In this study, the effect of resveratrol on stress markers and endogenous antioxidants in the hippocampus of Wistar rats administered ethanol at various concentrations was analyzed. It was observed that resveratrol had a protective effect for the elevation of nitrite production and lipid peroxidation products, mainly at high concentrations of ethanol, but it was not observed to have any specific effect on endogenous antioxidants. In various in vivo models, it has been shown that the continuous or periodic consumption of ethanol causes long-term brain problems in the adult brain by deregulating its redox balance, generating changes in various markers of oxidative stress such as NO and lipid peroxidation, that, depending on the pattern of consumption, the dose and the period of exposure to ethanol, a variety of structural and functional brain deficits can be observed. (27) At the end of the 1980s, a group of biomarkers for measuring oxidative stress was introduced, indicating the possible damage to macromolecules such as lipids and proteins, markers of physiological and cognitive functions, as well as the relationship with some specific pathology (28), which are used with the aim of developing new preventive, diagnostic and therapeutic strategies to prevent the appearance of various diseases (29).
The nitrite levels in the groups administered with resveratrol are lower than in the groups treated only with ethanol. Chronic treatment with resveratrol for a period of 2 months significantly reduces nitrite levels in the hippocampus of Wistar rats exposed to concentrations of 10–50% ethanol. This effect agrees with what was reported by Lorenz et al. (30) who found that treatment with resveratrol significantly attenuates nitrite levels in glial cells exposed to high concentrations of ethanol. Other studies carried out in recent years support the results found in this work, for example: Zima et al. (31) showed that NO metabolites (nitrites and nitrates) increase in alcoholic patients (34.3, SD: 2.6 vs 22.7, SD: 1.2 µmol/L), in addition, Si Yun & Lee, (32) found in vitro that high concentrations of NO diffuse into surrounding cells in the brain, which could be linked to increased cytotoxicity in neurons, glia, and myelin. Teixeira et al. (33) found a significant reduction in the number of neurons and glial cells with an increase in nitrite levels in young rats administered ethanol. One of several possible mechanisms for alcohol-induced neurotoxicity may be attributable to modulation of the hypothalamic-pituitary response to alcohol by nitric oxide generating systems. For example, alcohol can regulate NOS, a rate-limiting enzyme, so that NO increases its local levels, leading to neurotoxicity. (34)
The results obtained in this work showed that the effects of resveratrol could be beneficial for neurons, since it prevented the accumulation of NO and its metabolites in all the doses studied; the decrease in NO found would avoid the neurotoxic effect, suggesting that resveratrol has the capacity to prevent the toxic effects of ethanol by modifying nitrite levels, since these increase under conditions of high oxidative stress, in this case generated by the increase in ethanol concentration in the different groups.
Lipoperoxidation is a complex process that involves the formation of different products with various biological functions such as regulation of gene expression and cell signaling. (35) MDA and 4-HDA are generated after oxidation of biological membranes. These compounds are most calledlipoperoxides and are used as markers of oxidative stress in plasma and tissues. (36) Hippocampal atrophy is one of the most prominent manifestations of alcoholism, often caused by the experimental administration of ethanol. Ramezani et al. (37) found that chronic exposure to ethanol (35% orally ethanol solution) in rats significantly increased lipid peroxidation in the brain, as an indicator of oxidative stress in the cerebellum. Investigations carried out in the last decades maintain that the reduction of lipid peroxidation consequently allows favoring the neuroprotective effect of resveratrol. (38) The analysis in our study shows that ethanol gradually increases the levels of MDA + 4-HDA depending on the concentration administered, finding a significant increase of 150.91% (DE:8.17) in the group administered with 50% ethanol compared to the control group. The oral administration of (10 mg/kg/day) of resveratrol promoted a significant decrease in the levels of MDA + 4-HDA even in combination with high concentrations of ethanol, this protective activity agrees with results previously obtained in the laboratory where it is observed that the administration of resveratrol with a 10% ethanol solution significantly decreases lipid peroxidation in the same tissue. (39)
In vivo and in vitro studies have reported that chronic ethanol administration significantly increases MDA levels. (29) Nahar & Deepali, (40) demonstrated that chronic ethanol consumption caused a significant increase in MDA levels in rat brain synaptosomes. Similarly, Turkcu et al. (41) found that MDA levels in rat brain tissue samples increased significantly in animals treated with ethanol compared to the control and that when an antioxidant was administered, these metabolites returned to their normal levels, acting as a protective agent. These findings support the effect observed in the hippocampus of rats used in this investigation, where we observed that MDA increased with the different doses of ethanol administered for a period of 2 months compared to the control group, this increase was gradual and dependent on the dose, however, after the administration of resveratrol in all the doses studied, the MDA decreased its levels, exhibiting the protective activity that has already been reported for resveratrol.
A study conducted by Petrovic et al. (42), demonstrated that the marked increase in the product of lipid peroxidation (4-HDA) and the decrease in specific neuronal neurofilaments support the idea that the peroxidation process and the disruption of the cytoskeleton neurodegeneration could be initial steps in alcohol-associated neurodegeneration. It is known that 4-HDA is produced in less quantity compared to MDA during the lipid peroxidation process, in this investigation it was observed that the administration of resveratrol decreases the levels of 4-HDA even when it is administered in high concentrations of ethanol. On the other hand, it is shown that after administration of concentrations greater than 20% ethanol, the levels increase significantly when compared to the control group. The present study showed that exposure to high doses of ethanol causes the accumulation of this toxic aldehyde in the hippocampus of rats. However, when resveratrol was administered in combination with all the ethanol doses studied, it was observed that 4-HDA levels decreased, evidencing the protective effect of resveratrol. Although the dual role of 4-HDA is known, high concentrations are extremely toxic, leading to cell death. Prior to this event, many effects occur, such as rapid glutathione depletion, protein damage, increased lipid peroxidation, impaired calcium homeostasis, inhibition of DNA, RNA, and protein synthesis, inhibition of respiration and glycolysis, lactate release, and morphological changes. (43)
Regarding the effect of resveratrol on enzyme activity, Superoxide Dismutase did not show statistically significant changes in rats administered with any concentration of ethanol, in the administration of 50% ethanol where a an increase in activity of 34.78% (DE:23.73)compared to the control group, on the other hand, the group treated with resveratrol showed an increase in enzymatic activity in the doses of 40 and 50% of 21.24% (DE:13.86)and 56.9% (DE:25.6) respectively with respect to the group administered with ethanol. It is important to mention that although in the groups administered with 40 and 50% ethanol no significant difference was observed in the activity of this enzyme with respect to the control group, when using resveratrol, its activity did increase, showing a significant difference with respect to the ethanol group up to concentrations of 40 and 50%. This may be because, at concentrations lower than 40% ethanol, resveratrol acts as a scavenger, eliminating excess free radicals; however, at higher concentrations it is not capable of reducing stress levels and begins to act as an inducer of the enzymatic activity.
Regarding catalase, the administration of any concentrationof ethanol did not show significant changes, in the 50% of ethanol the enzymatic activity had an increase of 30.48% (DE: 12.65) compared to the control group, while with the administration of resveratrol showed a decrease in CAT enzymatic activity of 29.64% (DE:10.33) compared to the group treated only with 50% ethanol. It seems that at these concentrations this enzyme does not need to be activated or increase its activity, however, with the administration of 50% ethanol a significant increase is observed in various investigations that have shown greater catalase activity. The deficient activity of the endogenous enzymatic system leads to a greater production of free radicals and consequently greater cell damage. (44)