernatant) and levels of pro-IL-1β/pro-caspase-1 in cell lysates (Lysates) were analyzed by Western blotting to examine the effects of candidate drugs on NLRP3 inflammasome activation. (F) The chemical structure of S4737 (Psoralen). Levels of IL-1β and caspase-1 in SN and levels of pro-IL-1β/pro-caspase-1 in Lysates were analyzed by immunoblotting in BMDMs pretreated with different concentrations of Psoralen (0.01, 0.1 and 1 µM) followed by stimulation with LPS/ATP (G) or LPS/Nigericin (I). Phos-tag SDS-PAGE and quantification of the phosphorylation levels of NLRP3 in BMDMs pretreated with Psoralen (0.01, 0.1 and 1 µM) followed by stimulation with LPS/ATP (H) or LPS/Nigericin (J). (K) Immunofluorescence staining for NLRP3 (red) and ASC (green) in the LPS/ATP treated BMDMs. DAPI stains the nucleus (blue). The scale bar represents 50 μm. Enlarge vision: 10 μm. (L) The percentage of cells containing an ASC speck was quantified. 100 BMDMs per group were analyzed. Data were analyzed by one-way ANOVA, followed by Tukey post-tests. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001. ns: no significance