INFLUENCE OF FATTY ACIDS ON LIPID ACCUMULATION AND APOPTOSIS STATUS OF GRASS CARP CTENOPHARYNGODON IDELLUS HEPATOCYTE IN VITRO

To investigate the effects of fatty acids on hepatocyte lipid metabolism and health status of grass carp (Ctenopharyngodon idellus), the normal grass carp hepatocytes were cultured with different concentrations of oleic acid (0-1 mmol/L) for 24h to measure cell viability and lipids content by methylthiazolyl tetrazolium (MTT) assay and oil red O staining, respectively. The lipid droplets and nuclear of hepatocytes were observed with BODIPY and DAPI staining. Hepatocyte apoptosis was analyzed by flow cytometry. Real-time qPCR was performed to detect the mRNA expression of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein alpha (C/EBPα) and apoptosis related genesCaspase3a, Caspase-3b, Caspase-8, Caspase-9, apoptosis inducing factor (AIF), Bcl-2, Bax. The results showed that the cell viability and intracellular lipid accumulation of hepatocytes increased significantly after treatment with 0.6 and 0.4 mmol/L oleic acid (P < 0.05). The lowest hepatocytes apoptosis rate was in 0.4 mmol/L group and the highest was in 1 mmol/L oleic acid group (P < 0.05). The mRNA levels of Caspase-3b and Caspase-9 reduced significantly and the mRNA ration of Bcl-2/Bax was elevated by 0.4 mmol/L oleic acid (P < 0.05). Consistently, 0.4 mmol/L oleic acid significantly reduced caspase-3b, caspase-9 and AIF mRNA levels and increased the mRNA ratio of Bcl-2/Bax (P < 0.05) and these genes showed opposite expression pattern in hepatocytes treated with 0.8 mmol/L oleic acid (P < 0.05). These results demonstrated dosage-dependent role for fatty acids in grass carp hepatocytes viability and intracellular lipid accumulation via the regulation of the genes related to lipid metabolism and apoptosis.