Figure 4.

Cori ameliorated nonalcoholic fatty liver disease (NAFLD) by restoring autophagic flux in vitro. (A) AML12 cells were treated with 200 μM PA/OA in the absence or presence of Cori (10 or 20 μM) for 24 h. The relative expression levels of lipogenic genes (SREBP-1c, ACC1, and FASN), gene involved in β-oxidation of fatty acids (CPT1α, PPARα, ACOX1), and autophagy-related protein (Atg7, Atg5) were determined by RT-PCR assay. (B) Cells were exposed to 200 μM PA/OA and in the presence or absence of Cori (10 or 20 μM) for 24 h. 3-MA (5 mM) were add to serve as autophagic inhibitor. Western blot and semiquantitative analysis of autophagy related protein LC3A/B I, LC3A/B II, p62 in hepatocytes of each group. GAPDH was detected as a loading control. (C) Immunofluorescence stained with antibodies against LC3 (green) and the mitochondrial marker MitoTracker (red) in AML12 cells. (D) Quantitative analysis of yellow/orange positive fields represented LC3/MitoTracker double labeled mitochondria from (C). Images of cells were visualized by fluorescent microscope. (E) Cells were mixed with Ad-mCherryGFP-LC3B and then treated with Cori for 24 h, the mCherry (red) and GFP (green) were analyzed by fluorescence microscopy (Scale bar = 20 μm). Data were presented as mean ± SD of three independent experiments; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. NCD group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. high-fat diet (HFD) group. ^&p < 0.05 vs. HFD group.