Thus, empagliflozin prevented development of the pathological features of NASH, suggesting that it could become a simple new therapeutic strategy for NASH associated with diabetes. Authors contributions TJ, TT, TS, and KA collected the data. factor-, interleukin-6, and monocyte chemoattractant protein-1) was decreased IQGAP1 in the empagliflozin and linagliptin?+?empagliflozin groups compared with the vehicle group. The collagen deposition with Sirius reddish staining was significantly reduced in the linagliptin?+?empagliflozin group compared with the linagliptin or the empagliflozin group. Immunohistochemistry showed that expression of -easy muscle mass actin, a marker of myofibroblasts (fibrosis), was reduced in the linagliptin?+?empagliflozin group compared with the vehicle group, as was expression of type 1 and 3 collagen mRNA. Linagliptin?+?empagliflozin decreased expression of mRNAs for genes related to fatty acid synthesis, but did not increase mRNAs for -oxidation-related genes. Conclusions While empagliflozin alone attenuates development of NASH showing anti-steatotic and anti-inflammatory effects, combined administration of empagliflozin and linagliptin can synergistically ameliorates NASH with stronger anti-fibrotic effects. linagliptin; empagliflozin; glycated albumin; alanine aminotransferase *?P? ?0.05, ??P? ?0.01, ??P? ?0.001 vs. control; ?P? ?0.05, ||?P? ?0.01, ??P? ?0.001 vs. vehicle; #?P? ?0.05, **?P? ?0.01, ???P? ?0.001 vs. linagliptin alone Effect of empagliflozin and linagliptin around the liver/body weight ratio and hepatic triglyceride (TG) content The liver/body weight ratio was higher in the vehicle-treated group and the linagliptin-treated group than in the control group, while it was significantly lower in the empagliflozin group and the linagliptin?+?empagliflozin group than in the vehicle group or the linagliptin group (Fig.?1a). The hepatic TG content was higher in the vehicle group than in the control group, while BNC375 it was lower in the linagliptin, empagliflozin, and linagliptin?+?empagliflozin groups compared with the vehicle group (Fig.?1b). Open in a separate windows Fig.?1 Liver to body weight ratio (a) and liver triglyceride content (b) in the five groups. Data are mean??SE. *P? ?0.05, ?P? ?0.01, ?P? ?0.001 vs. control; P? ?0.05, ||P? ?0.01, ?P? ?0.001 vs. vehicle; #P? ?0.05 vs. Linagliptin alone Effect of empagliflozin and linagliptin around the histological NAFLD activity score (NAS) Examination of HCE stained liver sections revealed fatty degeneration, inflammatory cell infiltration, and hepatocellular ballooning, predominantly round the central veins, in mice from the vehicle group. The NAS score was significantly higher in the diabetic animals than in the non-diabetic control group (Fig.?2). The NAS score was significantly lower in the empagliflozin and linagliptin?+?empagliflozin groups compared with the vehicle group or the linagliptin BNC375 group. The scores of each component of NAS in all groups were shown in Table?2. Open in a separate windows Fig.?2 Representative microphotographs of liver sections stained with hematoxylin eosin and NAFLD activity score (nonalcoholic fatty liver disease (NAFLD) activity score Effect of empagliflozin and linagliptin on hepatic inflammation Immunohistochemical staining showed that expression of F4/80 antigen, a marker of macrophages, was increased in the livers of the vehicle-treated mice (Fig.?3a). Treatment with linagliptin significantly reduced F4/80 antigen expression in the peri-central zone of the liver compared with the vehicle group (Fig.?3a). Expression of F4/80 mRNA was increased in vehicle-treated NASH mice compared with control mice, while it was significantly decreased in the empagliflozin and linagliptin?+?empagliflozin groups compared with the vehicle group (Fig.?3c). Open in a separate windows Fig.?3 Representative microphotographs of immunohistochemical staining for F4/80 in liver sections (a) and percentage in area of positive immunostaining for F4/80 in the five groups (b). Normalized mRNA expression of F4/80 the liver of the five groups (c). Data are mean??SE. *P? ?0.05, ?P? ?0.001 vs. control; P? ?0.05, ?P? ?0.001 vs. vehicle Expression of TNF- mRNA was increased in vehicle-treated NASH mice compared with control mice (Fig.?4), while it was significantly decreased in the empagliflozin and linagliptin?+?empagliflozin groups compared with the vehicle group or the linagliptin group. Similarly, MCP-1 mRNA expression was significantly decreased in the empagliflozin group and BNC375 the linagliptin?+?empagliflozin group relative to the vehicle group or the linagliptin group (Fig.?4). Expression of SOCS3 mRNA was significantly decreased in the empagliflozin group (Fig.?4). Open in a separate windows Fig.?4 Gene expression.
