/80 immunostaining of the liver after 24 weeks of EPA treatment. Arrows indicate characteristic histological features termed “hepatic crown-like structures “. Immunofluorescent 
staining for F4/80 and CD11c. Hepatic mRNA expression of CD11c. TdT mediated dUTP-biotin nick end labeling immunostaining and the number of BCTC TUNEL-positive cells. Arrowheads indicate TUNEL-positive cells. Correlation PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19763960 of the number of hCLS and the fibrosis area and the number of TUNEL-positive cells. Scale bars, 50 m. P < 0.05; P < 0.01; n.d., not detected. WT-SD, n = 8; MC4R-control, n = 7; MC4R-EPA Pre, n = 10. doi:10.1371/journal.pone.0121528.g004 component did not reach statistic significance. Similar to the preventive protocol, hepatic mRNA expression of genes related to de novo lipogenesis, -oxidation, and fibrogenesis was decreased in EPA-treated MC4R-KO mice relative to control MC4R-KO mice. The number of hCLS was also significantly reduced in EPA-treated MC4R-KO mice relative to control MC4R-KO mice, along with down-regulation of CD11c mRNA expression. Furthermore, EPA treatment resulted in a significant reduction in the number of TUNEL-positive cells and TGF activation. These observations, taken together, suggest that EPA suppressed the progression of liver fibrosis in MC4R-KO mice after the mice developed NASH. 9 / 16 EPA Ameliorates NASH in MC4R-KO Mice Fig 5. Effect of EPA on hepatic TGF activation in MC4R-KO mice. Immmunostaining with anti-R58 LAP-DP antibody to determine TGF activation in the liver after 24 weeks of EPA treatment. Quantification of the R58 LAP-DP-positive area. Hepatic mRNA expression of urokinase-type plasminogen activator receptor. Active TGF protein levels in the liver. Scale bars, 50 m. P < 0.05; P < 0.01. WT-SD, n = 8; MC4R-control, n = 7; MC4R-EPA Pre, n = 10. doi:10.1371/journal.pone.0121528.g005 Fig 6. Histological analysis of the liver of MC4R-KO mice treated with EPA for 4 weeks after the development of NASH. Experimental protocol of therapeutic EPA treatment. Fibrillar collagen deposition evaluated by Masson-trichrome staining and fibrosis scores. Quantification of Sirius redpositive area. Scores of steatosis, lobular inflammation, ballooning degeneration and NAS. Scale bars, 50 m. P < 0.05; P < 0.01; n.s., not significant. MC4R-control, n = 7; MC4R-EPA Pre, n = 10. doi:10.1371/journal.pone.0121528.g006 10 / 16 EPA Ameliorates NASH in MC4R-KO Mice Discussion Using a variety of animal models through genetic, dietary, and/or pharmacologic approaches, many attempts have been made to identify novel therapeutic strategies for NASH, while their clinical efficacy is still unclear. It is partly because of the limited availability of appropriate animal models that reflect a liver condition of human NASH. For instance, dietary deficiency of methionine and choline develops steatosis and mild fibrosis in the liver, although without obesity and insulin resistance. Since NASH is considered as the hepatic phenotype of the metabolic syndrome, crosstalk among multiple organs should be involved in the pathophysiology of NASH. In this regard, MC4R-KO mice, a unique rodent model of NASH accompanied by obesity and systemic insulin resistance, would be useful for evaluating the effectiveness of novel drugs to PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19762901 treat NASH. This is the first report to evaluate drug efficacy using MC4R-KO mice. In this study, we demonstrate that EPA treatment effectively suppresses the development and progression of liver fibrosis along with marked reduction of hep
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