Resveratrol and fenofibrate ameliorate fructose-induced nonalcoholic steatohepatitis by modulation of genes expression

Abstract

Aim: To evaluate the effect of resveratrol, alone and in combination with fenofibrate, on fructose-induced metabolic genes abnormalities in rats.

Methods: Giving a fructose-enriched diet (FED) to rats for 12 wk was used as a model for inducing hepatic dyslipidemia and insulin resistance. Adult male albino rats (150-200 g) were divided into a control group and a FED group which was subdivided into 4 groups, a control FED, fenofibrate (FENO) (100 mg/kg), resveratrol (RES) (70 mg/kg) and combined treatment (FENO + RES) (half the doses). All treatments were given orally from the 9(th) week till the end of experimental period. Body weight, oral glucose tolerance test (OGTT), liver index, glucose, insulin, insulin resistance (HOMA), serum and liver triglycerides (TGs), oxidative stress (liver MDA, GSH and SOD), serum AST, ALT, AST/ALT ratio and tumor necrosis factor-α (TNF-α) were measured. Additionally, hepatic gene expression of suppressor of cytokine signaling-3 (SOCS-3), sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase (FAS), malonyl CoA decarboxylase (MCD), transforming growth factor-β1 (TGF-β1) and adipose tissue genes expression of leptin and adiponectin were investigated. Liver sections were taken for histopathological examination and steatosis area were determined.

Results: Rats fed FED showed damaged liver, impairment of glucose tolerance, insulin resistance, oxidative stress and dyslipidemia. As for gene expression, there was a change in favor of dyslipidemia and nonalcoholic steatohepatitis (NASH) development. All treatment regimens showed some benefit in reversing the described deviations. Fructose caused deterioration in hepatic gene expression of SOCS-3, SREBP-1c, FAS, MDA and TGF-β1 and in adipose tissue gene expression of leptin and adiponectin. Fructose showed also an increase in body weight, insulin resistance (OGTT, HOMA), serum and liver TGs, hepatic MDA, serum AST, AST/ALT ratio and TNF-α compared to control. All treatments improved SOCS-3, FAS, MCD, TGF-β1 and leptin genes expression while only RES and FENO + RES groups showed an improvement in SREBP-1c expression. Adiponectin gene expression was improved only by RES. A decrease in body weight, HOMA, liver TGs, AST/ALT ratio and TNF-α were observed in all treatment groups. Liver index was increased in FENO and FENO + RES groups. Serum TGs was improved only by FENO treatment. Liver MDA was improved by RES and FENO + RES treatments. FENO + RES group showed an increase in liver GSH content.

Conclusion: When resveratrol was given with half the dose of fenofibrate it improved NASH-related fructose-induced disturbances in gene expression similar to a full dose of fenofibrate.

Keywords: Adiponectin; Fatty acid synthase; Fructose; Leptin; Malonyl CoA decarboxylase; Nonalcoholic steatohepatitis; Sterol regulatory element binding protein-1c; Suppressor of cytokine signaling-3; Transforming growth factor-β; Tumor necrosis factor-α.

Figure 1

Effect of fenofibrate (100 mg/kg) and resveratrol (70 mg/kg) alone and in combination (half doses) on the body weight (A) and percentage of liver index (B) in fructose-induced NASH in rats. Values are means ± SE (SE was omitted in Figure 1A body weight for clarification). n = 8-12 rats. The significance of the difference between means was tested by ANOVA followed by Tukey Kramer multiple comparisons test. ^aP < 0.05 vs control; ^bP < 0.05 vs FED; ^cP < 0.05 vs FENO; ^dP < 0.05 vs RES. FED: Fructose enriched diet; FENO: Fenofibrate; RES: Resveratrol; Liver index: Liver weight/body weight.

Figure 2

Effect of fenofibrate (100 mg/kg) and resveratrol (70 mg/kg) alone and in combination (half doses) on OGTT (A), blood glucose (B), insulin (C) and HOMA (D) in fructose-induced NASH in rats. Values are mean ± SE (SE was omitted in Figure 2A OGTT for clarification). n = 8-12 rats.The significance of the difference between means was tested by ANOVA followed by Tukey Kramer multiple comparisons test. ^aP < 0.05 vs control; ^bP < 0.05 vs FED. FED: Fructose enriched diet; FENO: Fenofibrate; RES: Resveratrol; OGTT: Oral glucose tolerance test; HOMA: Homeostasis model of assessment.

Figure 3

Effect of fenofibrate (100 mg/kg) and resveratrol (70 mg/kg) alone and in combination (half doses) on serum TGs (A) and liver TGs (B) in fructose-induced NASH in rats. Values are mean ± SE, n = 8-12 rats. The significance of the difference between means was tested by ANOVA followed by Tukey Kramer multiple comparisons test. ^aP < 0.05 vs control; ^bP < 0.05 vs FED; ^cP < 0.05 vs FENO. FED: Fructose enriched diet; FENO: Fenofibrate; RES: Resveratrol; TGs: Triglycerides.

Figure 4

Effect of fenofibrate (100 mg/kg) and resveratrol (70 mg/kg) alone and in combination (half doses) on liver MDA (A), GSH (B) and SOD (C) in fructose-induced NASH in rats. Values are mean ± SE, n = 8-12 rats. The significance of the difference between means was tested by ANOVA followed by Tukey Kramer multiple comparisons test. ^aP < 0.05 vs control; ^bP < 0.05 vs FED; ^cP < 0.05 vs FENO; ^dP < 0.05 vs RES. FED: Fructose enriched diet; FENO: Fenofibrate; RES: Resveratrol; MDA: Malonaldehyde; GSH: Glutathione; SOD: Superoxide dismutase.

Figure 5

Effect of fenofibrate (100 mg/kg) and resveratrol (70 mg/kg) alone and in combination (half doses) on serum AST (A), ALT (B), AST/ALT ratio (C) and serum TNF-α (D) in fructose-induced NASH in rats. Values are mean ± SE, n = 8-12 rats. The significance of the difference between means was tested by ANOVA followed by Tukey Kramer multiple comparisons test. ^aP < 0.05 vs control; ^bP < 0.05 vs FED; ^cP < 0.05 vs FENO. FED: Fructose enriched diet; FENO: Fenofibrate; RES: Resveratrol; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; TNF-α: Tumor necrosis factor-α.

Figure 6

Effect of fenofibrate (100 mg/kg) and resveratrol (70 mg/kg) alone and in combination (half doses) on hepatic genes expression: SOCS-3 (A), SREBP-1c (B), FAS (C), MCD (D) and TGF-β1 (E) in fructose-induced NASH in rats. Gene expression (level of mRNA) was expressed in arbitrary units based on calculation expression level relative to the internal standard. Values are mean ± SE, n = 8-12 rats.The significance of the difference between means was tested by ANOVA followed by Tukey Kramer multiple comparisons test. ^aP < 0.05 vs control; ^bP < 0.05 vs FED; ^cP < 0.05 vs FENO; ^dP < 0.05 vs RES. FED: Fructose enriched diet; FENO: Fenofibrate; RES: Resveratrol; SOCS-3: Suppressor of cytokine signalling-3; SREBP-1c: Sterol regulatory element binding protein-1c; FAS: Fatty acid synthase; MCD: Malonyl Co-A decarboxylase.

Figure 7

Effect of fenofibrate (100 mg/kg) and resveratrol (70 mg/kg) alone and in combination (half doses) on adipose leptin (A) and adiponectin (B) genes expression in fructose-induced NASH in rats. Gene expression (level of mRNA) was expressed in arbitrary units based on calculation expression level relative to the internal standard. Values are mean ± SE, n = 8-12 rats. The significance of the difference between means was tested by ANOVA followed by Tukey Kramer multiple comparisons test. ^aP < 0.05 vs control; ^bP < 0.05 vs FED; ^cP < 0.05 vs FENO; ^dP < 0.05 vs RES. FED: Fructose enriched diet; FENO: Fenofibrate; RES: Resveratrol.

Figure 8

Representative photomicrographs of liver sections of control (A), FED (Bs), fenofibrate (100 mg/kg) (Cs), resveratrol (70 mg/kg) (Ds) and fenofibrate + resveratrol (half doses) (E) in fructose-induced NASH in rats. A: Control rat liver showing a central vein (C), hepatocytes (H) arranged in the form of plates separated from each other by irregular blood sinusoids (S). B1: Fructose-fed rat liver showing cellular infiltration (arrows) near the central vein (C); B2: Fructose-fed rat liver showing pyknotic hepatic nuclei (arrowheads), cytoplasmic vacuolations (V) and apoptotic hepatic cells (arrows); B3: Fructose-fed rat liver showing central vein (C) with macrovesicular steatosis (MS) compressing hepatic nuclei to the right side of the vein, and cytoplasmic vacuolations (V), pyknotic nuclei (P) and binucleated hepatic cells to the left side of the vein; C1: Fenofibrate-treated rat liver showing nearly normal liver. A central vein (C), hepatocytes (H) arranged in the form of plates separated from each other by irregular blood sinusoids (S); C2: Fenofibrate-treated rat liver showing cytoplasmic vacuolations (arrowheads) of hepatic cells; D1: Resveratrol-treated rat liver showing macrovesicular steatosis (MS); D2: Resveratrol-treated rat liver showing vacuolations (V) of the cytoplasm of the hepatic cells; E1: Fenofibrate + Resveratrol-treated rat liver showing macrovesicular steatosis (MS) not compressing hepatic nuclei; E2: Fenofibrate + Resveratrol-treated rat liver showing macrovesicular steatosis (MS) and vacuolations (V) within the cytoplasm of the hepatic cells (HE × 400).

Figure 9

Effect of fenofibrate (100 mg/kg) and resveratrol (70 mg/kg) alone and in combination (half doses) on steatosis area in fructose-induced NASH in rats. Values are mean ± SE, n = 400 images. The significance of the difference between means was tested by ANOVA followed by Tukey Kramer multiple comparisons test. ^aP < 0.05 vs control; ^bP < 0.05 vs FED; ^cP < 0.05 vs FENO; ^dP < 0.05 vs RES. FED: Fructose enriched diet; FENO: Fenofibrate; RES: Resveratrol.