Fig. 3

RFAs inhibited cholesterol induced NLRP3 inflammasome. Mouse BMDMs and Kuffer cells were pretreated with YCHT (5 μl/ml), artemisia capillaris (5 μl/ml), gardenia (5 μl/ml) and rhubarb (5 μl/ml) as well as RFAs (rhein/diacerein, emodin, aloe emodin, 1,8-dihydroxyanthraquinone, all 40 μM) for 30 min and then stimulated with LPS (100 ng/ml) for 4 h and cholesterol (100 μg/ml) for 12 h. In mouse BMDMs, IL-1β and TNF-α in cell culture supernatant were detected by ELISA (A, B) (n = 3/group), and the cleavage of procaspase-1influced by RFAs was detected by western blot (C). In mouse Kuffer cells, IL-1β and TNF-α in cell culture supernatant were detected by ELISA (D, E) (n = 3/group), and the ASC specks influenced by RFAs were detected by immunofluorescence (F, G). Mouse primary hepatocytes were pretreated with YCHT (5 μl/ml), artemisia capillaris (5 μl/ml), gardenia (5 μl/ml) and rhubarb (5 μl/ml) as well as RFAs (rhein/diacerein, emodin, aloe emodin, 1,8-dihydroxyanthraquinone, all 40 μM) for 30 min and then stimulated with LPS (1 μg/ml) for 4 h and cholesterol (100 μg/ml) for 12 h. IL-1β and TNF-α in cell culture supernatant were detected by ELISA and LDH were detected by LDH kits (H, I), ALT and AST were detected by ALT/AST kits (K, L), and the cleavage of procaspase-1 influenced by RFAs was detected by western blot (J). 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; ALT, alanine aminotransferase; ASC, apoptosis-associated speck-like protein containing CARD; AST, aspartate aminotransferase; BMDMs, bone marrow-derived macrophages; DAPI, 4′,6-diamidino-2-phenylindole; ELISA, enzyme linked immunosorbent assay; IL-1β, interleukin-1 beta; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-alpha