Original Article BtjJ01KOhcoCa980byreu.18iBon3grS20nt0N1ci-ona9605,cal-4h0iJl1oe/08aj2Aftc96py2b1CraGtfniloci.Fn0rlRei9Fgc-ra1el2eBe2RGioanFcdhRiceeamliRsterasyea bnrcdhNlJuaaptraitntioinon reduces steatohepatitis in mouse MCD diet model Motoki Inoue,1 Susumu Tazuma,2 Keishi Kanno,2,* Hideyuki Hyogo,1 Kazuhiko Igarashi3 and Kazuaki Chayama1 1Department of Medicine and Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, 123 Kasumi, Minamiku, Hiroshima 7348551, Japan 2Department of General Internal Medicine, Hiroshima University Hospital, 123 Kasumi, Minamiku, Hiroshima 7348551, Japan 3Department of Biochemistry, Tohoku University School of Medicine, 2 1 Seiryomachi, Aobaku, Sendai 9808575, Japan (Re3ceived 21 December, 2009; Accepted 11 February, 2010; Published online 10 April, 2010) svCBtiardci2TCeceha0hotd1te1ipsdv1tyihseireusiagsoaCehnrto,itrgmaod©inpinmsse2atcnorl0riniw1baps1uctocitJroiAekoCnsntsaBit,srlaNiapbrrentruiopdctiprloeernesrdsplyoirLsortcirdcioituebefcnudhtst.ieeod,mnuewinhodixaecnyrhgythepmeneateresmderim1iut ms(Ho,uOfpn1trho,ee- a.k.a. HSP32), which is an inducible enzyme and has antioxida tion/antiinflammatory properties shown in various models of organ injuries. Since oxidative stress plays a pivotal role in the pathogenesis of nonalcoholic steatohepatitis (NASH), HO1 induc tion would be expected to prevent the development of NASH. In this study, we investigated the influence of Bach1 ablation in mice on the progression of NASH in methioninecholine deficient (MCD) diet model. Bach1 ablation resulted in significant induction of HO1 mRNA and its activity in the liver. When fed MCD diet, Bach1−/− mice exhibited negligible hepatic steatosis compared to pronounced steatohepatitis in wild type mice with 6fold increase in hepatic triglyceride content. Whereas feeding of MCD diet decreased mRNA expressions of peroxisome proliferatoractivated receptor (PPAR) α and microsomal triglyceride transfer protein (MTP) in wild type mice, there were no change in Bach1−/− mice. In addition, hepatic concentration of malondialdehyde (MDA), a biomarker for oxidative stress as well as plasma alanine amino transferase (ALT) was significantly lower in Bach1−/− mice. These findings suggest that Bach1 ablation exerts hepatoprotective effect against steatohepatitis presumably via HO1 induction and may be a potential therapeutic target. Key Words: oxidative stress, steatohepatitis, nonalcoholic fatty liver disease, heme oxygenase1, peroxisome proliferatoractivated receptor α N NIonntarlocodhuoclticiofnatty liver disease (NAFLD) is the most major liver dysfunction in the world and its prevalence is increasing with epidemics of obesity, diabetes, and metabolic syndrome. NAFLD includes wide-ranging liver disorder from simple steatosis through steatohepatitis to cirrhosis and possibly hepatocellular carcinoma.(1,2) About one-third of NAFLD is regarded as non- alcoholic steatohepatitis (NASH) which shows activity of steato- hepatitis with the potential for a lethal outcome. In the develop- ment of NASH, “two hit theory” is assumed to be required, with the first hit being lipid accumulation in hepatocytes increasing the sensitivity of the liver to the second hit such as oxidative stress.(3) In this setting, emerging animal studies have demonstrated the protective effects of antioxidant (e.g., vitamin E, 1-aminobenzo- triazole, curcumin, N-acetylcysteine, angiotensin II type I receptor blockers) in experimental models of NASH.(4–7) In human, small clinical trials have revealed potential usefulness of vitamin E in the treatment of NASH.(8–10) Interestingly, administration of fer- mented green tea extract which exerts antioxidant activity reduced hepatic triglyceride content in addition to necro/inflammation and fibrosis in rat NASH model.(11) These suggest that oxidative stress plays a pivotal role in the pathogenesis of NASH and expected to be one of the promising targets in the treatment of this disease. Heme oxygenase (HO) is the rate limiting enzyme in the doi: 10.3164/jcbn.09122GFR ©2011 JCBN catabolism of heme, to yield biliverdin, carbon monoxide (CO), and free iron. HO exhibits antioxidant properties derived from the elimination of prooxidant heme as well as from biological activities of its reaction products, CO, biliverdin, bilirubin, and iron.(12) In mammals three isoforms, designated as HO-1, HO-2 and HO-3 have been identified in the liver. Among these HO isoforms, HO-3 is considered as processed pseudogenes derived from HO-2 transcripts.(13) While HO-2 is constitutively expressed mainly in parenchymal cells, HO-1 is prominent in Kupffer cells and is markedly induced in hepatocytes in the response to various stimuli. Interestingly, it has been reported that HO-1 expression was significantly increased in the liver from NASH patients and the increase reflected the severity of the disease.(14) These suggest that HO-1 functions as a defense system against oxidative stress in the liver. In fact, accumulating evidences have demonstrated hepatoprotective effects of HO-1. The induction HO-1 by cobalt protoporrphyrin (CoPP) protects human hepatocytes from ethanol- induced cytotoxicity.(15) Ginkgo biloba extract, a naturally occur- ring HO-1 inducer has been proved to ameliorate ethanol-induced sustained damage and redox imbalance on rat liver.(16) In addition, curcumin which has a beneficial effect on NASH has been recently proved to be an HO-1 inducer.(5,17) In addition to cytoprotective properties of HO-1, the regulatory mechanisms of this gene have been revealed. A heme-binding factor Bach1 functions as a transcriptional repressor of the gene encoding HO-1 (Hmox1). Bach1 forms complexes with small Maf proteins and competes against NF-E2 related factor (Nrf 2), the major transcriptional activator of Hmox1, for binding at the Maf recognition elements (MAREs) of Hmox1 enhancer. Consistently, mice lacking Bach1 exhibited constitutively high level of HO-1 expression in many tissues including the liver.(18) Further studies of Bach1−/− mice have revealed a hepatoprotective role of Bach1 ablation in LPS-induced liver injury.(19) In the current study, we investigated the influence of Bach1 disruption in animal NASH model. Accompanied with marked up- regulation of HO-1 in the liver, we observed reduced hepatic steatosis in Bach1−/− mice with altered expressions of key genes that regulate hepatic lipid metabolism. Consistent with these findings, the absence of Bach1 also exhibited hepatoprotection confirmed by serum liver enzyme and hepatic malondialdehyde (MDA) concentration. Collectively, these data suggest Bach1 as a potential target in the treatment of NASH presumably via induc- tion of HO-1. Materials and Methods Animals and experimental protocol. Male C57BL/6 mice *To whom correspondence should be addressed. Email: kkanno@hiroshimau.ac.jp J. Clin. Biochem. Nutr. | March 2011 | vol. 48 | no. 2 | 161–166