Resveratrol inhibits nonalcoholic fatty liver disease in rats

Nonalcoholic fatty liver disease (NAFLD) is characterized by histological changes similar to those seen in subjects with alcoholic hepatitis but in whom alcohol intake is absent or poorly significant. Factors promoting NAFLD development include obesity and diabetes. From 69% to 100% of patients with NAFLD are obese [1]. NAFLD prevalence in severe obesity is greater than 30% [2, 3]. Because of all these reasons, it is important to have animal models that allow us to study the effect of different substances on NAFLD.

Polyphenols have a variety of biological functions, including antioxidant, anti-inflammatory, and anticancer effects [4]. Resveratrol is a phytoalexin polyphenolic compound occurring in various plants, including grapes, berries, and peanuts. Multiple lines of compelling evidence suggest its beneficial effects on neurological, hepatic, and cardiovascular systems. The potential mechanisms responsible for its biological activities include downregulation of the inflammatory response through inhibition of the synthesis and release of pro-inflammatory mediators, modification of eicosanoid synthesis, inhibition of Kupffer cells and adhesion molecules, inhibition of inducible nitric oxide synthase and cyclooxygenase-2 (COX-2) via its inhibitory effects on nuclear factor (kappa)B (NF-kB) or the activator protein-1 (AP-1) (5,6). In our previous studies, resveratrol was seen to decrease the liver lesions and transaminase elevations caused by alcohol in mice [7].

Our purpose now was to investigate first whether resveratrol decreased hepatic steatosis in an animal model of steatosis, and second, whether this therapeutic approach resulted in a decrease in tumor necrosis factor α (TNF-α) production and oxidative stress.

Rat weight significantly increased in the control group (221 ± 10 g to 355 ± 16 g), remained similar in the group with fatty liver disease (222 ± 12 g to 226 ± 14 g), and decreased in the resveratrol group (218 ± 9 g to 201 ± 11 g). Hepatic index was 4.47 ± 0.63, 4.38 ± 0.34, and 3.9 ± 0.31 in the steatosis, resveratrol, and control groups respectively.

NAFLD represents a wide spectrum of disorders, the hallmark of which is hepatic steatosis. NAFLD was considered a benign condition, but is now increasingly recognized as a major cause of liver-related morbidity and mortality. Insulin resistance is the basis for accumulation of free fatty acids and triglyceride storage in hepatocytes or steatosis. Oxidative stress from steatotic hepatocytes leads to lipid peroxidation, impaired mitochondrial and peroxisomal oxidation of fatty acids, and cytokine release [11]. Endotoxins and endotoxin-inducible cytokines, particularly TNF-α, are required for the pathogenesis of NAFLD in experimental animals. Therefore the TNF-α plays an important role in NAFLD [12, 13].

In our study, hepatic steatosis was significantly decreased in rats treated with resveratrol. This effect was associated to a decreased TNF-α production. Different studies have shown that resveratrol decreases TNF-α production. We therefore think that the decreased liver damage in a model of liver steatosis could be related to its anti-TNF-α effect. Other studies [14‐16] have shown a relationship between NAFLD and TNF-α levels. Our study shows that TNF-α levels were increased in both the steatosis and resveratrol groups as compared to the control group, suggesting that TNF-α is an important factor in liver damage occurring in NAFLD. However, TNF-α levels were lower in the resveratrol group than in the steatosis group. We therefore think that TNF-α is an important factor for development of this condition. In agreement with other authors [17‐19], we did not consider administration of anti-TNF-α or use of another group given repeated TNF-α doses because adequate evidence was already available. Infliximab (anti-TNF-α) reverses the steatosis and the expression of the proinflammatory markers (TNF-α, IL6, IL-1B) and improves insulin signal trasduction in a model of steatosis in rats [20]. The presence of a control group (reference group) allows to compare the effects of the modified diet (high carbohydrate-fat free) on the liver (steatosis group) and the effect of the resveratrol (resveratrol group).

Bacterial translocation from the intestinal lumen to mesenteric lymph nodes is considered to be one of the main events in the pathogenesis of spontaneous bacterial peritonitis and other infections in cirrhosis. TNF-α is involved in the occurrence of bacterial translocation in rats with cirrhosis [21]. We therefore considered whether bacterial translocation acting as a stimulus for TNF-α production occurred in our model. Cultures of samples of portal blood, peripheral blood, and perihepatic lymph nodes were negative in all three groups tested, which rules out this mechanism as responsible for TNF-α elevation.

There are many models of NAFLD liver injuries in animals [22, 23]. In rats, cycles of feeding and fasting with hypertonic high calorie diets have been seen to induce fatty liver [8]. In this study, we used feeding and fasting cycles with a modified diet because it is a fast, easy procedure that results in pathological changes similar to those occurring in humans. In our model the deposit of fatty acids and triglyceride storage in hepatocytes is produced by insulin resistance [8, 11]. Insulin resistance is the basis for accumulation of free fatty acids and triglyceride storage in hepatocytes, and represents the "first hit" in the pathogenesis of NAFLD [11]. Body mass significantly decreased with the high carbohydrate-fat free diet and dietary restriction. This may be due to a metabolic imbalance of carbohydrate, protein, and fat. However, hepatic index was higher in the group with steatosis as compared to the resveratrol and control groups. Such higher hepatic index occurred despite the increased rat weight in the steatosis group, which means that resveratrol acts by decreasing fat accumulation in the liver and fat weight, and therefore decreases hepatic index.

ALT is a relatively liver-specific aminotransferase. Elevation of ALT activity in serum is the result of leakage from damaged cells and therefore reflects hepatocyte damage. Elevated transaminase levels correlated strongly with NAFLD [2]. ALT levels were significantly lower in the resveratrol group as compared to the control group (34 IU/L versus 40 IU/L) and similar to steatosis group levels. In the group treated with resveratrol, lower glucose levels and higher serum albumin levels as compared to untreated rats were also found. Studies have reported increases in serum TNF-α levels in humans with insulin resistance [24]. Hyperglycemia and insulin resistance are associated to the presence of NAFLD [2, 25]. Changes in plasma levels of markers predicting for the onset of diabetes occurred with a high carbohydrate-fat free diet [25]. Glucose levels were decreased in the resveratrol group [26], as occurred in our study. Other studies have also noted that resveratrol improved insulin sensitivity, lowered plasma glucose, and increased mitochondrial capacity in obese mice [27]. Insulin resistance was not analyzed in our study.

High hepatic MDA levels were found in the steatosis group, in agreement with other studies [28]. Resveratrol improved MDA levels. Oxidative stress is believed to play an important role in pathogenesis of NAFLD. It is likely to be involved in disease progression from steatosis to steatohepatitis and potentially cirrhosis. It has been shown that chronic oxidative stress, generated through oxidation of cytotoxic free fatty acids, may lead to cytokine upregulation and depletion of hepatic antioxidant levels [29, 30]. In addition, enhanced lipid peroxidation leads to the generation of by-products, such as MDA, which have been shown to further stimulate cytokine production. They are involved in hepatic stellate cell activation, fibrogenesis, and enhanced extracellular matrix protein deposition [28]. Resveratrol caused increased of hepatic antioxidant levels as superoxide dismutase, glutathione peroxidase and catalase and decreased nitric oxide synthase in the liver.

Fatty acid oxidation is an important source of reactive oxygen species in fatty livers. Some consequences of increased reactive oxygen species levels include an impaired protein stability, membrane destruction via lipid peroxidation, and release of proinflammatory cytokines (increased TNF-α levels) [31]. Reactive oxygen species may attack polyunsatured fatty acids and initiate lipid peroxidation within the cell, which results in MDA formation. Oxidants may not only act as toxic substances, but also as second messengers (activation of transcription factor NF-kB) [32]. Resveratrol increases insulin sensitivity and insulin-like growth factor-1 levels (IGF-1) [28]. Resveratrol would also act by decreasing lipid peroxidation and reactive oxygen species release, thereby decreasing inflammatory response and liver lesions [33, 34]

Recently, researches suggest that other mechanisms the resveratrol improve in NAFLD are by the activation of AMP-activated protein kinase and the activation of SIRT1 [35‐37].

In summary, our study shows that resveratrol decreases liver steatosis in rats and that its effect is mediated, at least partly, by TNF-α and antioxidant activities. In our model, bacterial translocation was not responsible for TNF-α elevation. Further studies are warranted to determine whether resveratrol decreases or prevents liver steatosis.