Low simvastatin concentrations reduce oleic acid-induced steatosis in HepG2 cells: An in vitro model of non-alcoholic fatty liver disease

Abstract

Abstract. Non-alcoholic fatty liver disease (NAFLD) is an inflammatory condition caused by hepatic lipid accumulation that is associated with insulin resistance, diabetes and metabolic syndrome. Although statins should be used with caution in liver diseases, they are increasingly investigated as a possible treatment for NAFLD. The present study recreated an in vitro model of NAFLD using HepG 2 cells exposed to oleic acid (OA), which was used to quantify OA-induced lipid accumulation in HepG 2 cells treated with various concentrations of simvastatin. In addition, the effect of simvastatin on HepG 2 cell morphology and microparticle generation as a marker of cell apoptosis was assessed. OA-induced lipid accumulation was quantified by Oil Red O staining and extraction for optical density determination. Stained lipid droplets were visualized using phase contrast microscopy. Furthermore, HepG 2 cell-derived microparticles were counted by flow cytometry subsequent to staining for Annexin V. HepG 2 cells treated with 0-1 mM OA showed dose-dependent lipid accumulation. Treatment of HepG 2 cells with increasing concentrations of simvastatin followed by treatment with 1 mM OA showed that low simvastatin concentrations (4-10 µM) were able to reduce lipid accumulation by ~40%, whereas high simvastatin concentrations (20 and 30 µM) induced apoptotic changes in cell morphology and increased the production of Annexin V + microparticles. This suggests that low simvastatin doses may have a role in preventing NAFLD. However, further investigations are required to confirm this action in vivo and to determine the underlying mechanism by which simvastatin reduces hepatic steatosis. IntroductionThe liver is a vital organ that is highly susceptible to fat accumulation, resulting in a condition known as fatty liver disease or hepatic steatosis (1). Although chronic alcohol consumption is a major cause of fatty liver disease (2), non-alcoholic fatty liver disease (NAFLD) is also common and is strongly associated with central obesity, insulin resistance, hyperlipidemia and the metabolic syndrome (3). Insulin resistance causes increased lipolysis and thus leading to high levels of plasma free fatty acids (FFAs), as well as increased FFA uptake by hepatocytes, which results in the formation of intracellular lipid droplets (4). Hepatic lipid accumulation can progress from simple steatosis to non-alcoholic steatohepatitis (NASH), which includes hepatocellular injury, inflammation and fibrosis (5). In addition, further severe complications may occur, such as liver cirrhosis and hepatocellular carcinoma (6).In order to prevent and treat NAFLD, lifestyle changes including weight reduction and increased physical activity are considered as the first-line approach (7). Although pharmacologic therapy is mainly directed toward increasing insulin sensitivity using insulin-sensitizing medications, such as metformin and pioglitazone (8), the use of other medications is increasingly investigated. Medications used to treat dyslipidemia, such as gemfibroz...

Protective Effects of Lactic Acid Bacteria Against TLR4 Induced Inflammatory Response in Hepatoma HepG2 Cells Through Modulation of Toll-Like Receptor Negative Regulators of Mitogen-Activated Protein Kinase and NF-κB Signaling

“…Therefore, a measurable in vitro steatosis model would increase our understanding of pathogenesis of liver disease and facilitate identification of novel therapeutic agents. Several studies have used oleic acid induced steatosis in HepG2 cells as an effective in vitro model to investigate the pathogenesis of fatty liver diseases ( 29 ). The present study showed that incubation of HepG2 cells with FFA induced steatosis, which was confirmed by Oil Red O staining and measurement of the optical density.…”

Effect of hepatic steatosis on the progression of chronic hepatitis B: A prospective cohort and in vitro study

“…In order to address the shortcomings, in vitro cell culture model was used in research of NAFLD with CHB. Recently, OA-induced lipid accumulation in HepG 2 cells may act as an in vitro model for studying NAFLD [ 23 ]. In this study, the HCC cells steatosis model was successfully established using OA.…”

Protective role of magnesium isoglycyrrhizinate in non-alcoholic fatty liver disease and the associated molecular mechanisms

“…A previous study indicated that OA was capable of decreasing cell viability and increasing cell apoptosis markedly (22). It has also been demonstrated that OA may induce the accumulation of lipid droplets in lipid-overloaded L02 cells (23).…”