Salsalate attenuates diet induced non-alcoholic steatohepatitis in mice by decreasing lipogenic and inflammatory processes

Abstract

Background and purpose: Salsalate (salicylsalicylic acid) is an anti-inflammatory drug that was recently found to exert beneficial metabolic effects on glucose and lipid metabolism. Although its utility in the prevention and management of a wide range of vascular disorders, including type 2 diabetes and metabolic syndrome has been suggested before, the potential of salsalate to protect against non-alcoholic steatohepatitis (NASH) remains unclear. The aim of the present study was therefore to ascertain the effects of salsalate on the development of NASH.

Experimental approach: Transgenic APOE*3Leiden.CETP mice were fed a high-fat and high-cholesterol diet with or without salsalate for 12 and 20 weeks. The effects on body weight, plasma biochemical variables, liver histology and hepatic gene expression were assessed.

Key results: Salsalate prevented weight gain, improved dyslipidemia and insulin resistance and ameliorated diet-induced NASH, as shown by decreased hepatic microvesicular and macrovesicular steatosis, reduced hepatic inflammation and reduced development of fibrosis. Salsalate affected lipid metabolism by increasing β-oxidation and decreasing lipogenesis, as shown by the activation of PPAR-α, PPAR-γ co-activator 1β, RXR-α and inhibition of genes controlled by the transcription factor MLXIPL/ChREBP. Inflammation was reduced by down-regulation of the NF-κB pathway, and fibrosis development was prevented by down-regulation of TGF-β signalling.

Conclusions and implications: Salsalate exerted a preventive effect on the development of NASH and progression to fibrosis. These data suggest a clinical application of salsalate in preventing NASH.

© 2015 The British Pharmacological Society.

Figures

Figure 1

Lipoprotein profile. Lipoprotein profiles of APOE*3Leiden.CETP mice fed a low‐fat diet (LFD; n = 5), a high‐fat and high‐cholesterol diet (HFC; n = 9) or an HFC diet supplemented with salsalate (1% w/v; HFC + S; n = 8) for 12 weeks. The distribution of cholesterol over the individual lipoprotein profiles in pooled plasma was determined after separation of lipoprotein profiles by fast‐performance liquid chromatography.

Figure 2

Histological photomicrographs and quantitative analysis of NASH in mice. Liver histological cross sections (A) and quantitative analysis (B–H) from APOE*3Leiden.CETP mice fed a low‐fat diet (LFD; n = 5), a high‐fat and high‐cholesterol diet (HFC; n = 9) or an HFC diet supplemented with salsalate (HFC + S; n = 8) for 12 weeks. Upper photomicrographs: haematoxylin and eosin (H&E); lower photomicrographs: Sirius Red staining; magnification 200×. Microvesicular (B) and macrovesicular (C) steatosis as percentage of total liver area, intrahepatic triglycerides (D), intrahepatic cholesterol esters (E) and intrahepatic‐free cholesterol (F), inflammatory foci per microscopic field (G) and fibrosis score (H) were analysed. *P < 0.05, ***P < 0.001, significantly different from HFC; #P < 0.05, ##P < 0.01, significantly different from LFD; Mann‐Whitney U‐test.

Figure 3

Enriched biological processes and hepatic fibrosis pathway analysis. A selection of significantly enriched biological processes (‐log (P‐value)) (A) and the sequence of events leading to fibrosis (B) and pathway analysis showing statistically significant gene expression changes in hepatic stellate cells (C) of APOE*3Leiden.CETP mice fed a HFC diet supplemented with salsalate for 12 weeks (n = 8) relative to HFC diet fed control group (n = 9). Red colour indicates up‐regulation and green colour indicates down‐regulation.

Figure 4

Gene expression of selected genes and plasma IL‐6, MCP‐1 and TIMP‐1. The gene expression of selected genes involved in fatty acid metabolism (A), inflammation (B) or fibrosis (C) by RT‐PCR of livers and plasma levels of IL‐6 (D), MCP‐1 (E) and TIMP‐1 (F) of APOE*3Leiden.CETP mice fed a low‐fat diet (LFD; n = 5), a high‐fat/high‐cholesterol diet (HFC; n = 9) or an HFC diet supplemented with salsalate (HFC + S; n = 8) for 12 weeks. *P < 0.05, **P < 0.01, significantly different from HFC; #P < 0.05, ##P < 0.01, significantly different from LFD; Mann‐Whitney U‐test.

Figure 5

Hepatic fibrosis pathway analysis. Pathway analysis showing statistically significant gene expression changes in activated hepatic stellate cells of APOE*3Leiden.CETP mice fed a HFC diet supplemented with salsalate for 12 weeks (n = 8) relative to HFC diet fed control group (n = 9). Red colour indicates up‐regulation, and green colour indicates down‐regulation.

Figure 6

Histological photomicrographs and quantitative analysis of NASH in mice, treated for longer (20 weeks) with a lower dose of salsalate. Liver histological cross sections (A) and quantitative analysis (B–F) from APOE*3Leiden.CETP mice fed a high‐fat and high‐cholesterol diet (HFC; n = 12) or an HFC diet supplemented with salsalate (0.33% w/v; HFC + S; n = 8) for 20 weeks. Upper photomicrographs: haematoxylin and eosin (H&E); lower photomicrographs: Sirius Red staining; magnification 200×. Microvesicular (B) and macrovesicular (C) steatosis as percentage of total liver area, inflammatory foci per microscopic field (D), percentage perisinusoidal fibrosis (E) and the hydroxyproline ; proline ratio (F) were analysed. *P < 0.05, **P < 0.01, significantly different from HFC; ***P < 0.001, significantly different from HFC; Mann‐Whitney U‐test.