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. 2019 Apr 2;24(7):1280.
doi: 10.3390/molecules24071280.

Silybin Modulates Collagen Turnover in an In Vitro Model of NASH

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Free PMC article

Silybin Modulates Collagen Turnover in an In Vitro Model of NASH

Beatrice Anfuso et al. Molecules. .
Free PMC article

Abstract

Silybin has been proposed as a treatment for nonalcoholic steatohepatitis (NASH). In this study, we assessed the effect of Silybin in a well-established in vitro coculture model of early-stage NASH. LX2 and Huh7 cells were exposed to free fatty acid (FFA) and Silybin as mono- or coculture (SCC). Cell viability, LX2 activation, collagen deposition, metalloproteinase 2 and 9 (MMP2-9) activity, and ROS generation were determined at 24, 96, and 144 h. Exposure to FFA induced the activation of LX2 as shown by the increase in cell viability and upregulation of collagen biosynthesis. Interestingly, while cotreatment with Silybin did not affect collagen production in LX2, a significant reduction was observed in SCC. MMP2-9 activity was reduced in FFA-treated Huh7 and SCC and cotreatment with Silybin induced a dose-dependent increase, while no effect was observed in LX2. Silybin also showed antioxidant properties by reducing the FFA-induced production of ROS in all the cell systems. Based on these data, Silybin exerts its beneficial effects by reducing LX2 proliferation and ROS generation. Moreover, MMP2-9 modulation in hepatocytes represents the driving mechanism for the net reduction of collagen in this NASH in vitro model, highlighting the importance of hepatic cells interplay in NASH development and resolution.

Keywords: NAFLD; NASH; Silybin; coculture model; fibrogenesis; hepatic stellate cells.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Cell viability and proliferation after Silybin exposure. Cytotoxic effects of Silybin were evaluated in the presence or absence of FFA in Huh7 (A), LX2 (B), and SCC (C) after 24, 96, and 144 h of treatment. # p < 0.05, ## p < 0.01 vs. CTRL; * p < 0.05, ** p < 0.01 vs. FFA.
Figure 2
Figure 2
Effect of Silybin on HSC (LX2) activation and collagen production. ACTA2 (α-SMA) gene expression upon FFA and Silybin exposure in SCC (A) and LX2 (B) vs. control. COL1A1 gene expression upon FFA and Silybin exposure in SCC (C) and LX2 (D) vs. control. Quantification of the total extracellular collagen upon FFA and Silybin exposure vs. control in SCC (E) and LX2 (F). # p < 0.05, ## p < 0.01, ### p < 0.001 vs. CTRL; * p < 0.05, ** p < 0.01 vs. FFA.
Figure 3
Figure 3
MMP2-9 activity. Enzymatic activity of active MMP2-9 was analyzed in the supernatant of Huh7 (A), LX2 (B), and SCC (C) treated with free fatty acids (FFAs) and Silybin for 96 and 144 h. Activity was normalized to the total proteins in the cell lysate and reported as percentage vs. CTRL sample. # p < 0.05, ## p < 0.01 vs. CTRL; * p < 0.05 vs. FFA.
Figure 4
Figure 4
ROS generation. Intracellular ROS was quantified for each condition after 1 h of exposure with Silybin alone (A) or in combination with FFA (B). Activity was normalized to the total proteins in the cell lysate and reported as a percentage vs. CTRL sample. H2O2 treatment was used as a positive CTRL. # p < 0.05, ## p < 0.01 vs. CTRL; * p < 0.05, ** p < 0.01 vs. FFA.
Figure 5
Figure 5
Scheme of the culture proceedings and the experimental checkpoints with the relative determinations.

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References

    1. NCD Risk Factor Collaboration (NCD-RisC) Trends in adult body-mass index in 200 countries from 1975 to 2014: a pooled analysis of 1698 population-based measurement studies with 19·2 million participants. Lancet Lond. Engl. 2016;387:1377–1396. doi: 10.1016/S0140-6736(16)30054-X. - DOI - PubMed
    1. Younossi Z., Anstee Q.M., Marietti M., Hardy T., Henry L., Eslam M., George J., Bugianesi E. Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention. Nat. Rev. Gastroenterol. Hepatol. 2018;15:11. doi: 10.1038/nrgastro.2017.109. - DOI - PubMed
    1. Araújo A.R., Rosso N., Bedogni G., Tiribelli C., Bellentani S. Global epidemiology of non-alcoholic fatty liver disease/non-alcoholic steatohepatitis: What we need in the future. Liver Int. Off. J. Int. Assoc. Study Liver. 2018;38:47–51. doi: 10.1111/liv.13643. - DOI - PubMed
    1. Buzzetti E., Pinzani M., Tsochatzis E.A. The multiple-hit pathogenesis of non-alcoholic fatty liver disease (NAFLD) Metabolism. 2016;65:1038–1048. doi: 10.1016/j.metabol.2015.12.012. - DOI - PubMed
    1. Angulo P., Kleiner D.E., Dam-Larsen S., Adams L.A., Bjornsson E.S., Charatcharoenwitthaya P., Mills P.R., Keach J.C., Lafferty H.D., Stahler A., et al. Liver Fibrosis, but No Other Histologic Features, Is Associated With Long-term Outcomes of Patients With Nonalcoholic Fatty Liver Disease. Gastroenterology. 2015;149:389–397. doi: 10.1053/j.gastro.2015.04.043. - DOI - PMC - PubMed
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