Vitamin C and Vitamin E in Prevention of Nonalcoholic Fatty Liver Disease (NAFLD) in Choline Deficient Diet Fed Rats

There are evidences that fatty liver, the most common hepatocellular change found in liver biopsies in humans, can play a role in the pathogenesis of chronic liver disease [1, 2]. Recently, some authors have considered that a more accurate denomination for this disease, which encloses the whole spectrum of fatty liver, nonalcoholic steatohepatitis (NASH) and eventually cirrhosis, is Nonalcoholic Fatty Liver Disease (NAFLD) [3, 4].

Although several predisposing factors have been related to NAFLD, such as obesity, diabetes, jejunoileal bypass, dyslipidemia, drugs and parenteral nutrition, the pathogenesis of NAFLD and its progression to fibrosis and chronic liver disease are still unclear [4]. As a consequence, the current treatment is largely conservative Some hypotheses have been implicated in the pathogenesis of NAFLD, mainly liver injury mediated either by oxidative stress [5, 6] or by endotoxins/cytokines [7‐9]. and hyperinsulinemia [10] Oxidative stress is considered to play a central role in the pathogenesis of NAFLD because the increased production of ROS is known to cause lipid peroxidation, followed by activation of the inflammatory response, and of stellate cells, leading to fibrogenesis [11‐13]. Lipid peroxidation usually leads to the formation of peroxyl radicals, which are the central species of the peroxidation chain reaction. The aim of this study was to evaluate whether this process can be prevented by antioxidant drugs, vitamin C or vitamin E using an animal model where NAFLD was induced by a choline deficient diet

We observed moderate macro- and microvesicular fatty change in the periportal zone in control and vitamin E-treated rats (Figure 1), whereas in vitamin C-treated rats did not develop liver steatosis in this model (Figure 2). These aspects were validated by SCHARLACH Rs stain (Figure 3 and 4). In both test groups, necroinflammatory activity was minimal and no fibrosis was seen in any of the groups. Following choline-deficient diet, the levels of AST and triglycerides were increased to a similar extent in vehicle, vitamin E or vitamin C-treated rats (Table 1). On the other hand, vitamin C prevented the rise of luminescence values in the animals fed a choline-deficient diet plus vitamin C (1080 ± 330 cpm/mg/minx10³) as compared to the animals fed a choline-deficient diet plus vitamin E or control (2247 ± 790; 2020 ± 407 cpm/mg/minx10³) (p < 0.05) (Table 2).

In this study, we managed to induce periportal macro- and microvesicular steatosis in Wistar rats fed a choline-deficient diet and evaluated the role of antioxidant drugs (vitamin C or vitamin E) in the prevention of fatty liver. Our results showed a marked effect of vitamin C in the prevention of NAFLD in Wistar rats fed a choline-deficient diet.

A number of studies showed that parameter of oxidative stress are increase and levels of endogenous antioxidants such as vitamin E and glutathione (GSH) are decreased in NAFLD [18, 19]. However, the use of antioxidants in the prevention of NAFLD is not established yet. The protective effect of vitamin C observed in our study is in line with the suggested role of oxidative stress in the pathogenesis of NAFLD. On the other hand, vitamin E another antioxidant drug neither reduced oxidative stress and nor prevented the development of NAFLD in this study.

Choline-deficient diet is a classical general model of NAFLD. However, this model does not lead liver inflammation and therefore does not produce a typical histological picture of NASH [20]. Confirming this finding, Teramoto et al did not observe histological aspects of NASH, but merely fatty change in rats fed 14, 28 and 42 days of a choline deficient diet [15]. Besides, in genetically obese mice in which spontaneous mutation (ob/ob) occurs, a second stimulus, such as the intraperitoneal injection of LPS (lipopolysacharide) is necessary to produce the histologic pattern of NASH [9]. Probably, this would parallel the human model in so far as a second stimulus such as toxins, alcohol or drugs would be needed to develop necrosis, inflammation and fibrosis in a fatty liver. Neverthless, once the pathogenesis of NASH and NAFLD share many common aspects, fatty change being their initial step, NAFLD would be appropriate to study the efficacy of antioxidant drugs.

This model indicated a strong effect in the vitamin C inhibit of fatty change. Vitamin C, a potent hydrosoluble antioxidant, surprisingly, inhibited the development of steatosis in the animals fed a choline-deficient diet and reduced the basal luminescence when it was administered orally daily. This protective mechanism of vitamin C could be suggested by antioxidant action because the observed reduction in the level of luminescence values reported here. Our group have demonstrated in previous studies [20] that the presence of steatosis showed some correspondence with the increase of superoxide anion generation in the animals fed a choline-deficient diet. Although some limitations of lucigenin-amplified chemiluminescence have been recently described, because lucigenin can undergo significant redox-cycling in the presence of reductases, generating itself artifactual superoxides [16], these findings were confirmed by the inhibition of luminescence by SOD administration. Neverthless, the oxidative hypothesis is by no means the only hypothesis that can be considered in the general context of the scope of use of antioxidants drugs in NAFLD. Vitamin E (α-tocopherol), a potent fat-soluble antioxidant with capacity to scavenge free radicals could not inhibit the development of steatosis in this model. Besides, in this study, vitamin E did not change the profile of biochemical and oxidative stress variables. Recently, Grattagliano et al showed in the same context, there is a depletion of endogenous antioxidants such as vitamin E and glutathione in liver tissue [18]. However, this does not imply that the administration of antioxidants would prevent fatty change. On the other hand, Lavine et al have demonstrated that vitamin E could reduce aminotransferases levels of obese children with NASH [21] and Hasegawa et al observed besides the reduction of aminotransferases, improvement of histological alterations. [22].

Moreover, curiously, vitamin C, a potent hydrosoluble antioxidant, could inhibit the development of steatosis in this model. The real action of vitamin C in its prevention is a question because, although this drug have reduced the basal luminescence, the use of another antioxidant, vitamin E, could not prevent the development of steatosis. This fact can suggest that vitamin C could inhibit the development of steatosis by another mechanism. Some studies have demonstrated that ascorbic acid could reduce plasma levels of cholesterol and triglyceride demonstrating anti-atherogenic action [23].

In conclusion, our results suggest that NAFLD may be associated with oxidative stress and that the treatment with vitamin C may block the development of and NAFLD, while vitamin E may not. Future investigations are necessary to elucidate the role of ascorbic acid in NAFLD prevention.