Fig. 4

Pinocembrin shows protective effects on IH-treated microglial cells and enhances BNIP3-mediated mitophagy. a Microglial cells were incubated with pinocembrin at different concentrations (0, 1, 3, 10, and 20 μM) under intermittent hypoxia (IH) condition for 24 h. Then, the viability of cells was measured by Cell Counting Kit-8 assay kit. b Pinocembrin exposure significantly reduced IH-induced cell apoptosis, and protein expressions of caspase-3, Bcl-2, and Bax were detected by immunoblotting. Pinocembrin at the concentration of 10 μM for BV2 cells with IH exposure showed optimal results in all. GAPDH worked as the loading control. c Western blot analysis of NLRP3, caspase-1, and ASC expression in microglial cells treated with different concentrations of pinocembrin (0, 1, 3, and 10 μM). d The relative optical density values of mentioned proteins to GAPDH were analyzed and demonstrated as statistical graphs, respectively. e Western blot was used for autophagic and mitophagic activation determination in groups with different concentrations of pinocembrin (0, 1, 3, and 10 μM). GAPDH worked as the internal control for whole cellular protein extraction. f The densitometric analysis of ATG7, P62, Beclin-1, ATG5, BNIP3/Nix, BNIP3, and LC3 expression. Pinocembrin exposure further increased autophagic and mitophagic protein expression. g Electron micrographs showed mitochondrial damages and mitophagosomes (red arrows) in microglial cells. Data represented as mean ± SEM from 3 independent experiments. ^#p < 0.05 vs. the Ctrl group; ^##p < 0.01 vs. the Ctrl group; *p < 0.05 vs. the IH group; **p < 0.01 vs. the IH group; ^†p < 0.05 vs. the IH + 10 μM group