Basic Study
Copyright ©The Author(s) 2016. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jul 14, 2016; 22(26): 6016-6026
Published online Jul 14, 2016. doi: 10.3748/wjg.v22.i26.6016
Silybin counteracts lipid excess and oxidative stress in cultured steatotic hepatic cells
Giulia Vecchione, Elena Grasselli, Adriana Voci, Francesca Baldini, Ignazio Grattagliano, David QH Wang, Piero Portincasa, Laura Vergani
Giulia Vecchione, Elena Grasselli, Adriana Voci, Francesca Baldini, Laura Vergani, DISTAV, Department of Earth, Environment and Life Sciences, University of Genova, 16132 Genova, Italy
Ignazio Grattagliano, Piero Portincasa, Department of Biomedical Sciences and Human Oncology, University School of Medicine, 70124 Bari, Italy
David QH Wang, Department of Internal Medicine, Division of Gastroenterology and Hepatology, Saint Louis, University School of Medicine, St. Louis, MO 63104, United States
Author contributions: Vergani L and Portincasa P designed research, analyzed data and wrote the paper; Vecchione G performed cell cultures and treatments, spectrophotometric and fluorimetric assays and drafted the manuscript; Baldini F performed cell staining and microscopy analyses; Voci A participated in cell cultures and treatments; Grasselli E performed qPCR experiments; Grattagliano I reviewed the data and revised the manuscript; Wang DQH participated in the study design and revised the manuscript; all authors read and approved the final manuscript.
Supported by MIUR-COFIN (Prot. 20089SRS2X_002), Compagnia San Paolo Torino, University of Genova; and Fondazione CARIGE.
Conflict-of-interest statement: None for all authors.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Dr. Laura Vergani, DISTAV, Department of Earth, Environment and Life Sciences, University of Genova, Corso Europa 26, 16132 Genova, Italy. laura.vergani@unige.it
Telephone: +39-10-03538403 Fax: +39-10-03538267
Received: March 30, 2016
Peer-review started: April 5, 2016
First decision: May 12, 2016
Revised: May 19, 2016
Accepted: June 13, 2016
Article in press: June 13, 2016
Published online: July 14, 2016
Abstract

AIM: To investigate in vitro the therapeutic effect and mechanisms of silybin in a cellular model of hepatic steatosis.

METHODS: Rat hepatoma FaO cells were loaded with lipids by exposure to 0.75 mmol/L oleate/palmitate for 3 h to mimic liver steatosis. Then, the steatotic cells were incubated for 24 h with different concentrations (25 to 100 μmol/L) of silybin as phytosome complex with vitamin E. The effects of silybin on lipid accumulation and metabolism, and on indices of oxidative stress were evaluated by absorption and fluorescence microscopy, quantitative real-time PCR, Western blot, spectrophotometric and fluorimetric assays.

RESULTS: Lipid-loading resulted in intracellular triglyceride (TG) accumulation inside lipid droplets, whose number and size increased. TG accumulation was mediated by increased levels of peroxisome proliferator-activated receptors (PPARs) and sterol regulatory element-binding protein-1c (SREBP-1c). The lipid imbalance was associated with higher production of reactive oxygen species (ROS) resulting in increased lipid peroxidation, stimulation of catalase activity and activation of nuclear factor kappa-B (NF-κB). Incubation of steatotic cells with silybin 50 μmol/L significantly reduced TG accumulation likely by promoting lipid catabolism and by inhibiting lipogenic pathways, as suggested by the changes in carnitine palmitoyltransferase 1 (CPT-1), PPAR and SREBP-1c levels. The reduction in fat accumulation exerted by silybin in the steatotic cells was associated with the improvement of the oxidative imbalance caused by lipid excess as demonstrated by the reduction in ROS content, lipid peroxidation, catalase activity and NF-κB activation.

CONCLUSION: We demonstrated the direct anti-steatotic and anti-oxidant effects of silybin in steatotic cells, thus elucidating at a cellular level the encouraging results demonstrated in clinical and animal studies.

Keywords: Non-alcoholic fatty liver disease, Steatotic hepatocytes, Silybin, Lipid metabolism, Oxidative stress, Lipid droplets, Mitochondrial β-oxidation

Core tip: FaO hepatic cells loaded with lipids by exposure to oleate/palmitate mixture represent a widely accepted cellular model of hepatic steatosis. FaO steatotic cells were used to investigate in vitro the possible direct effects of silybin as phytosome complex with vitamin E on lipid accumulation and metabolism and on oxidative stress. We demonstrated the ability of silybin in reducing fat accumulation and improving the oxidative imbalance caused by lipid excess. The results may elucidate at a cellular level the encouraging results demonstrated for silybin in previous clinical and animal studies.