Fig. 4.

Effect of SEA on the key metabolic signaling molecular in macrophage in vitro. a Immunoblots of protein extracts from RAW264.7 cells that were treated with SEA (20 μg/ml) for 24 h. Representative immunoblots of three independent experiments are shown, and then protein was subjected to SDS/PAGE, immunoblotting with antibodies to AMPK, p-AMPK, ACC, p-ACC, AKT, p-AKT, mTORC1, p-mTORC1, PI3K and p-PI3K, and quantification. Band density was measured by ImageJ software and normalized to β-actin (Student’s t-test: *P < 0.05, **P < 0.01, ***P < 0.001). RAW264.7 cells were treated with SEA (20 μg/ml) with or without phosphorylated AMPK inhibitor (dorsomorphin), phosphorylated AKT inhibitor (dihydrochloride) and phosphorylated mTOR inhibitor (rapamycin) for 24 h. After that, cell culture medium was collected for measurement of glucose concentrations (b), and intracellular lipid accumulation of cells was determined by oil red O staining (c) (lipid droplets vs the cell section area). Ten random fields of each group were chosen for the quantification of intracellular lipids by oil red O staining by using the ImageJ software (ANOVA/LSD: *P < 0.05, **P < 0.01, ***P < 0.001)