The Nutraceutic Silybin Counteracts Excess Lipid Accumulation and Ongoing Oxidative Stress in an In Vitro Model of Non-Alcoholic Fatty Liver Disease Progression.

Giulia Vecchione,Francesca Baldini,Vilma A Sardão,Elena Grasselli,Laura Vergani,Antonia Lanni,Federica Cioffi,Paulo J Oliveira,Katia Cortese,Adriana Voci,Piero Portincasa

doi:10.3389/fnut.2017.00042

Abstract

Non-alcoholic fatty liver disease (NAFLD) is a major cause of liver-related morbidity and mortality. Oxidative stress and release of pro-inflammatory cytokines, such as tumor necrosis factor α (TNFα), are major consequences of hepatic lipid overload, which can contribute to progression of NAFLD to non-alcoholic steatohepatitis (NASH). Also, mitochondria are involved in the NAFLD pathogenesis for their role in hepatic lipid metabolism. Definitive treatments for NAFLD/NASH are lacking so far. Silybin, the extract of the milk thistle seeds, has previously shown beneficial effects in NAFLD. Sequential exposure of hepatocytes to high concentrations of fatty acids (FAs) and TNFα resulted in fat overload and oxidative stress, which mimic in vitro the progression of NAFLD from simple steatosis (SS) to steatohepatitis (SH). The exposure to 50 µM silybin for 24 h reduced fat accumulation in the model of NAFLD progression. The in vitro progression of NAFLD from SS to SH resulted in reduced hepatocyte viability, increased apoptosis and oxidative stress, reduction in lipid droplet size, and up-regulation of IκB kinase β-interacting protein and adipose triglyceride lipase expressions. The direct action of silybin on SS or SH cells and the underlying mechanisms were assessed. Beneficial action of silybin was sustained by changes in expression/activity of peroxisome proliferator-activated receptors and enzymes for FA oxidation. Moreover, silybin counteracted the FA-induced mitochondrial damage by acting on complementary pathways: (i) increased the mitochondrial size and improved the mitochondrial cristae organization; (ii) stimulated mitochondrial FA oxidation; (iii) reduced basal and maximal respiration and ATP production in SH cells; (iv) stimulated ATP production in SS cells; and (v) rescued the FA-induced apoptotic signals and oxidative stress in SH cells. We provide new insights about the direct protective effects of the nutraceutic silybin on hepatocytes mimicking in vitro NAFLD progression.

Highlights

Hepatic steatosis is defined as the accumulation of triglycerides (TGs) exceeding 5% of liver weight
Chronic caloric overload initiates an inflammatory response originating from the adipose tissue with production of cytokines, such as tumor necrosis factor α (TNFα), that impairs lipid metabolism in remote tissues such as the liver promoting the progression of SS to SH [49]
Non-alcoholic fatty liver disease (NAFLD) progression was ascertained by assessing: (i) cell viability, which was not altered in SS cells but was significantly reduced in SH cells; (ii) IkBip expression, a classical liver damage marker, which increased in SS cells and further increased in SH cells; and (iii) caspase activity, a marker of apoptosis, which was stimulated in SS cells and further stimulated in SH cells

Summary

Introduction

Hepatic steatosis is defined as the accumulation of triglycerides (TGs) exceeding 5% of liver weight. The simple steatosis (SS) is named non-alcoholic fatty liver, which may progress to more severe conditions, such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) with hepatocyte injury and lobular and portal inflammation, and non-alcoholic liver cirrhosis with massive fibrosis and vascular remodeling [1], up to hepatocellular carcinoma [2, 3]. Following the epidemics of obesity, NAFLD has become the leading cause of liver disease in developed countries. FAs and cholesterol, especially when accumulated in the mitochondria, are “aggressive” lipids leading to tumor necrosis factor α (TNFα) and reactive oxygen species (ROS) production and acting as early “inflammatory” hits [6], which contribute to promote NASH [7]

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