Int J Pharm Pharm Sci, Vol 7, Issue 2, 417-424Original Article


EXPERIMENTAL EVIDENCES FOR THE PROMISING THERAPEUTIC ROLE OF VITIS VINIFERA SEED EXTRACT AGAINST NONALCOHOLIC STEATOHEPATITIS

HANAA H. AHMED1*, AHMED M. SALEM2, MOHAMED R. MOHAMED2, ABDELAATY A SHAHAT3, WAGDY KB KHALIL4, SAFAA H. MOHAMED1

1Hormones Department, National Research Centre, Dokki, Cairo, Egypt, 2Biochemistry Department, Faculty of Science, Ain Shams University, Cairo, Egypt, 3Phytochemistry Department, National Research Centre, Dokki, Cairo, Egypt, 4Cell Biology Department, National Research Centre, Dokki, Cairo, Egypt.
Email: hanaaomr@yahoo.com

Received: 23 Nov 2014 Revised and Accepted: 15 Dec 2014

Abstract

Objective: The present study was planned to investigate the possible therapeutic effect of methanolic extract of Vitis vinifera seeds on high fat diet-induced nonalcoholic steatohepatitis (NASH) in adult female Wistar rats.

Methods: The animals were divided into four groups, (G1)was served as healthy control group and the other three groups received high fat diet for 32 weeks for induction of NASH and then assigned as follow: (G2) in which the animals bearing NASH were left untreated, (G3) in which the animals bearing NASH were treated with Vitis vinifera seed extract in a dose of 0.28g/kg b. wt (GSH) and (G4) in which the animals bearing NASH were treated with Vitis vinifera seed extract in a dose of 0.14g/kg b. wt (GSL).

Results: The results revealed significant increase in serum ALT activity, plasma glucose, insulin levels, serum resistin, NF-κB, TNF-α, HGF levels, hepatic TNF-α and HGF gene expression levels. While, serum albumin, adiponectin levels and hepatic adiponectin gene expression level were decreased significantly in NASH group. Conversely, treatment of NASH groups with GSH or GSL resulted in significant decrease in serum ALT activity, plasma glucose, insulin levels, serum resistin, NF-κB, TNF-α, HGF levels, hepatic TNF-α and HGF gene expression levels. However, serum albumin, adiponectin levels and hepatic adiponectin gene expression level were increased significantly as a consequence of treatment with GSH or GSL.

Conclusion: The efficacy of Vitis vinifera extract against NASH might be attributed to its strong hepatoprotective potential and powerful anti-inflammatory activity in addition to its potent role in ameliorating insulin resistance indices.

Keywords: Nonalcoholic steatohepatitis, Vitis vinifera, Inflammation, insulin resistance, Rats.

INTRODUCTION

Nonalcoholic fatty liver disease (NAFLD) covers a wide spectrum of liver pathology from steatosis alone, through the necroinflammatory disorder of nonalcoholic steatohepatitis (NASH) to cirrhosis and liver cancer. NAFLD/NASH is mostly related with visceral adiposity, obesity, diabetes mellitus 2 and metabolic syndrome. Pathogenetic concepts of NAFLD include over nutrition and under activity, insulin resistance (IR) and genetic factors. The prevalence of NAFLD has been estimated to be 17-33% in some countries; NASH may be present in about 1/3 of such cases, while 20-25% of NASH cases could progress to cirrhosis. NAFLD/NASH is not a benign disease, progressive liver biopsy has shown histological progression of fibrosis in 32%, the estimated rate of cirrhosis development is 20% and a liver--related death is 12% over 10 years. No treatment has scientifically proved to ameliorate NAFLD or to avoid its progression. The various therapeutic alternatives are aimed at interfering with the risk factors involved in the pathogenesis of the disorder in order to prevent the progression to end-stage liver disease [1].

Underlying mechanisms responsible for the progression to NASH are till unclear. A ‘‘two-hit’’ model has been proposed; peripheral insulin-resistance may represent the ‘‘first hit’’ in the pathogenesis of NASH. Combined hyperglycemia and hyperinsulinemia promote de novo lipid synthesis and structural defects in mitochondria within hepatocytes. Moreover, insulin-resistance of adipose tissue leads to an enhanced free fatty acid flux to the liver that contributes to steatosis. Steatotic hepatocytes may be vulnerable to a ‘‘second hit’’ induced by cytokines such as tumor necrosis factor-α (TNF-α) and oxidative stress, which lead to the development of steatohepatitis and fibrosis [2]. Oxidative stress, in particular, plays an essential role by causing peroxidation of lipids in the hepatocyte membrane to initiate liver fibrosis. Lipid peroxidation and the generation of free radicals can result in cellular death and hepatic necrosis and contribute to impaired cellular function. Antioxidant supplements could potentially protect cellular structures against oxidative stress [3].

The genetic contribution to NASH remains to be fully elucidated, but wide-ranging research is ongoing. Indeed, the broad range of NASH phenotype found in individuals with similar metabolic characteristics points to a complex genetic contribution [4].

Grape seeds are waste products of the grape (Vitis vinifera) juice industry. These seeds contain lipid, protein and carbohydrates. The grape seed extract is rich in bioactive phytochemicals that possess inhibitory activity on the fat-metabolizing enzymes; pancreatic lipase and lipoprotein lipase. Grape seed compounds include flavonoids, polyphenols, anthocyanins, proanthocyanidins, procyanidines and the stilbene derivative resveratrol. Grape seeds extract has been reported to have a broad spectrum of pharmacological and therapeutic effects such as antioxidative, anti-inflammatory, and antimicrobial activities, as well as cardioprotective, hepatoprotective, and neuroprotective potentials [5]. The underlying mechanisms of the anti-inflammatory property of grape seed extracts are relevant to oxygen free radical scavenging, anti lipid peroxidation, inhibiting the formation of inflammatory cytokines, alterations in cell membrane receptors, intracellular signaling pathway proteins and modulation of gene expression [6]. The current study was constructed to investigate the possible therapeutic role of Vitis vinifera methanolic extract in management of nonalcoholic steatohepatitis induced in the experimental model with special reference to its mode of action.

Materials and Methods

Plant materials

Vitis vinifera seed (Grape seed) was purchased at the Harraz Herbal Drugstore (Cairo, Egypt). The plant was identified by Professor Ibrahim El-Garf, Botany Department, Faculty of Science, Cairo University.

Preparation of grape (Vitis vinifera) seed methanolic extract

Four kilograms (kgs) of dried seeds were ground to fine powder, then extracted with 80% methanol at room temperature for three times. The extract was then concentrated in a vacuum evaporator to obtain the crude aqueous methanolic extract (23% from the weight of the dried seeds).

Experimental animals

The present study was conducted on forty adult female Wistar rats weighing 120 - 150g obtained from the Animal House Colony of the National Research Centre, Cairo, Egypt. The animals were maintained on the standard laboratory diet and water ad libitum for two weeks before starting the experimental protocol. All animals received adequate human care and use according to the guidelines for Animal Experiments which were approved by the Ethical Committee of Medical Research, National Research Centre, Egypt. Nonalcoholic steatohepatitis (NASH) was induced in rats by using high fat diet which provided 30% of its energy from fat, 35% from carbohydrate and 35% from protein (casein) for 32 weeks. Supplements of vitamins and minerals were also included [7].

Experimental protocol

The animals were divided into four groups, ten animals each: (G1) Healthy control group which was fed ad-libitum with an isocaloric regular rat chow [8], (G2) Nonalcoholic steatohepatitis (NASH group) which was fed ad-libitum with high fat diet for 32 weeks and left untreated [7], (G3) NASH group treated orally with 0.28g/kg b. wt of Vitis vinifera seed extract daily for 8 weeks and was assigned as NASH+GSH and (G4) NASH group treated orally with 0.14g/kg b. wt of Vitis vinifera seed extract daily for 8 weeks and was assigned as NASH+GSL. The selected doses of Vitis vinifera seed extract were calculated from the chronic toxicity study for Vitis vinifera seed extract (data not shown).

At the completion of this round (40 weeks), the rats were fasted overnight and the blood samples were collected from the retro orbital venous plexus under diethyl ether anaesthesia [9]. Each blood sample was divided into two tubes, the first tube contains anticoagulant for separation of plasma and the second tube free from any anticoagulant for separation of serum. All blood samples were centrifuged using cooling centrifuge at 1800 xg for ten min. to obtain plasma and serum which were stored at -20°C until analysis. After blood collection, all animals were rapidly sacrificed and the liver tissues were dissected, immediately frozen in liquid nitrogen and stored at −80 °C prior to extraction for molecular genetics study.

Biochemical assays

Serum alanine transaminase (ALT) activity was estimated colorimetrically using kit purchased from Quimica Clinica Aplicada S. A. Co., Spain, according to the method of Reitman and Frankel [10]. Serum albumin level was measured colorimetrically using kit purchased from Stanbio Laboratory. Boerne, Texas, USA, according to the method of Dumas and Biggs [11]. Plasma glucose level was measured colorimetrically using kit purchased from Stanbio Laboratory, Boerne, Texas, USA, according to the method of Howantiz and Howantiz [12]. Plasma insulin level was estimated by enzyme linked immunosorbent assay (ELISA) procedure using DRG kit (Germany) according to the method of Temple et al. [13]. The homeostasis model assessment of basal insulin resistance (HOMA-IR) was used to calculate the index from the product of the fasting concentration of plasma glucose (mmol/L) and plasma insulin (mU/ml) divided by 22.5 according to the method of Duncan et al. [14]. Serum adiponectin concentration was measured by ELISA technique using kit purchased from Assay Pro, USA, according to the method of Pannacciulli et al. [15]. Serum resistin concentration was determined by ELISA technique using kit purchased from Glory Science Co., Ltd, Veterans Blvd, Suite, USA, according to the method of Schaffler et al. [16]. Serum nuclear factor kappa B-p56 (NF-κB-p56) concentration was determined by ELISA procedure using kit purchased from Glory Science Co., Ltd, Veterans Blvd, Suite, USA, according to the manufacturer’s instructions. Serum TNF-α concentration was measured by ELISA procedure using kit purchased from Ray Biotech Co., Georgia, USA, according to the method of Brouckaert et al. [17]. Serum hepatocyte growth factor (HGF) level was quantified by ELISA procedure using kit purchased from Glory Science Co., Ltd, Veterans Blvd, Suite, USA, according to the method of Plum et al. [18].

Semi-Quantitative real time PCR (sqRT-PCR) detection of adiponectin, TNF-α and HGF gene expression

Isolation of total RNA

Total RNA was isolated from liver tissue of female rats by the standard TRIzol® reagent extraction method (Invitrogen, USA). Then, the complete Poly(A)+ RNA was reverse transcribed into cDNA in a total volume of 20 µl using RevertAidTM First Strand cDNA Synthesis Kit (MBI Fermentas, Germany). An amount of total RNA (5µg) was used with a reaction mixture, termed as master mix (MM). The MM consisted of 50 mM MgCl2, 5x reverse transcription (RT) buffer (50 mM KCl; 10 mM Tris-HCl; pH 8.3; 10 mM of each dNTP, 50 µM oligo-dT primer, 20 U ribonuclease inhibitor (50 kDa recombinant enzyme to inhibit RNase activity) and 50 U M- MuLV reverse transcriptase.

Reverse transcription reaction and semi-quantitative real time PCR

The RT reaction was carried out at 25°C for 10 min, followed by 1 h at 42°C, and the reaction was stopped by heating for 5 min at 99°C. Afterwards, the reaction tubes containing RT preparations were flash-cooled in an ice chamber until being used for DNA amplification through semi-quantitative real time-polymerase chain reaction [19]. An iQ5-BIO-RAD Cycler (Cepheid, USA) was used to determine the rat cDNA copy number. PCR reactions were set up in 25 μl reaction mixtures containing 12.5 μl 1× SYBR® Premix Ex TaqTM (TaKaRa, Biotech. Co. Ltd.), 0.5 μl 0.2 M forward primer, 0.5 μl 0.2 M reverse primer, 6.5 μl distilled water, and 5 μl of cDNA template. Primer sequences were as follows: adiponectin: F: 5′-AGA TGG CAC CCC TGG TGA G-3′ and R: 5′-GGG TAC TCC GGT TTC ACC G-3′ [20], TNF-α: F: 5′-TCG TAG CAA ACC ACC AAG CA-3′ R: 5′-CCC TTG AAG AGA ACC TGG GAG TA-3′ [21]; HGF: F: 5′-AGCTCAGAACCGACCGGCTTGCAACAGGAT-3′, R: 5’-TTACCAATGATG CAATTTCTAATATAGTCT-3′ [22] and β-actin: F: 5′-CTGTCTGGCG GCACCACCAT-3′, R: 5′-GCAACTAAGTCATAGTCCGC-3′ [23]. The reaction program was allocated to 3 steps. First step was at 95.0°C for 3 min. Second step consisted of 40 cycles in which each cycle was divided into 3 steps: (a) denaturation at 95.0°C for 15 sec; (b) annealing at 60.4, 55.0, 60 and 60°C for 30, 5, 30 and 30 sec for (adiponectin, TNF-α, HGF and β-actin genes, respectively and (c) extension at 72.0°C for 30 sec. The gene expression level was calculated as follows:

First: the amplification efficiency (Ef) was calculated from the slope of the standard curve using the following formulae [24]

Ef = 10-1/slope

Efficiency (%) = (Ef – 1) x 100

The relative quantification of the target to the reference was determined by using the ΔCT method if E for the target (adiponectin, TNF-α and HGF) and the reference primers (β-actin) are the same:

Ratio (reference/ target gene) = EfCT(reference)–CT(target)

Statistical analysis

In the present study, all results were expressed as Mean + S. E of the mean. Data were analyzed by one way analysis of variance (ANOVA) using the Statistical Package for the Social Sciences (SPSS) program, version 14 followed by least significant difference (LSD) to compare significance between groups [25]. Difference was considered significant when P value was > 0.05.

Results

Table (1) illustrated the effect of treatment with different doses of Vitis vinifera seed extract on serum ALT activity and albumin level in NASH experimental model. The NASH group showed significant elevation (P<0.05) in serum ALT activity (71.7 %) associated with the significant decline (P<0.05) in serum albumin level (-68.75%) in comparison with the healthy control group. However, treatment with Vitis vinifera seed extract (0.28 or 0.14 g/kg b. wt) produced significant reduction in serum ALT activity (-36.01% and -39.88%, respectively) when compared with the untreated NASH group. Serum albumin level was increased significantly (P<0.05) by 173.3% and 180% in NASH group treated with Vitis vinifera seed extract (0.28 or 0.14 g/kg b. wt, respectively) as compared to the untreated NASH group.

Table 1: Effect of treatment with different doses of Vitis vinifera seed extract on serum ALT activity and albumin level in NASH experimental model

Albumin

(g/dl)

ALT

(U/L)

Parameters

Groups
4.8 ± 0.05 35.4± 3.2 Healthy control group (G1)

1.5 ± 0.04a

(-68.75%)

60.8±1.7a

(71.7%)

 NASH group (G2)

4.1 ± 0.08b

(173.3 %)

38.9±1.5b

(-36.01%)

 NASH + GSH group (0.28g/kg b. wt) (G3)

4.2 ± 0.06b

(180%)

36.6±1.4b

(-39.88%)

 NAFLD + GSL group (0.14g/kg b. wt) (G4)

a: Significant change at P < 0.05 in comparison with the healthy control group, b: Significant change at P < 0.05 in comparison with NASH group, (%): percent difference with respect to the corresponding control value

The effect of treatment with different doses of Vitis vinifera seed extract on plasma glucose and insulin levels in NASH experimental model is depicted in table (2). The data showed that NASH group reveals the significant increase (P<0.05) in plasma glucose and insulin levels (80.45% and 58%, respectively) in comparison with the healthy control group. Meanwhile, treatment of NASH group with Vitis vinifera seed extract in the different doses resulted in the significant decrease (P<0.05) in plasma glucose and insulin levels (-41.19% and -25.67%, respectively) for Vitis vinifera seed extract (0.28g/kg b. wt) and (-43.05% and -25.76%, respectively) for Vitis vinifera seed extract (0.14 g/kg b. wt) as compared to the untreated NASH group.

The data in table (2) also showed that the induction of NASH produced significant elevation (P<0.05) in insulin resistance (185.12%) in comparison with the healthy control group. On the other hand, treatment of NASH groups with different doses of Vitis vinifera seed extract produced significant depletion (P<0.05) in insulin resistance (-56.31%) for Vitis vinifera seed extract (0.28g/kg b. wt) and (-57.76%) for Vitis vinifera seed extract (0.14 g/kg b. wt) when compared with the untreated NASH group.

Table 2: Effect of treatment with different doses of Vitis vinifera seed extract on plasma glucose and insulin levels and insulin resistance value in NASH experimental model

insulin resistance value

Insulin

mU/ml

Glucose

(mg/dl)

Parameters

Groups
2.89±0.02 14.05±0.5 83.4 ± 1.2 Healthy control group (G1)

8.24±0.05a

(185.12%)

22.20±0.8b

(58%)

150.5 ± 2.3a

(80.45%)

 NASH group (G2)

3.6±0.03b

(-56.31%)

16.50±1.1b

(-25.67%)

88.5±2.0b

(-41.19%)

 NASH + GSH group (0.28g/kg b. wt) (G3)

3.48±0.03b

(-57.76%)

16.48±1.2b

(-25.76%)

85.7± 1.1b

(-43.05%)

 NAFLD + GSL group (0.14g/kg b. wt) (G4)

a: Significant change at P < 0.05 in comparison with the healthy control group, b: Significant change at P < 0.05 in comparison with NASH group, (%): percent difference with respect to the corresponding control value

The results in table (3) represented the effect of treatment with different doses of Vitis vinifera seed extract on serum adiponectin and resistin levels in NASH experimental model. Significant increase (P<0.05) in serum resistin level (79.2%) accompanied with the significant decrease (P<0.05) in serum adiponectin level (-33.6%) was recorded in NASH group as compared to the healthy control group. However, treatment of NASH group with Vitis vinifera seed extract (0.28 or 0.14 g/kg b. wt) resulted in the significant decrease (P<0.05) in serum resistin level (-40.5% and -42.39%, respectively).

The opposite was observed regarding serum adiponectin level, Vitis vinifera seed extracts produced significant increase (P<0.05) (36.23% for 0.28g/kg b. wt and 44.92% for 0.14g/kg b. wt) when compared with the untreated NASH group.

Table 3: Effect of treatment with different doses of Vitis vinifera seed extract on serum adiponectin and resistin levels in NASH experimental model

Resistin

(pg/ml)

Adiponectin

(ng/ml)

Parameters

Groups
30.8 ± 0. 25 10.4 ± 0.42 Healthy control group (G1)

55.2 ± 0.37a

(79.2 %)

6.9 ± 0.2a

(-33.6 %)

 NASH group (G2)

32.8 ± 0.29b

(-40.5 %)

9.4 ± 0.3b

(36.23%)

 NASH + GSH group (0.28g/kg b. wt) (G3)

31.5 ± 0.3b

(-42.93 %)

10.00 ± 0.2b

(44.92 %)

 NAFLD + GSL group (0.14g/kg b. wt) (G4)

a: Significant change at P < 0.05 in comparison with the healthy control group, b: Significant change at P < 0.05 in comparison with NASH group, (%): percent difference with respect to the corresponding control value.

The data in table (4) showed the effect of treatment with different doses of Vitis vinifera seed extract on serum NF-κBp56, TNF-α and HGF levels in NASH experimental model. Significant increase (P<0.05) in serum NF-κBp56, TNF-α and HGF levels (103.1%, 67.6% and 88.5%, respectively) was demonstrated in NASH group in comparison with the healthy control group.

On the other side, treatment of NASH group with Vitis vinifera seed extract (0.28 or 0.14 g/kg b. wt) induced significant decrease (P<0.05) in serum NF-κB p56 level (-44.6% and -49.2 %, respectively), TNF-α level (-34.49% and -37.16%, respectively) and HGF level (-34.77% and -37.25%, respectively) as compared to the untreated NASH group.

Table 4: Effect of treatment with different doses of Vitis vinifera seed extract on serum NF-κBp56, TNF-α and HGF levels in NASH experimental model

HGF

(ng/L)

TNF-α

(pg/ml)

NF-κB p56

(ng/ml)

Parameters

Groups
102.40 ± 1.6 58.1 ± 1.8 0.64 ± 0.04 Healthy control group (G1)
193.05 ± 1.4a(88.5 %)

97.4± 1.2a

(67.6 %)

1.3 ± 0.1a

(103.1 %)

 NASH group (G2)
125.70 ± 1.5b(-34.77 %)

63.8 ± 1.5b

(-34.49 %)

0.72± 0.02b

(-44.6 %)

 NASH + GSH group (0.28g/kg b. wt) (G3)
120.60 ± 1.1b(-37.52 %)

61.2 ± 1.2b

(-37.16 %)

0.66± 0.03b

(-49.2%)

 NAFLD + GSL group (0.14g/kg b. wt) (G4)

a: Significant change at P < 0.05 in comparison with the healthy control group, b: Significant change at P < 0.05 in comparison with NASH group, (%): percent difference with respect to the corresponding control value

The results in table (5) illustrated the effect of treatment with different doses of Vitis vinifera seed extract on hepatic adiponectin, TNF-α and HGF gene expression levels in NASH experimental model. The NASH group showed significant down-regulation (P<0.05) in hepatic adiponectin gene expression level by -35.66 % in comparison with the healthy control group. While, treatment of NASH group with Vitis vinifera seed extracts (0.28 or 0.14 g/kg b. wt) resulted in significant up-regulation (P<0.05) in hepatic adiponectin gene expression level by 50% and 52.17%, respectively, in comparison with the untreated NASH group. NASH group showed significant up-regulation (P<0.05) in hepatic TNF-α and HGF gene expression levels by 110.52 % and 54.28 %, respectively, when compared with the healthy control group. Meanwhile, treatment of NASH group with Vitis vinifera seed extract resulted in significant down-regulation (P<0.05) in hepatic TNF-α and HGF gene expression levels (-40.83% and -31.48%, respectively) for 0.28g/kg b. wt, and (-50% and -33.59%, respectively) for 0.14 g/kg b. wt as compared to the untreated NASH group.

Table 5: Effect of treatment with different doses of Vitis vinifera seed extract on hepatic adiponectin, TNF-α and HGF gene expression levels in NASH experimental model

Relative expression of HGF gene

(HGF/β-actin)

Relative expression of TNF-α gene

(TNF-α/β-actin)

Relative expression of adiponectin gene

(adiponectin/β-actin)

Parameters

Groups
1.05 ± 0.006 0.57 ± 0.003 1.43 ± 0.007 Healthy control group (G1)

1.62± 0.006a

(54.28 %)

1.2± 0.003a

(110.52 %)

0.92 ± 0.004a

(-35.66 %)

 NASH group (G2)

1.11 ± 0.005b

(-31.48 %)

0.71 ± 0.002b

(-40.83 %)

1.38± 0.002b

(50 %)

 NASH + GSH group (0.28g/kg b. wt) (G3)

1.07 ± 0.004b

(-33.59 %)

0.6 ± 0.002b

(-50 %)

1.40± 0.003b

(-52.17%)

 NAFLD + GSL group (0.14g/kg b. wt) (G4)

a: Significant change at P < 0.05 in comparison with the healthy control group, b: Significant change at P < 0.05 in comparison with NASH group, (%): percent difference with respect to the corresponding control value

Discussion

The results of the present study revealed the significant increase in serum ALT activity in NASH group which is in agreement with Hooper et al. [26]. Both aminotransferases (AST and ALT) are highly concentrated in the liver and the increasing serum ALT activity reflects the state of hepatocyte damage in NASH patients [27]. It has been demonstrated that oxidative stress status of the liver achieved in steato hepatitis promotes hepatocellular damage by inducing (1) severe oxidative alteration of biomolecules, with loss of their functions and impairment of cell viability; and (2) sustained activation of redox-sensitive transcription factors, such as NF-κB and AP-1, with consequent up-regulation of the expression of proinflammatory mediators at the Kupffer cell level [28]. A growing body of evidence supported the possibility that insulin resistance associated with adipose tissue inflammation and hepatic microvascular dysfunction may actually contribute to the development and/or progression of ALT activity in serum [29].

The present data showed significant reduction in serum albumin level in NASH group. This finding agrees with that of Farrelland Larter [30]. Albumin, the predominant protein in circulation, is mainly synthesized in the liver and it binds to a number of ligands. Amirtharaj et al. [31] stated that oxidative stress can cause modification of serum albumin, which influences ligand binding properties of the protein. This impact of oxidative stress might be the reason for decreasing serum albumin level in NASH group as shown in the current study. Many studies demonstrated that oxidative stress is implicated in conditions such as nonalcoholic fatty liver disease (NAFLD) and non alcoholic steatohepatitis (NASH) due to the increasing levels of free fatty acids with consequent increase in free radicals levels [32].

Treatment of NASH group with Vitis vinifera seed extract induced significant decrease in serum ALT activity associated with significant elevation in serum albumin level. The antioxidant and free radical scavenging property of Vitis vinifera seed extract may be responsible for this effect. Polyphenols present in the Vitis vinifera seed extract, especially (+)-catechin, possess strong antioxidant activity and they can also inhibit the oxidation of plasma lipids. Moreover, (-)-epicatechin is able to scavenge hydroxyl radicals, peroxyl radicals and superoxide radicals [33]. Furthermore, procyanidins, other active compounds of Vitis vinifera seed extract, are reported to have potent antioxidant activity both in vitro and in vivo [34]. Flavonoids represent the main active components in Vitis vinifera seed extracts; these compounds exhibit hepatoprotective activity due to their powerful antioxidant and anti-inflammatory properties in vitro and in vivo. These lead compounds can modulate mammalian enzyme activity, enhance intracellular signaling and strengthen membranes [35]. In addition, the active principles in the Vitis vinifera seed extract can prevent insulin resistance, improve insulin sensitivity and increase the activity of antioxidant enzymes [36]. In view of the above mentioned properties of Vitis vinifera seed extract, it could be suggested that this extract is able to maintain the structure integrity of hepatocytes and decrease serum ALT activity in the treated groups. This means that Vitis vinifera seed extract could restore the liver functions in NASH bearing rats. The elevated serum albumin level in Vitis vinifera seed extract treated groups in the present study supported the hypothesis of the ability of this extract to restore the liver to nearly normal condition. This effect of Vitis vinifera seed extract could be attributed to its bioavailability in addition to its potent antioxidant and anti-inflammatory effects [37]. Shin and Moon [5] reported that in chronic liver diseases, the serum albumin levels are reduced due to protein synthesis disruption in the liver and the Vitis vinifera treatment blocked this action via hepatoprotective effects of grape skin and seeds where the damaged hepatocytes are potent sources of reactive oxygen intermediates and Vitis vinifera has potent antioxidant activity. Thus, the hepatoprotective effects of Vitis vinifera seed extract possibly ascribed to the increasing serum albumin level in the treated groups.

In view of our results, there was significant increase in plasma glucose and insulin levels as well as insulin resistance value in NASH group as compared to the healthy control group. These results are in agreement with Yun et al. [38]. One of the most important functions of the liver is the homeostasis of blood glucose levels by taking up and storing glucose as glycogen (glycogenesis), breaking this down to glucose when needed (glycogenolysis) and forming glucose from noncarbohydratesources such as amino acids (gluconeogenesis) [39]. The presence of high lipolytic activity due to fat accumulation results in high free fatty acids mobilization to the liver. The elevated fatty acids flux to the liver accelerates gluconeogenesis and decreases the effect of insulin on peripheral glucose disposal [40] leading to the increased plasma glucose level. NAFLD is commonly associated with an increased risk of developing Type 2 diabetes and treatable features of insulin resistance such as dyslipidaemia and dysglycaemia. A growing body of evidence demonstrated that NAFLD exacerbates hepatic insulin resistance that often precedes glucose intolerance [41]. Hepatic insulin resistance confers the failure of insulin to stimulate glycogen synthesis and to suppress gluconeogenesis, thus, insulin fails to down-regulate hepatic glucose production [30]. Hyperinsulinemia is believed to play a key role in the pathogenesis of nonalcoholic steatohepatitis (NASH) and patients with NASH had hepatic insulin sensitivity. Decreased hepatic insulin clearance develops with the increase in liver fat accumulation. It appears to be largely driven by hepatic steatosis, whereas steato hepatitis is more closely associated with reduced whole-body insulin clearance [42].

In the healthy state, insulin stimulation of insulin receptor substrate-1(IRS-1) in muscle cells leads to activation of intracellular phosphoinositide 3-kinase (PI3K) which in turn activates glucose transporter-4 (GLUT) allowing glucose entry in the muscles. In NAFLD state, the increase in intracellular metabolites (diacylglycerol), leads to decreased phosphorylation of IRS-2 via activation of a serine kinase cascade [43]. This in turn, results in decreasing PI3K activity with consequent decrease in glucose cellular entry. It has been proposed that a process analogous to that in muscle cells occurs in hepatic cells [44].

Treatment of NASH groups with Vitis vinifera seed extract produced significant decrease in plasma glucose and insulin levels as well as insulin resistance value as shown in the current results. It has been demonstrated that the existence of antioxidant compounds in Vitis vinifera seed extract is responsible for this effect. These compounds play an important role in the restoration of pancreatic β-cell functions and thus they can decrease blood glucose level [45]. Vitis vinifera seed extract has been found to increase glucose uptake in β-cells under high-glucose conditions [46]. It has been reported that the reduction of plasma glucose levels is driven as a consequence of the enhanced adiponectin expression due to treatment with procyanidins [47].

Castell-Auví et al. [48] have shown that luteolin, one of the flavonoids present in Vitis vinifera seed extract decreases the gene expression of sterol regulatory element-binding protein 1c (Srebf1) and fatty acid synthase (Fasn) and it enhances the phosphorylation of AMP-activated protein kinase (AMPK), a transcription factor that activates the expression of several genes involved in free fatty acids and triglycerides synthesis, as well as it affects other components of the regulatory machinery of lipid metabolism, leading to a decrease in intracellular lipid levels. Thus, the decreased plasma insulin level due to Vitis vinifera seed extract administration could be at least in part explained by the lipid-lowering effect of this extract. In line with these evidences, Cedó et al. [46] stated that procyanidin causes a reduction in the Homeostasis Model Assessment for Insulin Resistance (HOMA-IR) index, suggesting the improvement of insulin resistance. Terra et al. [49] demonstrated that procyanidins significantly reduce blood glucose and insulin levels, as well as insulin resistance (HOMA-IR) values.

The present data revealed the significant decrease in serum adiponectin level as well as significant down-regulation of hepatic adiponectin gene expression level in NASH group. It has been demonstrated that normally adiponectin is found in relatively high circulating levels, but it is decreased in patients with NASH and in clinical manifestations associated with insulin resistance such as metabolic syndrome (MS) and type 2 diabetes mellitus [50]. In addition, plasma adiponectin levels have been found to be correlated inversely with the markers of systemic oxidative stress. And many studies hypothesized that oxidative stress contributes in conditions such as NAFLD and NASH due to the increased levels of free fatty acids and the consequent increased levels of free radicals [32]. It has been demonstrated that in cultured adipocytes, under oxidative stress condition, the suppressed mRNA expression and secretion of adiponectin are detected [51]. This explains the decreased gene expression level of hepatic adiponectin in NASH group as shown in the present results. Moreover, it has been found that in NASH, the local effects of adiponectin are limited due to (i) decreased adiponectin mRNA expression and (ii) decreased mRNA expression of hepatic adipoRII [20]. Furthermore, it was reported that TNF-α and adiponectin suppress each other’s production and are also able to antagonise each other’s action [52]. Therefore, the reduced adiponectin mRNA expression might be partially due to the suppressive effect of elevated TNF-α expression in NASH [53].

The current results demonstrated that serum resistin level shows the significant increase in NASH group. Pagano et al. [54] reported that patients with NASH are characterized by high serum resistin level. A major target organ of resistin is the liver, where resistin induces insulin resistance and increases glucose production. Therefore, resistin is related to hepatic fat content and insulin resistance [55] Thus, it has been suggested that resistin may contribute to hepatic steatosis via promoting insulin resistance and the increased resistin levels in NASH patients are related to histological severity of the disease [56]. Underlying liver damage and the progression of pure fatty liver to NASH and fibrosis, the hepatic stellate cells produce a variety of cytokines, including resistin. Moreover, it has been reported that a genetic polymorphism in the promoter region of the resistin gene may be an independent predictor of circulating resistin in humans. Hence, it is not possible to exclude that a gene polymorphism(s) of resistin which may be responsible for the high resistin levels in NAFLD patients [54].

Treatment of NASH group with Vitis vinifera seed extract led to significant increase in serum adiponectin level as well as up-regulation in hepatic adiponectin gene expression level. Décordé et al. [57] reported that Vitis vinifera seed markedly prevents hyperglycemia, reduces hyperinsulinemia, and increases adiponectin levels in hamsters fed a high-fat diet. Adiponectin is an adipokine that exerts a potent insulin-sensitizing effect by binding to its receptors such as AdipoR1 and AdipoR2, leading to activation of AMPK and PPARa thus, it consequently activates glucose uptake and reduces insulin resistance [58]. Therefore, Vitis vinifera seed extract may induce the significant increase in serum adiponectin level [36]. Thus, the observed increase in serum adiponectin levels could be directly related to the effect of Vitis vinifera seed extract consumption on adipocytes to secrete adiponectin or indirectly as a result of the reduction of body weight as the secretion of adiponectin, is increased by weight loss [59]. Moreover, Georgiev et al. [60] demonstrated that regular consumption of resveratrol-rich Vitis vinifera extract increased serum adiponectin, due to antioxidative effect beside its antiinflammatory activity.

The local enhancement in adiponectin gene expression level produced by the treatment of NASH groups with Vitis vinifera seed extract might be partly attributed to the reduced proinflammatory cytokine expression levels [35]. As hepatic adiponectin expression has been found to be regulated by different factors including the proinflammatory cytokines [49]. Moreover, grape seed extract contains bioactive compounds "procyanidins" which could enhance the gene expression of the antiinflammatory molecule "adiponectin" [35]. Furthermore, Vitis vinifera seed extract contains another bioactive compound called "resveratrol" which reverses mRNA expression of adiponectin [61]. Additionally, Meeprom et al. [62] reported that oxidative stress inhibits the gene expression of adiponectin. Vitis vinifera seed extract contains procyanidins and monomeric flavanols such as (+‏)-catechin and (-)-epicatechin, which have potent antioxidant activities. Thus, we could suggest that these powerful antioxidants present in Vitis vinifera seed extract help to replenish the decreased hepatic adiponectin gene expression level.

The present findings revealed that the treatment of NASH groups with Vitis vinifera seed extract results in significant reduction in serum resistin level. This result could be explained by ability of Vitis vinifera seed extract with its active compounds "procyanidins" to reduce insulin resistance. As it has been reported that resistin is involved in the pathogenesis of insulin resistance and the reduced resistin concentration by procyanidins in Vitis vinifera seed might contribute to a lower risk for the development of NASH [63].

The present data indicated that serum TNF-α, NF-κB and HGF levels display significant elevation in NASH group as compared to the healthy control group. Additionally, significant up-regulation in hepatic TNF-α and HGF gene expression levels has been recorded in NASH group when compared to the healthy control group. These findings could be explained by the oxidative stress and stimulation of kupffer cell as well as stellate cell to secrete inflammatory cytokines [64]. Mitochondrial dysfunction contributes to oxidative stress and NASH is associated with mitochondrial structural defects. Oxidative stress causes various types of functional and structural damage and commonly increases TNF-α production in NASH and enhanced expression of TNF-α mRNA [65]. A preponderance of evidence supports the hypothesis that the amounts of TNF-α mRNA and TNF-α released by adipose tissue are enhanced in NASH [66]. Moreover, it has been found that NAFLD patients have elevated plasma level of lipopolysaccharide-binding protein (LBP) which is further increased in patients with NASH. This increased LBP level is related to a rise in TNF-α gene expression in the hepatic tissue of NASH patients which supports a role of endotoxemia in the development of steatohepatitis [67]. It has been stated that the appearance of liver pathology is correlated with disruption of adipokines, evidence of insulin resistance, increased hepatic oxidative stress, and increased hepatic TNF-α expression. The commonalities between different experimental models suggest that these pathways are of fundamental importance underlying the development of liver pathology in NASH [68].

High oxidative stress status in the liver of NAFLD patients with steatohepatitis leads to modulation of Kupffer cell function, through activation of transcription factors such as NF-κB [69]. NF-κB, then translocates from the cytoplasm to the nucleus to activate the expression of inflammatory cytokines perturbing the inflammatory cycle [70]. This mechanism explains the significant increase in serum NF-κB level in NASH group in the present study.

The significant increase in serum HGF level in NASH group in our study is consistent with that of Koutsogiannis et al. [71]. HGF mRNA expression in nonparenchymal cells has been found to be increased in NASH patients [72]. In NASH, the activation of Kupffer cells and macrophages within liver tissue increased the production of NF-κB which induces the expression of HGF and consequently its level [4].

Treatment of NASH group with Vitis vinifera seed extract resulted in significant reduction in serum TNF-α, NF-κB and HGF levels in concomitant with significant down-regulation in TNF-α and HGF gene expression levels. Vitis vinifera seed contains procyanidins which could reduce the TNF-α plasma level as a result of the down-regulation of TNF-α gene expression level [73]. Furthermore, procyanidin could reduce TNF-α protein level in the mesenteric adipose tissue, indicating that the local inflammation in this tissue was prevented [35]. Weisberg et al. [74] demonstrated that adipose tissue macrophages are responsible for almost all adipose tissue TNF-α expression. Procyanidins has been reported to reduce the level of macrophage present in adipose tissue. Therefore, the inhibition of the cytokine expression including TNF-α might be due to a decrease in the number of macrophages [35].

Procyanidin has been found to decrease NF-κB level in liver, which is directly associated with the decreased hepatic expression of the other inflammatory molecules as TNF-α and C-reactive protein. Also, procyanidins have been hypothesized to reduce MCP-1 secretion in adipocytes, partially, due to the diminished TNF-α levels which might be a consequence of the inhibitory effects of procyanidins on NF-κB activation [35].

Furthermore, the existence of procyanidins in Vitis vinifera seed plays an important role in preventing low-grade inflammation in vivo, by adjusting adipose tissue cytokine imbalance, enhancing antiinflammatory molecules and diminishing proinflammatory mediators beside the down-regulation of TNF-α and IL-6 expression i. e., these compounds could inhibit NF-kB cascade [49]. Additionally, Vitis vinifera seed contains resveratrol which could reduce the expression of the inflammatory mediators (TNF-α, IL-6, and COX-2) in mature adipocytes, inhibit TNF- α -activated NF-kB signaling, and reverse the TNF-α secretion [61].

Serum HGF levels have been reported to be strongly associated with insulin resistance and all components of metabolic syndrome. Insulin resistance and metabolic syndrome are the most specific findings of NASH, and HGF might be the possible messenger of the disease between adipocytes and hepatocytes [75]. The antioxidant and free radical scavenging ability of Vitis vinifera seed extract active constituents (polyphenols) especially (+)-catechin may be responsible for the decreasing serum level of HGF in NASH-treated groups. (+) – catechin possesses antioxidant activity by inhibiting the oxidation of plasma lipids.

Moreover, (-)-epicatechin is able to scavenge hydroxyl radicals, peroxyl radicals, superoxide radicals [33]. Thus Vitis vinifera seed extract can ameliorate insulin resistance and improve insulin sensitivity [36]. Besides that, Vitis vinifera seed has been found to block NF-κB expression [35] and in turn it can indirectly inhibit the stimulant of HGF expression and consequently its level [4].

CONCLUSION

The current results provide a clear experimental evidence for the promising role of Vitis vinifera seed extract in management of nonalcoholic steatohepatitis (NASH). The active contituents of Vitis vinifera namely flavonoids, polyphenols, anthocyanins, proanthocyanidins, procyanidines and the stilbene derivative resveratrol may be responsible for this effect through their hepatoprotective activity, antioxidant capacity and anti-inflammatory property. Beside that, Vitis vinifera seed extract has proved its potential in modulating insulin resistance status associated with nonalcoholic steatohepatitis. Therefore, Vitis Vinifera seed extract could have potent therapeutic implication in chronic liver diseases accompanied with insulin resistance and severe inflammation.

Acknowledgment

Work is partially supported by Science and Technology Development Fund (STDF), Egyptian Academy of Scientific Research and Technology “ID# 245”.

CONFLICT OF INTERESTS

Declared None

References

  1. Raszeja-Wyszomirska J, Lawniczak M, Marlicz W, Miezyńska-Kurtycz J, Milkiewicz P. Non-alcoholic fatty liver disease--new view. Pol Merkur Lekarski 2008;24:568-71.
  2. Tran A, Gual P. Non-alcoholic steatohepatitis in morbidly obese patients. Clin Res Hepatol Gastroenterol 2013;37:17-29.
  3. Khoshbaten M, Aliasgarzadeh A, Masnadi K, Tarzamani MK, Farhang S, Babaei H, et al. N-acetylcysteine improves liver function in patients with non-alcoholic Fatty liver disease. Hepat Mon 2010;10:12-6.
  4. Preiss D, Sattar N. Non-alcoholic fatty liver disease: an overview of prevalence, diagnosis, pathogenesis and treatment considerations. Clin Sci 2008;115:141–50.
  5. Shin M, Moon J. Effect of dietary supplementation of grape skin and seeds on liver fibrosis induced by dimethylnitrosamine in rats. Nutr Res Pract 2010;4:369-74.
  6. Kris-Etherton PM, Lefevre M, Beecher GR, Gross MD, Keen CL, Etherton TD. Bioactive compounds in nutrition and health-research methodologies for establishing biological function: the antioxidant and anti-inflammatory effects of flavonoids on atherosclerosis. Annu Rev Nutr 2004;24:11–38.
  7. Tipoe GL, Ho CT, Liong EC, Leung TM, Lau TYH, Fung ML, Nanji AA. Voluntary oral feeding of rats not requiring a very high fat diet is a clinically relevant animal model of non-alcoholic fatty liver disease (NAFLD). Histol Histopathol 2009;24:1161-9.
  8. Reeves PG, Nielsen FH, Fahey GC. AIN-93 purified diets for laboratory rodents: final report of the american institution ad hoc. writing committee on the reformulation of the AIN-76A rodent diet. J Nutr 1993;123:1939-51.
  9. Sandford HS. Method for obtaining venous blood from the orbital sinus of the rat or mouse. Sci 1954;119:100.
  10. Reitman S, Frankel S. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am J Clin Path 1957;28:56-63.
  11. Dumas BT, Biggs HG. In Standard Methods of Clinical Chemistry. Vol. 7. Acadamic Press: New York; 1972. p. 175.
  12. Howanitz PJ, Howantiz JH. In Clinical Diagnosis and Management by Laboratory Methods, 17th ed. JB Henry, Ed. WB Saunders Philadelphia; 1984. p. 168.
  13. Temple R, Clark PMS, Hales CN. Measurement of insulin secretion in type 2 diabetes: problems and pitfalls. Diabetic Med 1992;9:503-12.
  14. Duncan MH, Singh BM, Wise PH, Carter G, Alaghband ZJ. A simple measure of insulin resistance. Lancet 1995;346:120-1.
  15. Pannacciulli N, Vettor R, Milan G, Granzotto M, Catucci A, Federspil G, et al. Anorexia nervosa is characterized by increased adiponectin plasma levels and reduced nonoxidative glucose metabolism. J Clin Endocrinol Metab 2003;88:1748-52.
  16. Schäffler A, Büchler C, Müller-Ladner U, Herfarth H, Ehling A, Paul G, et al. Identification of variables influencing resistin serum levels in patients with type 1 and type 2 diabetes mellitus. Horm Metab Res 2004;36:702-7.
  17. Brouckaert P, Libert C, Everaerdt B, Takahashi N, Cauwels A, Fiers W. Tumor necrosis factor, its receptors and the connection with interleukin-1 and interleukin-6. Immunobiol 1993;187:317-29.
  18. Plum L, Lin HV, Dutia R, Tanaka J, Aizawa KS. The obesity susceptibility gene carboxypeptidase e links foxO1 signaling in hypothalamic pro–opiomelanocortin neurons with regulation of food intake. Nat Med 2009;15(10):1195-201.
  19. Ali FKh, El-Shafai SA, Samhan FA, Khalil WKB. Effect of water pollution on expression of immune response genes of Solea aegyptiaca in Lake Qarun. Afr J Biotechnol 2008;7:1418-25.
  20. Kaser S, Moschen A, Cayon A, Kaser A, Crespo J, Pons-Romero F, et al. Adiponectin and its receptors in non-alcoholic steatohepatitis. Gut 2005;54:117–21.
  21. Liu J, Lei D, Waalkes MP, Beliles RP, Morgan DL. Genomic analysis of the rat lung following elemental mercury vapor exposure. Toxicol Sci 2003;74:174–81.
  22. Hata J, Ikeda E, Uno H, Asano S. Expression of hepatocyte growth factor mRNA in rat liver cirrhosis induced by N-nitrosodimethylamine as evidenced by in situ RT-PCR. J Histochem Cytochem 2002;50:1461–8.
  23. Gaytan F, Barreiro ML, Caminos JE, Chopin LK, Herington AC, Morales C, et al. Expression of ghrelin and its functional receptor, the type 1a growth hormone secretagogue receptor, in normal human testis and testicular tumors. J Clin Endocrinol Metab 2004;89:400-9.
  24. Bio-Rad Laboratories Inc: Real-Time PCR applications guide. Bull 2006;5279:101.
  25. Armitage P, Berry G. Comparison of several groups. In: statistical method in medical research 2nd Ed. Blockwell significant publication. Oxford; 1987. p. 186-213.
  26. Hooper AJ, Adams LA, Burnett JR. Genetic determinants of hepatic steatosis in man. J Lipid Res 2011;52:593-617.
  27. Yang RZ, Park S, Reagan WJ, Goldstein R, Zhong S, Lawton M, et al. Alanine aminotransferase isoenzymes: molecular cloning and quantitative analysis of tissue expression in rats and serum elevation in liver toxicity. Hepatol 2009;49(2):598–607.
  28. Videla L. Oxidative stress signaling underlying liver disease and hepatoprotective mechanisms. World J Hepatol 2009;1:72-8.
  29. Jacobs M, Van Greevenbroek MMJ, Van der Kallen CJH, Ferreira I, Feskens EJM, Jansen EHJM, et al. The association between the metabolic syndrome and alanine amino transferase is mediated by insulin resistance via related metabolic intermediates (the Cohort on Diabetes and Atherosclerosis Maastricht [CODAM] study). Metab 2011;60:969–75.
  30. Farrell GC, Larter CZ. Nonalcoholic fatty liver disease: from steatosis to cirrhosis: liver failure and liver disease. Hepatol 2006;43:99-112.
  31. Amirtharaj GJ, Natarajan SK, Mukhopadhya A, Zachariah UG, Hegde SK, Kurian G, et al. Fatty acids influence binding of cobalt to serum albumin in patients with fatty liver. Biochim Biophys Acta 2008;1782:349–54.
  32. Lewis GF, Carpentier A, Adeli K, Giacca A. Disordered fat storage and mobilization in the pathogenesis of insulin resistance and type 2 diabetes. Endocr Rev 2002;23:201–29.
  33. Yilmaz Y, Toledo RT. Major flavonoids in grape seeds and skins: antioxidant capacity of catechin, epicatechin, and gallic acid. J Agric Food Chem 2004;52:255–60.
  34. Simonetti P, Ciappellano S, Gardana C, Bramati L, Pietta P. Procyanidins from Vitis vinifera seeds: in vivo effects on oxidative stress. J Agric Food Chem 2002;50:6217–21.
  35. Terra X, Pallarés V, Ardèvol A, Bladé C, Fernández-Larrea J, Pujadas G, et al. Modulatory effect of grape-seed procyanidins on local and systemic inflammation in diet-induced obesity rats. J Nutr Biochem 2011;22:380-7.
  36. Suwannaphet W, Meeprom A, Yibchok-Anun S, Adisakwattana S. Preventive effect of grape seed extract against high-fructose diet-induced insulin resistance and oxidative stress in rats. Food Chem Toxicol 2010;48:1853–7.
  37. El-Shafeey M, El-Adawi H, Al-Azhari D, Abd El-Wahab A, Abdel-Mohsen. Synergistic effect of milk thistle and grape seed extracts on the recovery of fumonisin b1 toxicity in rats. Egypt Acad J Biol Sci 2012;4:63-85.
  38. Yun JW, Cho YK, Park JH, Kim HJ, Park D, Sohn C, et al. Abnormal glucose tolerance in young male patientswith nonalcoholic fatty liver disease. Liver Int 2009;29:1478-3223.
  39. Giannini EG, Testa R, Savarino V. Liver enzyme alteration: a guide for clinicians. Can Med Assoc J 2005;172:367-79.
  40. Ginsberg HN, Stalenhoef AF. The metabolic syndrome targeting dyslipidemia to reduce coronary risk. J Cardiovasc Risk 2003;10:121-8.
  41. Adams LA, Angulo P. Recent concepts in non-alcoholic fatty liver disease. Diabetic Med 2005;22:1129-33.
  42. Bril F, Lomonaco R, Orsak B, Ortiz-Lopez C, Webb A, Tio F, et al. Relationship between disease severity, hyperinsulinemia, and impaired insulin clearance in patients with nonalcoholic steatohepatitis. Hepatology 2014;59:2178-87.
  43. Jung HL, Kang HY. Effects of endurance exercise and high-fat diet on insulin resistance and ceramide contents of skeletal muscle in sprague-dawley rats. Korean Diabetes J 2010;34:244–52.
  44. Wilcox G. Insulin and insulin resistance. Clin Biochem Rev 2005;26:19–39.
  45. Rostamian V, Shakeri F, Estakhr J. The effect of hydro-alcoholic extract of grape seed (Vitis vinifera) on sugar and lipids in serum of diabetic rats. Res J Biol Sci 2011;6:547-9.
  46. Cedó L, Castell-Auví A, Pallarès V, Blay M, Ardévol A, Pinent M. Grape seed procyanidin extract improves insulin production but enhances bax protein expression in cafeteria-treated male rats. Int J Food Sci 2013;2013:7.
  47. Cho SY, Park PJ, Shin HJ, Kim YK, Shin DW, Shin ES, et al. (-)-Catechin suppresses expression of Kruppel-like factor 7 and increases expression and secretion of adiponectin protein in 3T3-L1 cells. Am J Physiol Endocrinol Metab 2007;292:E1166-72.
  48. Castell-Auví A, Cedó L, Pallarès V, Blay M, Pinent M, Ardévol A. Grape seed procyanidins improve β-cell functionality under lipotoxic conditions due to their lipid-lowering effect. J Nutr Biochem 2013;24:948–53.
  49. Terra X, Ardèvol A, Bladé C, Fernández-Larrea J, Pujadas G, Salvadó J, et al. Inhibitory effects of grape seed procyanidins on foam cell formation in vitro. J Agric Food Chem 2009;57:2588-94.
  50. Pagano C, Soardo G, Esposito W. Plasma adiponectin is decreased in nonalcoholic fatty liver disease. Eur J Endocrinol 2005;152:113–8.
  51. Furukawa S, Fujita T, Shimabukuro M, Iwaki M, Yamada Y, Nakajima Y, et al. Increased oxidative stress in obesity and its impact on metabolic syndrome. J Clin Invest 2004;114:1752–61.
  52. Yokota T, Oritani K, Takahashi I, Ishikawa J, Matsuyama A, Ouchi N, et al. Adiponectin, a new member of the family of soluble defense collagens, negatively regulates the growth of myelomonocytic progenitors and the functions of macrophages. Blood 2002;96:1723–32.
  53. Crespo J, Cayón A, Fernández-Gil P, Hernández-Guerra M, Mayorga M, Domínguez-Díez A, et al. Gene expression of tumor necrosis factor alpha and TNF-receptors, p55 and p75, in nonalcoholic steatohepatitis patients. Hepatology 2001;34:1158–63.
  54. Pagano C, Soardo G, Pilon C, Milocco C, Basan L, Milan G, et al. Increased serum resistin in nonalcoholic fatty liver disease is related to liver disease severity and not to insulin resistance. J Clin Endocrinol Metab 2006;91:1081–6.
  55. Bajaj M, Suraamornkul S, Hardies LJ, Pratipanawatr T, DeFronzo RA. Plasma resistin concentration, hepatic fat content, and hepatic and peripheral insulin resistance in pioglitazone-treated type II diabetic patients. Int J Obes Relat Metab Disord 2004;28:783–9.
  56. Murad A, Hassan H, Husein H, Ayad A. Serum resistin levels in nonalcoholic fatty liver disease and their relationship to severity of liver disease. J Endocrinol Metab Diabetes South Africa 2010;15:53-6.
  57. Décordé K, Teissèdre PL, Sutra T, Ventura E, Cristol JP, Rouanet JM. Grape-seed procyanidins prevent low-grade inflammation by modulating cytokine expression in rats fed a high-fat diet. Mol Nutr Food Res 2009;53:659–66.
  58. Kadowaki T, Yamauchi T, Kubota N, Hara K, Ueki K, Tobe K. Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome. J Clin Invest 2006;116:1784–92.
  59. Barona J, Blesso CN, Andersen CJ, Park Y, Lee J, Fernandez ML. Grape consumption increases anti-inflammatory markers and upregulates peripheral nitric oxide synthase in the absence of dyslipidemias in men with metabolic syndrome. Nutr 2012;4:1945–57.
  60. Georgiev V, Ananga A, Tsolova V. Recent advances and uses of grape flavonoids as nutraceuticals. Nutr 2014;6:391–415.
  61. Gonzalez-Castejon M, Rodriguez-Casado A. Dietary phytochemicals and their potential effects on obesity: a review. Pharmacol Res 2011;64:438–55.
  62. Meeprom A, Sompong W, Suwannaphet W, Yibchok-anun S, Adisakwattana S. Grape seed extract supplementation prevents high-fructose diet-induced insulin resistance in rats by improving insulin and adiponectin signaling pathways. Br J Nutr 2011;106:1173–81.
  63. Graf D, Seifert S, Jaudszus A, Bub A, Watzl B. Anthocyanin-Rich juice lowers serum cholesterol, Leptin, and Resistin and Improves plasma fatty acid composition in fischer rats. PLoS ONE 2013;8(6):e66690.
  64. Wigg AJ, Roberts-Thomson IC, Dymock RB, McCarthy PJ, Grose RH, Cummins AG. The role of small intestinal bacterial overgrowth, intestinal permeability, endotoxaemia, and tumor necrosis factor a in the pathogenesis of non-alcoholic steatohepatitis. Gut 2001;48:206–11.
  65. Lieber CS, Leo MA, Mak KM, Xu Y, Cao Q, Ren C, et al. Model of nonalcoholic steatohepatitis. Am Soc Clin Nutr 2004;79:502–9.
  66. Fain JN, Bahouth SW, Madan AK. TNF [alpha] release by the nonfat cells of human adipose tissue. Int J Obes Relat Metab Disord 2004;28:616–22.
  67. Ruiz AG, Casafont F, Crespo J, Cayón A, Mayorga M, Estebanez A, et al. Lipopolysaccharide-binding protein plasma levels and liver TNF-alpha gene expression in obese patients: evidence for the potential role of endotoxin in the pathogenesis of non-alcoholic steatohepatitis. Obes Surg 2007;17:1374-80.
  68. Baumgardner JN, Shankar K, Hennings L, Badger TM, Ronis MJJ. A new model for nonalcoholic steatohepatitis in the rat utilizing total enteral nutrition to overfeed a high-polyunsaturated fat diet. Am J Physiol Gastrointest Liver Physiol 2008;294:G27–G38.
  69. Baeuerle PA, Henkel T. Function and activation of NF-kB in the immune system. Ann Rev Immunol 1994;12:141–79.
  70. Tian N, Moore RS, Braddy S, Rose RA, Gu JW, Hughson MD, et al. Interactions between oxidative stress and inflammation in salt-sensitive hypertension. Am J Physiol Heart Circ Physiol 2007;293:H3388–H3395.
  71. Koutsogiannis D, Summers K, George B, Adams P, Marotta P, Chakrabarti S. Identification of serum biomarkers in end stage liver disease. Open Biomarkers J 2010;3:1-6.
  72. Fausto N, Laird AD, Webber EM. Role of growth factors and cytokines in hepatic Regeneration. FASEB J 1995;9:1527-36.
  73. Zhong JY, Cong HQ, Zhang LH. Inhibitory effects of grape procyanidins on free radical-induced cell damage in rat hepatocytes in vitro. World J Gastroenterol 2007;13:2752-5.
  74. Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Ferrante Jr. Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest 2003;112:1796–808.
  75. Balaban YH, Sumer H, Simsek H, Us D, Tatar G. Metabolic syndrome, non-alcoholic steatohepatitis (NASH), and hepatocyte growth factor (HGF). Ann Hepatology 2006;5:109-14.