Figure 5. URMC099-treated mice are protected against FFC diet–induced liver injury and fibrosis.

WT C57BL/6J mice were fed either chow or a diet high in saturated fat, fructose, and cholesterol (FFC) for 24 weeks, and URMC099 or vehicle (Veh) was given during the last 2 weeks. (A) Serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), bile acid, and total bilirubin were measured (n = 4–7). (B) Hepatocyte apoptosis was assessed by the TUNEL assay. Brown-stained apoptotic nuclei (black arrowheads) were quantified by counting nuclei in 10 random ×20 microscopic fields, and averaged for each animal (n = 3). Scale bars: 50 μm. (C) Huh7 cells were treated with either Veh or 20 μM lysophosphatidylcholine (LPC) with or without URMC099 for 16 hours. Apoptosis was assessed by DAPI staining and counting of apoptotic nuclei. (D) Fixed liver tissue sections were stained by Sirius red. Sirius red chromogen was quantified by morphometry using ImageJ software in 15 random ×20 microscopic fields, and averaged for each animal (n = 3.) Scale bars: 50 μm. (E) Frozen liver tissue sections were examined by second harmonic generation (SHG) microscopy using a ×25 objective. Scale bars: 50 μm. Total RNA was extracted from the liver tissues. The mRNA expression of (F) the profibrogenic markers osteopontin, collagen 1α1, and TIMP1 were evaluated by real-time PCR (n = 3–5). Fold change was determined after normalization to GAPDH mRNA expression, and expressed relative to that observed in chow-fed, vehicle-treated mice. Data represent mean ± SEM. Differences between the groups were compared using 1-way ANOVA followed by Bonferroni’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. ns, non-significant.