Effects of daphnetin on lipid metabolism, insulin resistance and oxidative stress in OA‑treated HepG2 cells.

Abstract

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease, and has high rates of morbidity and mortality worldwide. Daphnetin (DAP) possesses notable antioxidative, anti-inflammatory and anticoagulant activities; DAP is an active ingredient extracted from Daphne Koreana Nakai. To investigate the effects and the underlying mechanism of DAP on NAFLD, we treated HepG2 cells with oleic acid (OA) and DAP simultaneously and non-simultaneously. In the simultaneous treatment condition, HepG2 cells were co-treated with 0.5 mM OA and DAP (5, 20, and 50 µM) for 24 h. In the non-simultaneous treatment conditions, HepG2 cells were pretreated with 0.5 mM OA for 24 h, and then treated with DAP (5, 20 and 50 µM) for 24 h. Following the aforementioned treatments, the biochemical indexes associated with NAFLD were measured as follows: i) The intracellular contents of triglyceride (TG), reactive oxygen species (ROS) and fluorescent glucose 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxyglucose were analyzed with corresponding detection kits; and ii) the cellular expression levels of glycolipid metabolism- and oxidative stress-related genes, including 5′AMP-activated protein kinase (AMPK), sterol regulatory element-binding protein-1C (SREBP-1C), patatin-like phospholipase domain-containing protein 3 (PNPLA3), peroxisome proliferator-activated receptor α (PPARα), phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), nuclear factor-like 2 (Nrf2), cytochrome P450 (CYP) 2E1 and CYP4A were determined by reverse transcription-quantitative polymerase chain reaction and western blotting. The results revealed the potential mechanism underlying the effects of DAP on NAFLD in vitro: i) By increasing the phosphorylation of AMPK, DAP inhibited the expression of SREBP-1C and PNPLA3, and induced that of PPARα. Lipid accumulation within hepatocytes was reduced; ii) by upregulating PI3K expression and pAKT/AKT levels, DAP may alleviate insulin resistance and promote hepatocellular glucose uptake; and iii) by upregulating the expression of Nrf2, DAP downregulated the expression of CYP2E1 and CYP4A, and the levels of reactive oxygen species in hepatocytes.