Digestive Diseases and Sciences

, Volume 63, Issue 12, pp 3398–3408 | Cite as

Silybin Alleviates Hepatic Steatosis and Fibrosis in NASH Mice by Inhibiting Oxidative Stress and Involvement with the Nf-κB Pathway

  • Qiang Ou
  • Yuanyuan Weng
  • Siwei Wang
  • Yajuan Zhao
  • Feng ZhangEmail author
  • Jianhua ZhouEmail author
  • Xiaolin WuEmail author
Original Article


Background and Aim

Silybin is the major biologically active compound of silymarin, the standardized extract of the milk thistle (Silybum marianum). Increasing numbers of studies have shown that silybin can improve nonalcoholic steatohepatitis (NASH) in animal models and patients; however, the mechanisms underlying silybin’s actions remain unclear.


Male C57BL/6 mice were fed a methionine-choline deficient (MCD) diet for 8 weeks to induce the NASH model, and silybin was orally administered to the NASH mice. The effects of silybin on lipid accumulation, hepatic fibrosis, oxidative stress, inflammation-related gene expression and nuclear factor kappa B (NF-κB) activities were evaluated by biochemical analysis, immunohistochemistry, immunofluorescence, quantitative real-time PCR and western blot.


Silybin treatment significantly alleviated hepatic steatosis, fibrosis and inflammation in MCD-induced NASH mice. Moreover, silybin inhibited HSC activation and hepatic apoptosis and prevented the formation of MDBs in the NASH liver. Additionally, silybin partly reversed the abnormal expression of lipid metabolism-related genes in NASH. Further study showed that the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway played important roles in the silybin-derived antioxidant effect, as evidenced by the upregulation of Nrf2 target genes in the silybin treatment group. In addition, silybin significantly downregulated the expression of inflammation-related genes and suppressed the activity of NF-κB signaling.


Silybin was effective in preventing the MCD-induced increases in hepatic steatosis, fibrosis and inflammation. The effect was related to alteration of lipid metabolism-related gene expression, activation of the Nrf2 pathway and inhibition of the NF-κB signaling pathway in the NASH liver.


Silybin Nonalcoholic steatohepatitis Oxidative stress NF-κB signaling pathway 



This work was supported by the National Natural Science Foundation of China (81670129 to Xiaolin Wu).

Compliance with ethical standards

Conflict of interest

There is no conflict of interest to disclose.


  1. 1.
    Harmon RC, Tiniakos DG, Argo CK. Inflammation in nonalcoholic steatohepatitis. Expert Rev Gastroenterol Hepatol. 2011;5:189–200.CrossRefGoogle Scholar
  2. 2.
    Bellentani S, Marino M. Epidemiology and natural history of non-alcoholic fatty liver disease (NAFLD). Ann Hepatol. 2009;8:S4–S8.PubMedGoogle Scholar
  3. 3.
    Fan JG, Farrell GC. Epidemiology of non-alcoholic fatty liver disease in China. J Hepatol. 2009;50:204–210.CrossRefGoogle Scholar
  4. 4.
    Wree A, Broderick L, Canbay A, Hoffman HM, Feldstein AE. From NAFLD to NASH to cirrhosis-new insights into disease mechanisms. Nat Rev Gastroenterol Hepatol. 2013;10:627–636.CrossRefGoogle Scholar
  5. 5.
    Day CP, James OF. Steatohepatitis: a tale of two “hits”? Gastroenterology. 1998;114:842–845.CrossRefGoogle Scholar
  6. 6.
    Takaki A, Kawai D, Yamamoto K. Multiple hits, including oxidative stress, as pathogenesis and treatment target in non-alcoholic steatohepatitis (NASH). Int J Mol Sci. 2013;14:20704–20728.CrossRefGoogle Scholar
  7. 7.
    Abenavoli L, Capasso R, Milic N, Capasso F. Milk thistle in liver diseases: past, present, future. Phytother Res. 2010;24:1423–1432.CrossRefGoogle Scholar
  8. 8.
    Aghazadeh S, Amini R, Yazdanparast R, Ghaffari SH. Anti-apoptotic and anti-inflammatory effects of Silybum marianum in treatment of experimental steatohepatitis. Exp Toxicol Pathol. 2011;63:569–574.CrossRefGoogle Scholar
  9. 9.
    Cacciapuoti F, Scognamiglio A, Palumbo R, Forte R, Cacciapuoti F. Silymarin in non alcoholic fatty liver disease. World J Hepatol. 2013;5:109–113.CrossRefGoogle Scholar
  10. 10.
    Marino Z, Crespo G, D’Amato M, et al. Intravenous silibinin monotherapy shows significant antiviral activity in HCV-infected patients in the peri-transplantation period. J Hepatol. 2013;58:415–420.CrossRefGoogle Scholar
  11. 11.
    Hajiaghamohammadi AA, Ziaee A, Oveisi S, Masroor H. Effects of metformin, pioglitazone, and silymarin treatment on Nonalcoholic Fatty liver disease: a randomized controlled pilot study. Hepat Mon. 2012;12:e6099.CrossRefGoogle Scholar
  12. 12.
    Yao J, Zhi M, Gao X, Hu P, Li C, Yang X. Effect and the probable mechanisms of silibinin in regulating insulin resistance in the liver of rats with Nonalcoholic fatty liver. Braz J Med Biol Res. 2013;46:270–277.CrossRefGoogle Scholar
  13. 13.
    Solhi H, Ghahremani R, Kazemifar AM, Hoseini Yazdi Z. Silymarin in treatment of Nonalcoholic steatohepatitis: A randomized clinical trial. Caspian J Intern Med. 2014;5:9–12.PubMedPubMedCentralGoogle Scholar
  14. 14.
    Wah Kheong C, Nik Mustapha NR, Mahadeva S. A randomized trial of silymarin for the treatment of nonalcoholic steatohepatitis. Clin Gastroenterol Hepatol. 2017;15:1940–1949. e1948.CrossRefGoogle Scholar
  15. 15.
    Carpino G, Morini S, Ginanni Corradini S, et al. Alpha-SMA expression in hepatic stellate cells and quantitative analysis of hepatic fibrosis in cirrhosis and in recurrent chronic hepatitis after liver transplantation. Dig Liver Dis. 2005;37:349–356.CrossRefGoogle Scholar
  16. 16.
    Eguchi A, De Mollerat Du, Jeu X, Johnson CD, Nektaria A, Feldstein AE. Liver Bid suppression for treatment of fibrosis associated with NonalcoholicNonalcoholic steatohepatitis. J Hepatol. 2016;64:699–707.CrossRefGoogle Scholar
  17. 17.
    Guicciardi ME, Gores GJ. Apoptosis as a mechanism for liver disease progression. Semin Liver Dis. 2010;30:402–410.CrossRefGoogle Scholar
  18. 18.
    Banner BF, Savas L, Zivny J, Tortorelli K, Bonkovsky HL. Ubiquitin as a marker of cell injury in nonalcoholic steatohepatitis. Am J Clin Pathol. 2000;114:860–866.CrossRefGoogle Scholar
  19. 19.
    Kayacetin S, Basaranoglu M. Mallory-Denk bodies: correlation with steatosis, severity, zonal distribution, and identification with ubiquitin. Turk J Gastroenterol. 2015;26:506–510.CrossRefGoogle Scholar
  20. 20.
    Abdelmegeed MA, Banerjee A, Yoo SH, Jang S, Gonzalez FJ, Song BJ. Critical role of cytochrome P450 2E1 (CYP2E1) in the development of high fat-induced Nonalcoholic steatohepatitis. J Hepatol. 2012;57:860–866.CrossRefGoogle Scholar
  21. 21.
    Teufel U, Peccerella T, Engelmann G, et al. Detection of carcinogenic etheno-DNA adducts in children and adolescents with Nonalcoholic steatohepatitis (NASH). Hepatobiliary Surg Nutr. 2015;4:426–435.PubMedPubMedCentralGoogle Scholar
  22. 22.
    Lee LY, Kohler UA, Zhang L, et al. Activation of the Nrf2-ARE pathway in hepatocytes protects against steatosis in nutritionally induced Nonalcoholic steatohepatitis in mice. Toxicol Sci. 2014;142:361–374.CrossRefGoogle Scholar
  23. 23.
    Marin V, Gazzin S, Gambaro SE, et al. Effects of oral administration of silymarin in a juvenile murine model of Nonalcoholic steatohepatitis. Nutrients 2017;9.CrossRefGoogle Scholar
  24. 24.
    Rinella ME, Elias MS, Smolak RR, Fu T, Borensztajn J, Green RM. Mechanisms of hepatic steatosis in mice fed a lipogenic methionine choline-deficient diet. J Lipid Res. 2008;49:1068–1076.CrossRefGoogle Scholar
  25. 25.
    Marcolin E, Forgiarini LF, Tieppo J, Dias AS, Freitas LA, Marroni NP. Methionine- and choline-deficient diet induces hepatic changes characteristic of Nonalcoholic steatohepatitis. Arq Gastroenterol. 2011;48:72–79.CrossRefGoogle Scholar
  26. 26.
    Phung N, Pera N, Farrell G, Leclercq I, Hou JY, George J. Pro-oxidant-mediated hepatic fibrosis and effects of antioxidant intervention in murine dietary steatohepatitis. Int J Mol Med. 2009;24:171–180.PubMedGoogle Scholar
  27. 27.
    Schattenberg JM, Galle PR. Animal models of Nonalcoholic steatohepatitis: of mice and man. Dig Dis. 2010;28:247–254.CrossRefGoogle Scholar
  28. 28.
    Higashi T, Friedman SL, Hoshida Y. Hepatic stellate cells as key target in liver fibrosis. Adv Drug Deliv Rev. 2017;121:27–42.CrossRefGoogle Scholar
  29. 29.
    Liu H, Gong M, French BA, Li J, Tillman B, French SW. Mallory-Denk Body (MDB) formation modulates Ufmylation expression epigenetically in alcoholic hepatitis (AH) and Nonalcoholic steatohepatitis (NASH). Exp Mol Pathol. 2014;97:477–483.CrossRefGoogle Scholar
  30. 30.
    Xiao P, Yang Z, Sun J, et al. Silymarin inhibits adipogenesis in the adipocytes in grass carp Ctenopharyngodon idellus in vitro and in vivo. Fish Physiol Biochem. 2017;43:1487–1500.CrossRefGoogle Scholar
  31. 31.
    Jha P, Knopf A, Koefeler H, et al. Role of adipose tissue in methionine-choline-deficient model of Nonalcoholic steatohepatitis (NASH). Biochim Biophys Acta. 2014;1842:959–970.CrossRefGoogle Scholar
  32. 32.
    Aubert J, Begriche K, Knockaert L, Robin MA, Fromenty B. Increased expression of cytochrome P450 2E1 in nonalcoholic fatty liver disease: mechanisms and pathophysiological role. Clin Res Hepatol Gastroenterol. 2011;35:630–637.CrossRefGoogle Scholar
  33. 33.
    Weltman MD, Farrell GC, Hall P, Ingelman-Sundberg M, Liddle C. Hepatic cytochrome P450 2E1 is increased in patients with nonalcoholic steatohepatitis. Hepatology. 1998;27:128–133.CrossRefGoogle Scholar
  34. 34.
    Maher J. The CYP2E1 knockout delivers another punch: first ASH, now NASH. Alcoholic steatohepatitis Nonalcoholic steatohepatitis. Hepatology. 2001;33:311–312.CrossRefGoogle Scholar
  35. 35.
    Das SK, Mukherjee S. Biochemical and immunological basis of silymarin effect, a milk thistle (Silybum marianum) against ethanol-induced oxidative damage. Toxicol Mech Methods. 2012;22:409–413.CrossRefGoogle Scholar
  36. 36.
    Zhang W, Hong R, Tian T. Silymarin’s protective effects and possible mechanisms on alcoholic fatty liver for rats. Biomol Ther (Seoul). 2013;21:264–269.CrossRefGoogle Scholar
  37. 37.
    Vecchione G, Grasselli E, Voci A, et al. Silybin counteracts lipid excess and oxidative stress in cultured steatotic hepatic cells. World J Gastroenterol. 2016;22:6016–6026.CrossRefGoogle Scholar
  38. 38.
    Zhang X, Shen J, Man K, et al. CXCL10 plays a key role as an inflammatory mediator and a non-invasive biomarker of Nonalcoholic steatohepatitis. J Hepatol. 2014;61:1365–1375.CrossRefGoogle Scholar
  39. 39.
    Marra F, Tacke F. Roles for chemokines in liver disease. Gastroenterology. 2014;147:577–594. e571.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.The Eighth People’s Hospital of ShanghaiShanghaiChina
  2. 2.Department of Clinical Laboratory, Core FacilityQuzhou People’s HospitalQuzhouChina
  3. 3.The Central Laboratory of the Eighth People’s Hospital of ShanghaiShanghaiChina

Personalised recommendations