Isosilybin regulates lipogenesis and fatty acid oxidation via the AMPK/SREBP-1c/PPARα pathway

It is well known that the excessive accumulation of lipid in hepatocytes is one of the important causes of non-alcoholic fatty liver disease (NAFLD). The purpose of this study was to explore the effects of isosilybin on lipid metabolism in free fatty acids (FFAs) or TO901317-induced HepG2 cells. Cells were treated with FFAs (oleic acid: palmitic acid, 2:1) or TO901317 to induce steatosis in vitro. Intracellular triglyceride (TG) content was quantified using commercial assay kits. The mRNA and protein expression of genes involved in fatty acid uptake, synthesis and oxidation were analyzed by RT-qPCR and western blotting. Selected biological pathways regulated by isosilybin treatment were determined by GO and KEGG analysis. The results showed that isosilybin significantly reduced TG levels in FFAs- and TO901317-induced HepG2 cells. Further studies showed that isosilybin treatment decreased the mRNA and protein expression of lipid synthesis genes Srebp-1c, Pnpla3, Acc and Fas, as well as the mRNA expression of fatty acid uptake gene CD36, whereas increased the mRNA levels of lipid oxidation genes Pparα, Acox1 and Cpt1α, as well as the mRNA expression of lipid export gene Mttp, in FFAs-induced HepG2 cells. Moreover, TO901317 was employed to induce endogenous lipid synthesis and steatosis, and the expression of Srebp-1c and its target genes in TO901317-induced hepatocytes was basically similar to that in FFAs-induced hepatocytes following isosilybin treatment. We also observed the increased level of phosphorylated AMP kinase (AMPK) after isosilybin treatment, while this effect was reversed after further treatment with AMPK inhibitor, compound C. The results of GO and KEGG analysis indicated that the pathways of fatty acid and TG metabolism were regulated by isosilybin. Interestingly, we found that treatment with the diastereoisomer A of isosilybin increased TG level, while exposure to the diastereoisomer B of isosilybin decreased TG level in FFAs-induced HepG2 cells. The above results suggest that isosilybin can inhibit lipid synthesis and activate lipid oxidation through AMPK signaling pathway, thereby improving steatosis of hepatocytes, and isosilybin B is the basis of its active substance.