Volume 138, Issue 2, Pages 694-704.e1 (February 2010) Heme Oxygenase-1 Protects Against Steatohepatitis in Both Cultured Hepatocytes and Mice  Jun Yu, Eagle S.H. Chu, Ruizhi Wang, Shiyan Wang, Chung W. Wu, Vincent W.S. Wong, Henry L.Y. Chan, Geofferey C. Farrell, Joseph J.Y. Sung  Gastroenterology  Volume 138, Issue 2, Pages 694-704.e1 (February 2010) DOI: 10.1053/j.gastro.2009.09.058 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 (A) Effect of heme oxygenase-1 (HO-1) inducer (hemin) on HO-1 protein expression in cultured immortalized mice hepatocytes (AML-12). AML-12 hepatocytes were exposed to control medium or methionine- and choline-deficient (MCD) medium in 96-well plates and treated with hemin (0, 3, 5 10, 15 μM) for 24 hours. (B) Effect of adenovirus vector that expressed HO-1 (Ad-HO-1) infection on expression of HO-1 protein in cultured AML-12. AML-12 were infected with Ad-HO-1 (1, 5, 10, 100 multiplicities of infection [MOI]) or Ad-LacZ (control) for 5 days and then incubated in MCD culture medium or control medium for an additional 24 hours. Protein expression was measured by Western blotting. Gastroenterology 2010 138, 694-704.e1DOI: (10.1053/j.gastro.2009.09.058) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Effects of hemin or adenovirus vector that expressed heme oxygenase-1 (Ad-HO-1) infection in AML-12 hepatocytes on (A) triglyceride level in culture medium and (B) alanine aminotransferase (ALT) level in culture medium. Data are mean ± standard deviations (SD) of 3 to 5 experiments. (C) Lipoperoxide level of AML-12 hepatocytes. Data are mean ± SD of 3 to 5 experiments. *P < .001 methionine- and choline-deficient (MCD) vs control; #P < .05; ##P < .001 AML-12 cells treated with hemin and/or Ad-HO-1 vs MCD. SnMP, stannic mesoporphyrin. Gastroenterology 2010 138, 694-704.e1DOI: (10.1053/j.gastro.2009.09.058) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Effect of treatment with hemin on methionine- and choline-deficient (MCD) diet-induced steatohepatitis in mice. (A) Hematoxylin and eosin-stained liver sections from mice fed: (A1) the control diet—appearances are normal. (A2) MCD diet, liver shows numerous foci of necroinflammation and macrovesicular fat droplets. (A3) In mice fed the MCD diet, treatment with hemin (25 mg/kg/d, 3 times per week), largely ameliorated steatohepatitis. (B) The severity of hepatic steatosis and necroinflammation were scored as described in Methods. Duration of this experiment is 4 weeks. Values of hepatic steatosis and necroinflammation are mean ± standard deviation (n = 5/group). *P < .0001 control vs MCD diet, #P < .05; ##P < .01 MCD vs MCD + hemin-treated mice. Gastroenterology 2010 138, 694-704.e1DOI: (10.1053/j.gastro.2009.09.058) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Effects of the methionine- and choline-deficient (MCD) diet and treatment with hemin on: (A1) serum alanine aminotransferase (ALT), (A2) serum aspartate aminotransferase (AST), (B) hepatic lipoperoxide content measured as thiobarbituric acid-reactive substrances (TBARs), and (C) hepatic triglyceride. Mice fed the control, MCD diet, or MCD diet treated with hemin are expressed as mean ± standard deviation (n = 5−6/group). *P < .0001, MCD vs control diet-fed mice. #P < .05; ##P < .01, MCD with hemin vs MCD diet alone for 4 weeks. Gastroenterology 2010 138, 694-704.e1DOI: (10.1053/j.gastro.2009.09.058) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Hepatic heme oxygenase-1 (HO-1) protein expression and HO-1 activity in mice. (A) Immunohistochemistry for HO-1 in livers of mice: (A1) Mice fed the control diet, few HO-1−positive cells are seen. (A2) Mice fed the methionine- and choline-deficient (MCD) diet: the number of HO-1 staining cells is increased. (A3) Treatment of MCD-fed mice with hemin: strong HO-1 staining is presented. Experimental duration is 4 weeks. Slides are representative of 5 to 6 separate experiments (original magnification ×200). (B) Hepatic HO-1 protein and (C) HO-1 activity were measured in mice fed the control diet, MCD diet, or MCD diet with hemin for 4 weeks. Hepatic HO-1 protein was determined by Western blotting, bands were quantitated, and HO-1 protein level was expressed as relative amounts normalized to β-actin. Data are mean ± standard deviation with 5 to 6 animals in each group. *P < .05; **P < .01, MCD vs control diet-fed mice. #P < .01, MCD with hemin vs MCD diet alone for 4 weeks. Gastroenterology 2010 138, 694-704.e1DOI: (10.1053/j.gastro.2009.09.058) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 Schematic diagram for the mechanisms of heme oxygenase-1 (HO-1) protection against nutritional steatohepatitis. (A) HO-1 induction by hemin-ameliorated steatohepatitis was associated with several biological effects: (1) up-regulating genes of reactive oxygen species metabolism system including superoxide dismutase 1 (Sod1), glutathione peroxidase (Gpx) 5, Gpx2, Gpx1, peroxiredoxin (Prdx) 2, and glutathione S-transferase μ1 (Gstm), as well as potent ROS scavenger neuroglobin (Ngb), inhibiting oxidative stress; (2) increasing the expression of antioxidant crystallin α-B (Cryab) and fanconi anemia C (Fancc), which in turn not only suppressed oxidative stress directly but also exerted anti-inflammatory effect; (3) down-regulating pro-inflammatory cytokines interferon-γ, (IFN-γ) interferon-γ inducible protein-10 (IP-10), keratinocyte-derived cytokine (KC), and macrophage inflammatory protein (MIP)-1α and up-regulating protective cytokine interleukin (IL)-22. (B) HO-1 induction blunted hepatic steatosis through suppression of lipogenesis by down-regulating sterol regulatory element binding protein isoform-1c (SREBP-1c) and induction of lipolysis by up-regulating peroxisome proliferator−activated receptor-α (PPARα), acyl-CoA oxidase (ACO), cytochrome P450 (Cyp) 4a10, and Cyp4a14, which led to fatty acid oxidation. Gastroenterology 2010 138, 694-704.e1DOI: (10.1053/j.gastro.2009.09.058) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1 Effects of the MCD diet and hemin on levels of hepatic cytokine production. Mice were fed the control, MCD diet, or MCD diet treated with hemin for 4 weeks and hepatic cytokine profiling was assayed. Data are mean ± SD (n = 4–6/group). *P < .05, **P < .01, MCD vs control diet-fed mice. #P < .05, ##P < .01, MCD with hemin vs MCD diet alone. Gastroenterology 2010 138, 694-704.e1DOI: (10.1053/j.gastro.2009.09.058) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 2 Effects of the MCD diet and hemin on DNA binding activity of SREBP-1c and PPARα. DNA binding activity of SREBp-1c and PPARα was determined by an ELISA-based assay. Nuclear SREBp-1c and PPARα binding activity were quantified by using specific antibodies. Data are mean ± SD, *P < .05, MCD vs control diet-fed mice. #P < .05, ##P < .01, MCD with hemin vs MCD diet alone. Gastroenterology 2010 138, 694-704.e1DOI: (10.1053/j.gastro.2009.09.058) Copyright © 2010 AGA Institute Terms and Conditions