Fig. 4

DS inhibited NLRP3 inflammasome activation and promoted mitophagy in microglia in vitro. BV-2 microglia were first pre-stimulated by LPS (100 ng/ml) for 3.5 h, then treated for 1h with indicated concentrations of DS followed by stimulation with nigericin (5 μM) for 30 min. a, b Immunoblot analyses of caspase-1 p10, IL-1β p17 in culture supernatants (SN) and NLRP3, pro-caspase-1, pro-IL-1β in lysates (LYS) were shown. c The level of IL-1β in supernatants was analyzed by ELISA method. After pre-stimulated with LPS, adding autophagy inhibitor 3-MA (5 mM) before DS and incubated for 0.5 h, and finally stimulated with nigericin for 12 h. d, e Immunoblot analyses of NLRP3, LC3II/I, Beclin 1 and p62 in LYS and c ELISA analysis of IL-1β in SN. Data are expressed as means ± SEM (n = 3 in each group). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc analysis where ^##P < 0.01, ^###P < 0.001 vs. control; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. LPS + Nigericin group; ^$P < 0.05, ^$$P < 0.01, vs. LPS + Nigericin + 30 μM DS group. e Mitochondria and lysosome were respectively labeled with the mito-tracker (green) and lyso-tracker (red), images were captured using Leica TCS SP8 confocal microscope