This study investigated the capability of folic acid, melatonin or the combination of these two compounds to protect against CCl4-induced hepatotoxicity. Our findings confirmed the previous observations that melatonin  and folic acid  effectively reduce oxidative stress, restore the normal concentrations of anti-oxidant enzymes, and exhibit antihyperlipidemic activity. In addition, the antioxidant activity of natural products has a number of beneficial effects in the treatment of various types of diseases .
The hepatotoxicity of CCl4 includes the production of free radicals and the activation of Kupffer cells and macrophages, which generate inflammatory and profibrogenic mediators. The overproduction of free radicals is the initial step in a chain of events that eventually leads to membrane lipid peroxidation and ultimately to cellular apoptosis and necrosis . The potential mechanisms of chemical-induced liver apoptosis include an increase in cytokine concentrations and/or oxidative stress . In addition, lipid peroxidation, a ROS-mediated mechanism, has been implicated in the pathogenesis of various liver injuries and the subsequent liver fibrogenesis that is observed in experimental animals .
We found that the concentration of MDA and hydroperoxide, which are indices of oxidative stress, were elevated in CCl4-challenged rats. However, decreased concentrations of these two compounds were found in the liver tissues of those CCl4-rats that were treated with either melatonin, folic acid or the combination of melatonin and folic acid, which suggests that these compounds can effectively protect against lipid peroxidation. Melatonin exerts antioxidant protection in different cell organelles, both in vitro and in vivo. The body has an effective defense mechanism to prevent and neutralize damage that is induced by free radicals. These enzymes constitute a mutually supportive team of defense against ROS . Melatonin and folic acid were found to significantly restore the normal concentrations of GSH and the catalase activity. The catalase enzyme, which exists in all aerobic cells, is a hemeprotein that metabolizes the decomposition of H2O2 to form oxygen and water. GSH acts as a non-enzymatic anti-oxidant that reduces the amount of H2O2, hydroperoxides and xenobiotic toxicity . The significant decrease of hydroperoxide in the blood and hepatic tissues confirmed that the pre-treatment with folic acid and melatonin could effectively protect against the hepatic lipid peroxidation that is induced by CCl4. The anti-oxidant activity and antilipidemic effects of melatonin may enhance the modulation of blood pressure and most likely play the most important role in the amelioration of the damage to the target organ . GSH is a crucial determinant for cell survival or death in oxidative stress conditions  and, thus, GSH is critical in reducing the toxic effects of CCl4. Moreover, the increase in hepatic GSH concentrations in rats treated with either folic acid, melatonin or their combination may be due to an increase in the amount of GSH synthesis or regeneration.
ROS upregulates NF-κB, which is required for the induction of pro-inflammatory cytokines, such as IL-1ß, TNF-α and IL-6 . TNF-α is a key mediator of the immune and inflammatory responses and controls the expression of the inflammatory gene network. Therefore, the overproduction of TNF-α contributes significantly to the pathological complications observed in many inflammatory diseases; for example, pro-inflammatory cytokines can increase the risk of schizophrenia . Similarly, hepatic injury is associated with the upregulation of TNF-α gene expression that was observed in the CCl4 group; this result is therefore in accordance with previous studies . Consequently, the overproduction of TNF-α contributed to the manifestation of the systemic inflammatory response and ultimately to the development of organ failure.
We found that the upregulation of TNF-α expression was accompanied by the upregulation of the Fas genes in CCl4-induced liver injury. The Fas protein is a type I membrane receptor that belongs to the TNF-receptor superfamily. Mita et al.  found that the expression of FasL by macrophages plays a role in their pathogenesis.
In the present study, Fas mRNA expression was significantly upregulated in CCl4- injected rats. Similarly, Zhang et al.  found that the expression of Fas is increased in CCl4-induced liver fibrosis. Activation of the Fas receptor by the FasL induces apoptosis via the activation of the caspase cascade . Therefore, the upregulation of both TNF-α and Fas clearly explains the hepatic tissue damage and dysfunction that was observed in the CCl4 group. Furthermore, hepatic injury in rats leads to elevations of serum AST and ALT and an increased incidence and severity of histopathological hepatic lesions. The present study revealed a significant increase in the concentrations of AST, ALT and ALP upon exposure to CCl4, which indicates considerable hepatocellular injury. In addition, an increase in serum AST and ALT concentrations by CCl4 has been attributed to hepatic structural damage because these entities are normally localized to the cytoplasm and are only released into the circulatory system after cellular damage has occurred .
The oxidative stability that is induced by the combination of melatonin and folic acid may mediate a downregulation of NF-κB activation, which results in the suppression of the inflammatory cascade and the low concentrations of TNF-α that were observed. Thus, the hepatic injury markers were significantly retarded in the animals that received any of these treatments. In fact, folic acid and melatonin significantly attenuated the increased concentrations of the serum liver enzymes that were induced by CCl4 and therefore led to the subsequent restoration of these to normal concentrations. The effect of folic acid and melatonin was further confirmed through histopathological examinations. It was found that pre-treatment with the combination of melatonin and folic acid had broad anti-inflammatory effects and attenuated the allergic inflammation in the CCl4- challenged rats. This amelioration of the hepatic tissues by melatonin and folic acid seemed to be mediated by the inhibition of oxidative stress and therefore the suppression of NF-κB, the key regulator of inflammatory production, which results in the decreased production of pro-inflammatory cytokines.
The Akt1 signal is critical for cell survival that is triggered by growth factors, the extracellular matrix, and other stimuli . Therefore, there is impaired Akt and eNOS activation in cirrhotic livers . We found a downregulation of Akt1 gene expression in CCl4-challenged rats. The concentration of Akt1 mRNA expression was markedly restored in CCl4-rats that were pre-treated with the combination of melatonin and folic acid. Previous studies have shown that Myr-Akt gene therapy can restore Akt activation and NO production in cirrhotic livers, which suggests that this therapy may be helpful in treating portal hypertension . The restorative capacity of melatonin and folic acid was also confirmed in the complete restoration of the IFN- γ mRNA expression to normal concentrations. The proteins EMSY and BRAC2 repress a number of IFN-stimulated genes; however, an Akt1-dependent pathway contributes to the full activation of IFN-stimulated genes by relieving this repression . Similar data establishes that Akt activity is essential for the upregulation of key IFN-α- and IFN-γ-inducible proteins, which have important functional consequences in the induction of IFN responses . In addition, the retrovirus-mediated expression of activated Akt in primary T cells from CD28-deficient mice is capable of selectively restoring the production of IL-2 and IFN-γ . We proved that the activation and upregulation of Akt1 was accompanied by an improvement in the biomarkers of hepatic damage, the histological architecture and liver function.
We confirmed the hypolipidemic effect of melatonin and folic acid in CCl4-treated rats, which was manifested by the low concentrations of cholesterol, triglycerides, and LDL and the increased HDL concentrations. Similar findings were also reported for other experimental models, which observed a decline in LDL and total cholesterol and an increase in HDL concentrations in animals treated with melatonin . This effect may be related to the enhancement of the catabolism of cholesterol to form bile acids  and the inhibition of cholesterol synthesis and LDL receptor activity .
HDL plays an essential role in the transport of cholesterol to the liver for excretion into bile acids , which are cytoprotective in hepatocytes because of their ability to activate phosphatidylinositol-3-kinase and its downstream signal Akt . We confirmed the upregulation of the Akt1 signal by the combination of melatonin and folic acid. The combination treatment was better able to restore the elevated concentrations of cholesterol, triglycerides and inflammatory indicators than the individual treatments. A significant increase in the arterial elasticity index, a significant improvement in glucose and lipid metabolism, and a significant increase in HDL cholesterol have also been observed in patients treated with anti-oxidants . Additionally, the beneficial effect of anti-oxidants on LDL oxidation has been previously demonstrated .
Our findings provide evidence of the potential anti-oxidant and anti-inflammatory effects of melatonin and folic acid. This combination restored normal oxidative stress concentrations, which might inhibit NF-κB and thus downregulate TNF-α and Fas mRNA expression. In addition, melatonin and folic acid markedly upregulated the cell survival signal, Akt1, and the IFN-γ concentrations. Furthermore, the restoration of the survival signaling genes that was induced by melatonin and folic acid also resulted in significant improvements to the liver function and the histological architecture. There are currently no data on the treatment of CCl4-induced hepatic injury with melatonin and folic acid that would indicate whether this intervention has a significant clinical impact. We have elucidated a potential role for these substances in the treatment of hepatic injury that needs to be intensively investigated in future research that focuses on patient oriented outcomes.