Histological Analysis of Liver

Fresh liver tissues were fixed with 4% paraformaldehyde solution for 24 h, embedded in paraffin, and sliced into 4-μm sections for hematoxylin-eosin (H&E) staining and Sirius Red staining. The NAFLD activity score (NAS) was calculated according to the guidance provided by the Pathology Committee of the NASH Clinical Research Network (Kleiner et al., 2005): steatosis (<5% = 0, 5–33% = 1, 33–66% = 2, >66% = 3), lobular inflammation (none = 0, <2 foci = 1, 2–4 foci = 2, >4 foci = 3), and hepatocellular ballooning (none = 0, few = 1, prominent = 2). All features were scored in a blinded manner based on six fields of view per sample. Individual scores for each field of view were summed to calculate the NAS for each animal. Histological assessments were performed by a pathologist who was blinded to the treatment.