Fig. 2

RFAs inhibited both the transcription and assembly of NLRP3 inflammasome. Mouse BMDMs were pretreated by rhein, diacerein, emodin, aloe emodin and 1,8-dihydroxyanthraquinone with the concentrations of 40 μM, 20 μM, 10 μM, and 5 μM for 30 min and then stimulated with LPS (100 ng/ml) for 4 h. NLRP3, ASC and pro-caspase-1 (A) were detected by western blot. Moue BMDMs were pretreated with rhein, diacerein, emodin, aloe emodin, 1,8-dihydroxyanthraquinone (all 40 μM) for 30 min and then stimulated with LPS (100 ng/ml) for 1 h. NLRP3 mRNA (B) was detected by RT-qPCR (n = 3/group). Mouse BMDMs were pretreated by rhein, diacerein, emodin, aloe emodin and 1,8-dihydroxyanthraquinone (all 40 μM) for 30 min and then stimulated with LPS (100 ng/ml) for 0 min, 20 min and 40 min. The phosphorylation of P65, P38, JNK and ERK (C) were detected by western blot. Mouse BMDMs were stimulated by LPS (100 ng/ml) for 4 h and then changed the media. Rhein, diacerein, emodin, aloe emodin and 1,8-dihydroxyanthraquinone with the concentrations of 40 μM, 20 μM, 10 μM, and 5 μM were added for 30 min and then the BMDMs were stimulated with nigericin (2.5 μM) for 2 h. The protein expression of NLRP3, ASC and pro-caspase-1 as well as the cleavage of pro-caspase-1 (D) were detected by western blot. Mouse BMDMs were stimulated by LPS (100 ng/ml) for 4 h before the media was changed. Rhein, diacerein, emodin, aloe emodin and 1,8-dihydroxyanthraquinone (all 40 μM) were added for 30 min and then stimulated with nigericin (2.5 μM) for 2 h. IL-1β (E) in cell culture supernatant was detected by ELISA (n = 3/group); The ASC specks (F, G) was detected by immunofluorescence; the combination of NLRP3 and ASC (H) was detected by immunoprecipitation. Data are presented as mean ± SEM. For multiple comparisons, one-way ANOVA coupled with LSD’s post hoc testing was performed. &: p < 0.05; &&: p < 0.01; &&&: p < 0.001. ANOVA, analysis of variance; ASC, apoptosis-associated speck-like protein containing CARD; BMDMs, bone marrow-derived macrophages; DAPI, 4',6-diamidino-2-phenylindole; ELISA, enzyme linked immunosorbent assay; IL-1β, interleukin-1 beta; LPS, lipopolysaccharide; RT-qPCR, quantitative reverse transcription polymerase chain reaction; TNF-α, tumor necrosis factor-alpha