Fig. 3. Protective effect of Bach1 ablation on MCD dietinduced liver injury. (A) Serum ALT levels were determined at indicated time points by 8 w in wild type mice and Bach1−/− mice fed either regular chow or MCD diet (n = 5/each group). (B) Hepatic MDA concentration was assessed with standarlization of protein concentration in wild type (open bar) and Bach1−/− (closed bar) mice (n = 5/each group). *p<0.05, regular chow vs MCD diet. †p<0.05, wild type vs Bach1−/− mice. contrast to 4-fold increase in MDA level in wild type mice on MCD diet. Influence of Bach1−/− ablation on hepatic fibrogenesis. As previously reported,(23) rodents fed MCD diet develops steato- hepatitis mimicking human NASH. Fig. 4A depicts mild peri- cellular fibrosis with fat deposition in the liver from wild type mice fed MCD diet (upper panel). In contrast, Bach1−/− mice dis- played no significant change in the liver (lower panel). Consistent with these morphological changes, hepatic mRNA expression of α smooth muscle actin (αSMA) which is predominantly expressed in activated stellate cells was up-regulated in the liver of wild type mice on MCD diet while it remained unchanged in Bach1−/− mice. Discussion The aim of this study was to explore the influence of Bach1 ablation in the development of NASH. As demonstrated in Fig. 1, Bach1 ablation in mice leads to significant up-regulation of HO-1 mRNA as well as its activity. This was in keeping with previous data that hepatic expressions of HO-1 mRNA and protein were constitutively high in the absence of Bach1.(18) To date, HO-1, β- globin, ferritin, thioredoxin reductase1 and NADP(H) quinine (oxido) reductase have been identified as a target genes of Bach1, 164 but other downstream genes of Bach1 have not been fully under- stood.(24) HO-1 has been reported to have cytoprotective, anti- inflammatory and anti-oxidative properties. Emerging reports have suggested protective roles of HO-1 in experimental-induced hepatic damage including lipopolysaccharide-induced injury, immune liver fibrosis model, and hepatic ischemia-reperfusion injury.(12,19,25–27) In addition, pathophysiological importance of HO- 1 has also been suggested by previous findings that HO-1 is induced under various etiological conditions including human NASH.(14) The pathogenesis of NASH is widely recognized as two hit theory, in which the first hit is an initial metabolic disturbance causes steatosis and second pathogenic stimulus such as free radical and cytokines triggers necro/inflammation leading to the progression of steatohepatitis. The current study was undertaken on the basis of our initial hypothesis that HO-1 induction by Bach1 ablation may ameliorate oxidative stress and inflammation, the second hit in NASH pathogenesis and may repress the transition from bland steatosis to steatohepatitis. Surprisingly, in addition to reduction in hepatic damage, Bach1 ablation significantly diminished hepatic steatosis following MCD feeding. This was explained by the finding in this study that hepatic expression of PPARα and MTP, that are involved in hepatic fatty acid oxidation and hepatic secretion of triglyceride-rich VLDL, remained un- changed in MCD diet-fed Bach1−/− mice in comparison to signifi- cant reduction in wild type mice. These changes were inversely paralleled with hepatic MDA concentrations, suggesting the possi- bility that relief from oxidative damage in Bach1−/− mice con- served hepatic lipid metabolism. Along a similar line, the previous study of ob/ob mice fed MCD diet has revealed that YHK, a naturally derived herbal medicine that is reported to reduce reac- tive oxygen species declined hepatic steatosis via induction of hepatic mRNAs of PPARα and MTP.(28,29) This suggests a potential mechanism of the previous findings that antioxidant agents have beneficial effects not only on hepatic necro/inlflam- mation but also on hepatic steatosis.(4,28) Since mice assigned to high fat diet or dietary choline restriction exhibited mitochondrial dysfunction which negatively affects the bioenergenetics of liver, antioxidative status observed in Bach−/− mice might preserve the integrity of the mitochondria from oxidative damage and help to maintain cellular functions including hepatic lipid metabo- lism.(30,31) However, the current study lacks the direct evidence that hepatoprotective property against NASH in Bach1−/− mice was due to reduction in oxidative stress attributed to HO-1 induction, and further investigations are needed. As shown in Fig. 4, MCD diet caused modest pericellular fibrosis with significant induction of αSMA mRNA in wild type mice whereas Bach1−/− mice exhibited almost normal in liver histology as well as αSMA expression. Reduction in hepatic fibrosis is likely to be elicited by relief of hepatic damage and inflammation in Bach1−/− mice. However, previous report has demonstrated that HO-1 is expressed in human hepatic myofibro- blast and induction of HO-1 leads to inhibition of collagen synthesis and to reduction in proliferation of myofibroblast.(32) This suggests direct role for HO-1 induction against the develop- ment of hepatic fibrosis and might be a possible explanation for our finding. In the current study, MCD dietary model was utilized as a NASH animal model. Although this model is widely used on the basis that morphological changes of the liver resembles human NASH with induction of oxidative stress, this does not entirely reflect the etiological background of human NASH such as obesity and insulin resistance. Alternative dietary models for NASH include high fat diet, which develops obesity and insulin resistance but not remarkable steatohepatitis as seen in MCD diet model.(33) Interestingly, recent study has reported that feeding rats with choline-deficient fat-rich diet for 8 w lead to marked steato- hepatitis.(34) doi: 10.3164/jcbn.09122GFR ©2011 JCBN