ince unbalanced production of pro- and anti-inflammatory adipocytokines in obesity has been implicated in the pathogenesis of NASH, we examined serum adipocytokine concentrations and found that EPA treatment significantly increased serum adiponectin concentrations in MC4R-KO mice. On the other hand, EPA treatment did not affect serum concentrations of leptin in MC4R-KO mice. 5 / 16 EPA Ameliorates NASH in MC4R-KO Mice WT, wildtype; SD, standard diet; BG, blood glucose; TG, triglyceride; FFA, free fatty acid; TC, total cholesterol; ALT, alanine aminotransferase. Data are expressed as the mean SE. P < 0.01 vs. WT-SD P < 0.01 vs. MC4R-Control. n = 710 doi:10.1371/journal.pone.0121528.t001 Effect of EPA on the development of liver fibrosis in MC4R-KO mice After 24 weeks, the livers from MC4R-KO mice fed control diet exhibited micro- and macrovesicular steatosis, ballooning degeneration, massive infiltration of inflammatory cells and pericellular fibrosis as reported previously. On the other hand, steatotic changes and ballooning degeneration were markedly suppressed in EPA-treated MC4R-KO mice. The fibrosis score and fibrosis area were also significantly decreased by EPA treatment. Although the inflammation score was unchanged, the scores for steatosis and ballooning degeneration were decreased by EPA treatment, so that there PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19763871 was a significant reduction in NAS in EPA-treated MC4R-KO mice relative to control MC4R-KO mice. In this study, mRNA expression of genes related to de novo lipogenesis and stearoyl-CoA desaturase-1 ) and -oxidation ) was markedly increased in the liver of control MC4R-KO mice relative to wildtype mice as reported, which was significantly suppressed by EPA treatment. There was no apparent change in mRNA expression of proinflammatory genes such 
as ). On the other hand, mRNA expression of TGF1-target genes such as collagen 1, tissue inhibitor of metalloproteinase-1 and matrix metalloproteinase-2 was significantly suppressed, although EPA treatment did not affect mRNA expression of TGF1. These observations, taken together, suggest that EPA treatment effectively prevents the development of liver fibrosis in MC4R-KO mice. Effect of EPA on hCLS formation and hepatocyte apoptosis in PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19761586 MC4R-KO mice We have recently reported a unique histological structure or hCLS in the liver of MC4R-KO mice, where dead hepatocytes are surrounded by CD11c-positive macrophages. Our data also suggest that hCLS promotes liver fibrosis during the order Oleandrin progression from simple steatosis to NASH. We found that EPA treatment effectively suppresses hCLS formation in MC4R-KO mice, In this study, the F4/80-positive area was roughly comparable between the treatments. Double immunofluorescent staining of F4/80 and CD11c revealed that hCLS-constituting macrophages are positive for CD11c in EPA-treated MC4R-KO mice 6 / 16 EPA Ameliorates NASH in MC4R-KO Mice Fig 2. Effect of EPA on liver injury and fibrosis in MC4R-KO mice. Fibrillar collagen deposition evaluated by Masson-trichrome staining and fibrosis scores at 24 weeks. Inset: Representative image of hepatocyte ballooning. Sirius red staining and quantification of Sirius red-positive area. Scores of steatosis, lobular inflammation, ballooning degeneration and non-alcoholic fatty liver disease activity score . Scale bars, 50 m. P < 0.05; P < 0.01; n.s., not significant; n.d., not detected. WT-SD, n = 8; MC4R-control, n = 7; MC4R-EPA Pre, n = 10. doi:10.1371/journal.pone.0121528.g002 , whereas hepatic mRNA express