Comparative Effects of Pterostilbene and Its Parent Compound Resveratrol on Oxidative Stress and Inflammation in Steatohepatitis Induced by High-Fat High-Fructose Feeding

Abstract

Different studies have revealed that oxidative stress and inflammation are crucial in NAFLD (Non-alcoholic fatty liver disease). The aim of this study is to analyze whether pterostilbene and resveratrol are able to either avoid or delay the progression of non-alcoholic liver steatosis towards steatohepatitis. This has been performed by examining their effects on oxidative stress, inflammation, fibrosis and pre-carcinogenic stages. Rats were distributed into five experimental groups and were fed with either a standard diet or a high-fat high-fructose diet, supplemented or not with pterostilbene (15 or 30 mg/kg/d) or resveratrol (30 mg/kg/d), for 8 weeks. Liver histological analysis was carried out by haematoxylin-eosin staining. Serum and hepatic oxidative stress-related parameters were assessed using spectrophotometry, and the expression of genes related to inflammation, fibrosis and cancer by qRT-PCR. The dietary model used in this study led to the development of steatohepatitis, where rats displayed oxidative stress, inflammation and ballooning, although not fibrosis. It also modified the expression of hepatocarcinoma-related genes. The results show, for the first time, that pterostilbene was able to partially prevent these alterations, with the exception of changes in hepatocarcinoma-related genes, mainly at 30 mg/kg/d. Pterostilbene was more effective than its parent compound resveratrol, probably due to its high bioavailability and higher anti-oxidant and anti-inflammatory activities, attributable to its different chemical structure.

Keywords: (poly)phenols; hepatocarcinoma; inflammation; liver steatohepatitis; liver steatosis; oxidative stress; pterostilbene; rat; resveratrol.

Figure 1

Representative H&E stained histological liver samples (magnification ×20). (A) healthy liver from CC group, (B) liver from HFHF group showing moderate inflammation and ballooning degeneration, (C) liver from PT15 group showing moderate inflammation and ballooning degeneration, (D) liver from PT30 group showing mild inflammation and ballooning degeneration and (E) liver from RSV30 group showing mild–moderate inflammation and ballooning degeneration. White arrows indicate inflammation.

Figure 2

Lipid peroxidation (MDA content), ORAC, rGSH, activities of SOD, CAT and GPx, and gene expression of Nox4 and P22phox in liver from the rats fed with a control diet (CC group), a high-fat high-fructose diet (HFHF group), a high-fat high-fructose diet supplemented with pterostilbene at a dose of 15 mg/kg body weight/d (PT15 group), a high-fat high-fructose diet supplemented with pterostilbene at a dose of 30 mg/kg body weight/d (PT30 group) or a high-fat high-fructose diet supplemented with resveratrol at a dose of 30 mg/kg body weight/d (RSV30 group) (A–H). Values are presented as mean ± SEM. Bars not sharing common letters a–c are significantly different (p < 0.05). MDA: malondialdehyde; ORAC: Oxygen Radical Antioxidant Capacity; rGSH: reduced glutathione; SOD: superoxide dismutase; CAT: catalase; GPx: glutathione peroxidase; Nox4: NADPH oxidase 4; P22phox: cytochrome B-245 alpha chain.

Figure 3

Il-1β, Tnfα and Crp mRNA levels (A), F4/80, Mcp1 and Cd206 mRNA levels (B) and Tlr-2, Tlr-4 and MyD88 mRNA levels (C) in the livers from the rats fed with a control diet (CC group), a high-fat high-fructose diet (HFHF group), a high-fat high-fructose diet supplemented with pterostilbene at a dose of 15 mg/kg body weight/d (PT15 group), a high-fat high-fructose diet supplemented with pterostilbene at a dose of 30 mg/kg body weight/d (PT30 group) or a high-fat high-fructose diet supplemented with resveratrol at a dose of 30 mg/kg body weight/d (RSV30 group). Values are presented as mean ± SEM. Bars not sharing common letters are significantly different (p < 0.05). Il-1b: interleukin 1b; Tnfα: tumor necrosis factor α; Crp: C-reactive protein; Mcp1: monocyte chemoattractant protein 1; Cd206: mannose receptor C; Tlr-2 and Tlr-4: toll-like receptor 2 and 4; MyD88: myeloid differentiation factor 88 MyD88.

Figure 3

Il-1β, Tnfα and Crp mRNA levels (A), F4/80, Mcp1 and Cd206 mRNA levels (B) and Tlr-2, Tlr-4 and MyD88 mRNA levels (C) in the livers from the rats fed with a control diet (CC group), a high-fat high-fructose diet (HFHF group), a high-fat high-fructose diet supplemented with pterostilbene at a dose of 15 mg/kg body weight/d (PT15 group), a high-fat high-fructose diet supplemented with pterostilbene at a dose of 30 mg/kg body weight/d (PT30 group) or a high-fat high-fructose diet supplemented with resveratrol at a dose of 30 mg/kg body weight/d (RSV30 group). Values are presented as mean ± SEM. Bars not sharing common letters are significantly different (p < 0.05). Il-1b: interleukin 1b; Tnfα: tumor necrosis factor α; Crp: C-reactive protein; Mcp1: monocyte chemoattractant protein 1; Cd206: mannose receptor C; Tlr-2 and Tlr-4: toll-like receptor 2 and 4; MyD88: myeloid differentiation factor 88 MyD88.

Figure 4

Acta2, Col1a1, Timp1 and Tgfβ1 mRNA levels in the livers from the rats fed with a control diet (CC group), a high-fat high-fructose diet (HFHF group), a high-fat high-fructose diet supplemented with pterostilbene at a dose of 15 mg/kg body weight/d (PT15 group), a high-fat high-fructose diet supplemented with pterostilbene at a dose of 30 mg/kg body weight/d (PT30 group) or a high-fat high-fructose diet supplemented with resveratrol at a dose of 30 mg/kg body weight/d (RSV30 group). Values are presented as mean ± SEM. Bars not sharing common letters a,b are significantly different (p < 0.05). Acta2: α-smooth muscle actin; Col1a1: collagen 1; Timp: tissue inhibitor of matrix metalloproteases; Tgfβ1: transforming growth factor beta1.

Figure 5

Gstm2, Tp53, Tert, Sirt1, Birc5, Tgf β1, Alex3 and Fgf21 mRNA levels in the livers from the rats fed with a control diet (CC group), a high-fat high-fructose diet (HFHF group), a high-fat high-fructose diet supplemented with pterostilbene at a dose of 15 mg/kg body weight/d (PT15 group), a high-fat high-fructose diet supplemented with pterostilbene at a dose of 30 mg/kg body weight/d (PT30 group) or a high-fat high-fructose diet supplemented with resveratrol at a dose of 30 mg/kg body weight/d (RSV30 group) (A–H). Values are presented as mean ± SEM. Bars not sharing common letters a,b are significantly different (p < 0.05). Alex3: ARM protein lost in epithelial cancers on chromosome X 3; Birc5: survivin; Fgf21: fibroblast growth factor 21; Gstm2: glutathione s-transferase mu2; Sirt1: sirtuin 1; Tert: telomerase reverse transcriptase; Tgf β1: transforming growth factor beta 1; Tp53: tumor protein P53.