ed. The presence of F4/80-positive macrophages was detected immunohistochemically using the rat monoclonal anti-mouse F4/ 80 antibody described elsewhere. Proteolytic activation of latent TGF was detected with antibody against R58 latency associated protein degradation products . Apoptotic cells were detected by TdT mediated dUTP-biotin nick end labeling assay using ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit. The Sirius redpositive and R58 LAP-DP-positive areas were measured using the software WinROOF. TUNEL-positive cells were counted in the whole area of each section and expressed as the mean number/mm2. The liver histology was assessed by two investigators without knowledge of the origin of the slides according to the NASH clinical research network scoring system. Quantitative Real-Time PCR Total RNA was extracted from the liver using Sepasol reagent. Quantitative real-time PCR was performed with StepOnePlus Real-time PCR MedChemExpress Vorapaxar system using Fast SYBR Green Master Mix Reagent as described previously. Primers used in this study were described in S2 Statistical Analysis Data are presented as 
mean SE, and P < 0.05 was considered statistically significant. Statistical analysis was performed using analysis of variance followed by Scheffe's test. Differences between two groups were compared using Student t-test. Pearson correlation coefficient was employed to investigate the correlation among the numbers of hCLS and TUNEL-positive cells, and the extent of fibrosis. Results Preventive effect of EPA on hepatic lipid accumulation in MC4R-KO mice PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19764104/ First, we examined whether EPA treatment prevents the development of NASH using our mouse model of NASH. Wildtype mice were fed SD and MC4R-KO mice were fed control diet, WD plus 5% weight palmitate or with the diet, in which 5% weight palmitate was replaced to EPA for 24 weeks. The amount of food intake was comparable between control and EPA-treated MC4R-KO mice. The MC4R-KO mice fed control diet showed accelerated body weight gain relative to 4 / 16 EPA Ameliorates NASH in MC4R-KO Mice Fig 1. Body weight and tissue weights in MC4R-KO mice treated with EPA for 24 weeks. Experimental protocol of preventive EPA treatment. Growth curve and weights of the subcutaneous, epididymal and mesenteric white adipose tissues and liver of male MC4R-KO and wildtype mice. WT-SD, WT mice fed standard PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19761586 diet; MC4R-control, MC4R-KO mice fed Western diet supplemented with 5% palmitate; MC4R-EPA Pre, MC4R-KO mice fed WD supplemented with 5% EPA for 24 weeks. Open circle, WT-SD; Open triangle, MC4R-control; closed square, MC4R-EPA Pre. Liver triglyceride content at 24 weeks. Insulin tolerance test at 12-week WD feeding. Open triangle, MC4R-control; closed square, MC4R-EPA Pre. P < 0.01; n.s., not significant. WT-SD, n = 8; MC4R-control, n = 7; MC4R-EPA Pre, n = 10. doi:10.1371/journal.pone.0121528.g001 wildtype mice fed SD, along with increased weights of adipose tissue and liver as reported. EPA treatment showed no appreciable or only marginal effect on body weight and adipose tissue weights. On the other hand, the liver weight and the hepatic TG content were markedly reduced in EPA-treated MC4R-KO mice relative to control MC4R-KO mice. Hepatic fatty acid composition analysis revealed increased hepatic EPA content and decreased arachidonic acid content. EPA treatment also reduced serum concentrations of TC, FFA, and ALT in MC4R-KO mice, whereas EPA treatment did not affect glucose metabolism and insulin resistance. S
