Journal of Gastroenterology

, Volume 47, Issue 8, pp 924–935 | Cite as

Nrf2 inhibits hepatic iron accumulation and counteracts oxidative stress-induced liver injury in nutritional steatohepatitis

  • Kosuke Okada
  • Eiji Warabi
  • Hirokazu Sugimoto
  • Masaki Horie
  • Katsutoshi Tokushige
  • Tetsuya Ueda
  • Nobuhiko Harada
  • Keiko Taguchi
  • Etsuko Hashimoto
  • Ken Itoh
  • Tetsuro Ishii
  • Hirotoshi Utsunomiya
  • Masayuki Yamamoto
  • Junichi ShodaEmail author
Original Article—Liver, Pancreas, and Biliary Tract



The transcription factor nuclear factor-E2-related factor-2 (Nrf2) is a key regulator for induction of hepatic antioxidative stress systems. We aimed to investigate whether activation of Nrf2 protects against steatohepatitis.


Wild-type mice (WT), Nrf2 gene-null mice (Nrf2-null) and Keap1 gene-knockdown mice (Keap1-kd), which represent the sustained activation of Nrf2, were fed a methionine- and choline-deficient diet (MCDD) for 13 weeks and analyzed.


In Keap1-kd fed an MCDD, steatohepatitis did not develop over the observation periods; however, in Nrf2-null fed an MCDD, the pathological state of the steatohepatitis was aggravated in terms of fatty change, inflammation, fibrosis and iron accumulation. In WT mice fed an MCDD, Nrf2 and antioxidative stress genes regulated by Nrf2 were potently activated in the livers, and in Keap1-kd, their basal levels were potently activated. Oxidative stress was significantly increased in the livers of the Nrf2-null and suppressed in the livers of the Keap1-kd compared to that of WT, based on the levels of 4-hydroxy-2-nonenal and malondialdehyde. Iron accumulation was greater in the livers of the Nrf2-null mice compared to those of the WT mice, and it was not observed in Keap1-kd. Further, the iron release from the isolated hepatocyte of Nrf2-null mice was significantly decreased. Sulforaphane, an activator of Nrf2, suppressed the pathological states and oxidative stress in the livers.


Nrf2 has protective roles against nutritional steatohepatitis through inhibition of hepatic iron accumulation and counteraction against oxidative stress-induced liver injury. Nrf2 activation by pharmaceutical intervention could be a new option for the prevention and treatment of steatohepatitis.


Nrf2 gene-knockout mouse Keap1 gene-knockdown mouse Methionine- and choline-deficient diet Iron metabolism 



Alpha-smooth muscle actin


Alkaline phosphatase


Alanine aminotransferase


Aspartate aminotransferase




γ-Glutamylcysteine synthetase




Glutathione S-transferase


Hepcidin gene




Kelch-like Ech-associated protein 1


Methionine- and choline-deficient diet




Non-alcoholic steatohepatitis


Nuclear factor-E2-related factor-2


NAD(P)H: quinone oxidoreductase 1


Reactive oxygen species




Transferrin receptor


Transforming growth factor


Wild type



This work was supported in part by a Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (19791054) and Grants-in-Aid from Nakayama Cancer Research Institute (Tokyo, Japan).

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Torres DM, Harrison SA. Diagnosis and therapy of nonalcoholic steatohepatitis. Gastroenterology. 2008;134:1682–98.PubMedCrossRefGoogle Scholar
  2. 2.
    Vuppalanchi R, Naga C. Nonalcoholic fatty liver disease and nonalcoholic steatohepatitis: selected practical issues in their evaluation and management. Hepatology. 2009;49:306–17.PubMedCrossRefGoogle Scholar
  3. 3.
    Falch-Ytter Y, Younossi ZM, Marchesini G, McCullough AJ. Clinical features and natural history of nonalcoholic steatosis syndromes. Semin Liver Dis. 2001;21:17–26.CrossRefGoogle Scholar
  4. 4.
    Adams LA, Lymp JF, Sauver JS, Sanderson SO, Lindor KD, Feldstein A, et al. The natural history of nonalcoholic fatty liver disease: a population based cohort study. Gastroenterology. 2005;129:113–21.PubMedCrossRefGoogle Scholar
  5. 5.
    Malaguarnera L, Madeddu R, Palio E, Arena N, Malaguarnera M. Heme oxygenase-1 levels and oxidative stress-related parameters in non-alcoholic fatty liver disease patients. J Hepatol. 2005;42:585–91.PubMedCrossRefGoogle Scholar
  6. 6.
    Sumida Y, Nakashima T, Yoh T, Furutani M, Hirohama A, Kakisaka Y, et al. Serum thioredoxin levels as a predictor of steatohepatitis in patients with nonalcoholic fatty liver disease. J Hepatol. 2003;38:32–8.PubMedCrossRefGoogle Scholar
  7. 7.
    Tomita K, Oike Y, Teratani T, Taguchi T, Noguchi M, Suzuki T, et al. Hepatic AdipoR2 signaling plays a protective role against progression of nonalcoholic steatohepatitis in mice. Hepatology. 2008;48:458–73.PubMedCrossRefGoogle Scholar
  8. 8.
    Yu J, Chu ES, Wang R, Wang S, Wu CW, Wong VWS, et al. Heme oxygenase-1 protects against steatohepatitis in both cultured hepatocyte and mice. Gastroenterology. 2010;138:694–704.PubMedCrossRefGoogle Scholar
  9. 9.
    Leonarduzz G, Scavazza A, Biasi F, Chiarpotto E, Camandola S, Vogel S, et al. The lipid peroxidation end product 4-hydroxy-2,3-nonenal up-regulates transforming growth factor beta expression in the macrophages lineage: a link between oxidative injury and fibrosclerosis. FASEB J. 1997;11:851–7.Google Scholar
  10. 10.
    Matsuzawa N, Takamura T, Kurita S, Nisu H, Ota T, Ando H, et al. Lipid-induced oxidative stress causes steatohepatitis in mice fed an atherogenic diet. Hepatology. 2007;46:1392–403.PubMedCrossRefGoogle Scholar
  11. 11.
    Zhang DD, Hannink M. Distinct cysteine in Keap1 are required for Keap1-dependent ubiquitination on Nrf2 and for stabilization of Nrf2 by chemopreventive agents and oxidative stress. Mol Cell Biol. 2003;23:8137–51.PubMedCrossRefGoogle Scholar
  12. 12.
    Kwak MK, Itoh K, Yamamoto M, Sutter TR, Kensler TW. Role of transcription factor Nrf2 in the induction of hepatic phase 2 and antioxidative enzymes in vivo by the cancer chemoprotective agent, 3H-1, 2-dithiole-3-thione. Mol Med. 2001;7:135–45.PubMedGoogle Scholar
  13. 13.
    Okada K, Shoda J, Taguchi K, Maher JM, Ishizaki K, Inoue Y, et al. Ursodeoxycholic acid stimulates Nrf2-mediated hepatocellular transport, detoxification, and antioxidative stress systems in mice. Am J Physiol Gastrointest Liver Physiol. 2008;295:G735–47.PubMedCrossRefGoogle Scholar
  14. 14.
    Okada K, Shoda J, Taguchi K, Maher JM, Ishizaki K, Inoue Y, et al. Nrf2 counteracts cholestatic liver injury via stimulation of hepatic defense systems. Biochem Biophys Res Commun. 2009;389:431–6.PubMedCrossRefGoogle Scholar
  15. 15.
    Sugimoto H, Okada K, Shoda J, Warabi E, Ishige K, Ueda T, et al. Deletion of nuclear factor-E2-related factor-2 leads to rapid onset and progression of nutritional steatohepatitis in mice. Am J Physiol Gastrointest Liver Physiol. 2010;298:G283–94.PubMedCrossRefGoogle Scholar
  16. 16.
    Chowdhry S, Nazmy MH, Meakin PJ, Dinkova-Kostova AT, Walsh SV, Tsujita T, et al. Loss of Nrf2 markedly exacerbates nonalcoholic steatohepatitis. Free Radic Biol Med. 2010;48:357–71.PubMedCrossRefGoogle Scholar
  17. 17.
    Zhang YKJ, Yeager RL, Tanaka Y, Klaassen CD. Enhanced expression of Nrf2 in mice attenuated the fatty liver produced by a methionine- and choline-deficient diet. Toxicol Appl Pharmacol. 2010;245:326–34.PubMedCrossRefGoogle Scholar
  18. 18.
    Harada N, Kanayama M, Maruyama A, Yoshida A, Tazumi K, Hosoya T, et al. Nrf2 regulates ferroportin 1-mediated iron efflux and counteracts lipopolysaccharide-induced ferroportin 1 mRNA suppression in macrophages. Arch Biochem Biophys. 2011;508:101–9.PubMedCrossRefGoogle Scholar
  19. 19.
    Nemeth E, Tuttle MS, Powelson J, Vaughn MB, Donovan A, Ward DM, et al. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science. 2004;306:2090–3.PubMedCrossRefGoogle Scholar
  20. 20.
    George DK, Goldwurm S, Macdonald GA, Cowley LL, Walker NI, Ward PJ, et al. Increased hepatic iron concentration in nonalcoholic steatohepatitis is associated with increased fibrosis. Gastroenterology. 1998;114:311–8.PubMedCrossRefGoogle Scholar
  21. 21.
    Shinkai Y, Sumi D, Fukami I, Ishii T, Kumagai Y. Sulforaphane, an activator of Nrf2, suppresses cellular accumulation of arsenic and its cytotoxicity in primary mouse hepatocytes. FEBS Lett. 2006;580:1771–4.PubMedCrossRefGoogle Scholar
  22. 22.
    Foy AL, Williams HL, Cortell S, Conrad ME. A modified procedure for the determination of non-heme iron in tissue. Anal Biochem. 1967;18:559–63.CrossRefGoogle Scholar
  23. 23.
    Imeryuz N, Tahan V, Sonsuz A, Eren F, Uraz S, Yuksel M, et al. Iron preloading aggravates nutritional steatohepatitis in rats by increasing apoptotic cell death. J Hepatol. 2007;47:851–9.PubMedCrossRefGoogle Scholar
  24. 24.
    Garrick MD. Human iron transporters. Genes Nutr. 2011;6:45–54.PubMedCrossRefGoogle Scholar
  25. 25.
    Sanyal AJ, Chalasani N, Kowdley KV, McCullough A, Diehl AM, Bass NM, et al. Pioglitazone, vitamin E, or placebo for nonalcoholic steatohepatitis. N Engl J Med. 2010;362:1675–85.PubMedCrossRefGoogle Scholar

Copyright information

© Springer 2012

Authors and Affiliations

  • Kosuke Okada
    • 1
  • Eiji Warabi
    • 2
  • Hirokazu Sugimoto
    • 3
  • Masaki Horie
    • 1
  • Katsutoshi Tokushige
    • 4
  • Tetsuya Ueda
    • 5
  • Nobuhiko Harada
    • 6
  • Keiko Taguchi
    • 7
  • Etsuko Hashimoto
    • 4
  • Ken Itoh
    • 6
  • Tetsuro Ishii
    • 2
  • Hirotoshi Utsunomiya
    • 8
  • Masayuki Yamamoto
    • 7
  • Junichi Shoda
    • 1
    Email author
  1. 1.Field of Basic Sports Medicine, Sports Medicine, Faculty of Medicine, Graduate School of Comprehensive Human SciencesThe University of TsukubaTsukubaJapan
  2. 2.Biomedical Sciences, Faculty of Medicine, The University of TsukubaTsukubaJapan
  3. 3.Department of Gastroenterology, Faculty of MedicineThe University of TsukubaTsukubaJapan
  4. 4.Department of Internal Medicine and GastroenterologyTokyo Women’s Medical UniversityTokyoJapan
  5. 5.Drug Development Service Division, Pharmacodynamics Group, Medi-Chem Business SegmentMitsubishi Chemical Medience CorporationTokyoJapan
  6. 6.Department of Stress Response ScienceHirosaki University Graduate School of MedicineHiroksakiJapan
  7. 7.Department of Medical BiochemistryTohoku University Graduate School of MedicineSendaiJapan
  8. 8.Department of Strategic Surveillance for Functional Food and Comprehensive Traditional MedicineWakayama Medical UniversityWakayamaJapan

Personalised recommendations