The mechanism underlying pain symptoms in fibromyalgia (FM) is not fully understood. Oxidative stress has emerged as pathophysiological event occurring during the development of the disease. The present study aimed at investigating the efficacy of resveratrol associated with rice bran oil on fibromyalgia-like mice model. Subcutaneous injection of reserpine (0.25 mg/Kg) during 3 days produced fibromyalgia-like symptoms. Resveratrol and/or rice oil or pregabalin were administered through oral route in therapeutic (single dose) and preventive (four doses) schemes. In both schemes, treatment with resveratrol associated with rice bran oil and pregabalin significantly reduced mechanical allodynia and thermal hyperalgesia in animals. The preventive scheme displayed antidepressant effect which was demonstrated by the forced swimming test as well as reduced reactive species in the cerebrospinal fluid of reserpinized animals. Taken together, our data provide evidences that the intake of resveratrol associated with rice bran oil plays antinociceptive and antidepressant actions probably through reducing reactive species and suggests the involvement of oxidative stress in this model of FM as possible underlying mechanism of pathogenesis of the disease.
Fibromyalgia is a complex painful disorder associated with other symptoms, leading to a multidisciplinary approach for its treatment . Pharmacological management of FM is often associated with nonpharmacological approaches. The available drugs to treat FM symptoms might include several classes of analgesics, sedatives, antidepressants, and other drugs . Nevertheless, not all are well tolerated [47, 48] and they do not cover the broad range of FM-related symptoms , thereby raising the necessity of finding new drugs. Addressing this approach, Nagakura et al.  validated a rat model of fibromyalgia and de Souza et al.  adapted the model for mice. In an attempt to find new compounds to treat FM, previously reported mice model of FM has been used. There is much evidence in the literature showing the benefits of RSV in various diseases, which led us to search for possible effects of RSV in the FM model.
Considering the involvement of impaired antioxidant defenses of the organism in the development of diseases such as cardiovascular , inflammatory , tumorigenic , neurodegeneration , and neuropathic  ones, similar oxidative stress processes might be involved in the pathological events underlying FM. To assess this possible mechanism, the potential role of nutraceutical antioxidant compounds RSV and RO isolated or combined in reverting behavioral changes induced in FM-like model and measuring RS levels was tested.
In this circumstance acute and chronic effects of the administration of RSV, RO, or RO-combined RSV in mice on nociceptive and depressive-like behavior in a model of FM were accessed. The acute administration of RSV produced an increase in the mechanical allodynia threshold, but not in the hot plate test compared to the administration of saline in the reserpine-injected mice. On the other hand, the analgesic effects of chronic administration of RSV displayed a different pattern: RSV increased either the mechanical allodynia or hot plate test thresholds, similar to the positive drug control PGB. These data indicate the antinociceptive role of RSV for the treatment of painful symptoms of FM. The antinociceptive effects of RSV in acute and chronic inflammatory pain models in rodents have already been reported [21, 22], suggesting a preventive analgesic role for this compound; additionally, it was also demonstrated that RSV is able to relieve diabetic neuropathic pain .
There is a lack of data investigating whether ROper se displays antinociceptive effects. In the present FM model, it has been noticed that RO is not able to produce analgesic effects, which was demonstrated through mechanical allodynia and hot plate tests. However, the chronic administration of RO itself almost reversed thermal hyperalgesia in the hot plate test ( compared to RES-SAL group). Interestingly, Souto et al.  demonstrated a synergistic therapeutic effect of RSV and RO against an acute model of inflammation and a model of polyarthritis induced in rats. Then, we assessed whether the RSV transported in RO displays analgesic effects in the FM model. It has been noted that both acute and chronic schemes of administration of RO-combined RSV exhibited antinociceptive effects, demonstrated by the increased mechanical and thermal threshold levels performed through the mechanical allodynia and hot plate tests, indicating the potential therapeutic effect of the combination of RSV and RO to treat FM-painful related symptoms.
The locomotive activity of treated mice in the open field test was also assessed. It was observed that neither acute nor chronic treatment was able to reverse the locomotive behavior induced by the repeated injections of reserpine. The depressive behavior associated with FM only in the chronic scheme of administration was assessed and it was observed that the depressive behavior was prevented by the repeated administration of RSV and RO-combined RSV, returning to the control situation; this did not happen with the repeated administration of RO only. The immobility time data are in accordance with the previous data published by Souto et al. , in which the depressive behavior in rats induced by inflammatory states was prevented by the chronic administration of RSV and RO-combined RSV.
The results concerning the use of RSV transported in RO represent a promising therapeutic alternative for the treatment of symptoms related to several diseases, including FM, as we have presented here. The therapeutic approach of RO-combined RSV is reinforced by data published by other groups, in which the effects of the RSV were improved by lipid core nanocapsules [30, 54].
In an attempt to provide information concerning the mechanism of action for the antinociceptive effect of RSV and RO in this model, the antioxidant potential of both nutraceutical compounds was analyzed . For this purpose, the RS levels in the CSF of the animals have been assessed, using the DCFH assay. Interestingly, when dosed daily after reserpine injections, all treatments prevented the rise in the RS levels present in the reserpine-injected mice receiving only saline solution. These data reinforce the antioxidant ability of the RSV, corroborating a wide range of works which show the beneficial antioxidant effects of RSV in cardiovascular  and inflammatory disease , hepatic steatosis , cancer , diabetic neuropathy , and antiageing alterations  and the antioxidant ability of RO as hypolipidemic agent , anti-inflammatory , and others.
The involvement of oxidative stress has been related to the symptom of fatigue , a coexistent symptom in addition to widespread pain in FM patients. Some researchers investigated the role of oxidative stress in the pathological processes underlying FM. Akbas et al.  have observed elevated superoxide dismutase (SOD) antioxidant enzyme activity in patients with FM compared to healthy control patients, suggesting an increased oxidative stress. Furthermore, data has been published showing low total antioxidant status associated with high total oxidant status and oxidative stress index in patients with FM according to control groups . Remarkably, mitochondrial dysfunction was demonstrated to be present in patients with FM, through the expression of transcription factor A mitochondrial (TFAM) and peroxisome proliferator-activated receptor gamma-coactivator 1-alpha (PGC-1α), the mitochondrial factors involved in mitochondrial biogenesis . Although we have not investigated mitochondrial biogenesis, we can deduce that mitochondrial pathways are involved in the present fibromyalgia-like model, since the mitochondria is responsible for RS production, and changes were observed in this parameter with the increased RS levels in reserpinized mice prevented by the antioxidant compounds RSV and RO.
Moreover, the CSF increased levels of RS present in reserpine-injected mice observed here are in accordance with the results noted by other researchers, in which the RS production is involved in persistent pain arising from injury  or inflammatory insult . Based on the ability of the association of RSV and RO in preventing increased RS levels observed here, focusing on antioxidant compounds can be an alternative for holding the oxidative stress. Thus, we might assign a relationship between RS levels and nociceptive behavior in reserpine-injected mice. Although further investigation should be made about the underlying pathophysiological mechanism of FM, it was noted that the association of RSV and RO might be a therapeutic option for FM, since they present preventive antinociceptive and antioxidant actions on the fibromyalgia-like model.
The animal model used in this study is based on the ability of reserpine in depleting biogenic amine (serotonin, glutamate, and dopamine) in the central nervous system . A question which has not been explored yet concerning reserpine is the impact of the repeated administration of this drug for liver functional parameters. In order to answer this question, the serum levels of ALT and AST obtained from mice were assessed. ALT and AST are transaminases enzymes, with increment in serum levels that indicate hepatic lesion . Results showed no alteration in serum transaminases levels of reserpine-injected mice, with similar values to those observed in control group. This data suggests that the reserpine injections cause no change in liver function according to biochemical activity, which could be noted at least in a subchronic scheme of administration.
Strikingly, data presented here show that treatment with RSV transported in RO (10 mg/kg +10 mL/kg), in a dose 10-fold lower than treatment with RSV only (100 mg/kg), reversed nociceptive thresholds back to control levels and had higher thresholds than the RSV only group. Based on this, we suggest at least an additive antinociceptive effect of combining RSV and RO. This effect may be due to delayed delivery of RSV into the system when it is in an oil-blend composition. The effects of RSV and RO in the fibromyalgia-like model may be due to diminishment of oxidative stress and reduced RS levels in CSF in the mice. RS has been implicated in the development of persistent pain resulting from injury or inflammatory insult . Agents reducing RS have been demonstrated displaying antihyperalgesic action . Bazzo et al.  suggest that RSV might be a viable alternative in pain management through its powerful antioxidant activity. Antioxidant compounds, such as polyphenols, tocopherols, and tocotrienols, have been proposed to have beneficial effects on human health by preventing cellular damage and the development of chronic diseases [27, 34]. Additionally, recent data shows that the serotoninergic system contributed to the antinociceptive and antidepressant action of RSV in a mouse model of neuropathic pain . It is possible that both of the above-mentioned mechanisms are involved in the beneficial activity of RSV on fibromyalgia pathology.
In conclusion, we demonstrated herein a novel possible mechanism involved in the model of reserpine-induced fibromyalgia additional to the depletion of biogenic amine, despite further studies being needed. Reserpine was not shown to alter the hepatic function. Moreover, the potential of analgesic action of the RSV and RO association in treating fibromyalgia-related symptoms in a mouse model was also demonstrated. Because of the broad range of applicability of RSV and RO as a nutraceutical product and its relevant antioxidant and antinociceptive activities, the approach presented by us can be easily applicable in rheumatologic clinics as a pharmacological option for the treatment of FM.