Fig. 7

JNK-ERK AMPK pathway was involved in the protective effect of pinocembrin which activated BNIP3-mediated mitophagy. BV2 cells were pretreated with SP600125 (JNK inhibitor, 10 μM) or FR 108204 (ERK inhibitor, 10 μM) for 30 min before the pinocembrin administration. Pinocembrin was added to the culture medium for 1 h before exposing to NA (normal air) or IH (intermittent hypoxia). a Total proteins from the microglia were immediately separated by SDS-PAGE gel and analyzed by western blot. Blots were probed with specific primary antibodies as indicated. b Quantitation of pinocembrin-activated ERK1/2 and JNK MAPK pathways. Depicted is the weighted average density of bands (p-JNK and p-ERK) normalized to JNK or ERK at 24-h exposure with NA or IH, respectively. c Proliferation of BV2 cells in different groups was analyzed by CCK-8 assay. d Flow cytometric analysis of microglia for mtROS with MitoSOX. e Western analysis showed pinocembrin induced Bcl-2 expression under IH exposure, but reduced by JNK or ERK inhibitor. GAPDH was shown as a loading control. f Representative images of NLRP3 (red) and ASC (green) by immunofluorescent staining of BV2 cells. Data are representative of at least 3 independent experiments and presented as mean ± SEM. ^†p < 0.05 vs. the NA group; ^#p < 0.05 vs. the IH group; *p < 0.05 vs. the IH + PIN group