Fibromyalgia (FM) is a multifactorial chronic syndrome [1
] that occurs in up to 6% of the population [3
]; it is one of the most common pathological conditions seen in primary health care [2
]. FM is a musculoskeletal condition characterized by chronic pain and tenderness (generalized allodynia/hyperalgesia) accompanied by other somatic and psychological symptoms including fatigue, sleep disturbances, irritable bowel, restless leg, tension or migraine headaches, dysmenorrhea and cognitive difficulties including memory problems, concentration difficulties and psychological and emotional distress [6
Despite significant developments in understanding its pathophysiology, the etiology of FM is still unknown [8
]. Liptan [9
] underlined the role of a generalized inflammation of the muscle fascia leading to widespread pain and central sensitization and proposed that the fascial dysfunction in FM could be caused by inadequate deep sleep and insufficient growth hormone release. Furthermore, a possible peripheral pathogenic factor involved in FM etiopathogenesis is an imbalance between pro- and anti-inflammatory cytokines [10
], as this derangement seems, in turn, to play an important role in induction and maintenance of chronic pain [8
], fatigue [12
] and muscle stiffness [13
]. Sprott and colleagues [14
] demonstrated that patients with FM are characterized by abnormalities in muscle tissue that include increased DNA fragmentation, impaired expression of constitutive enzymes and changes in the number and size of mitochondria. Regarding this hypothesis, cyclooxygenase-1 (COX-1) seems to be a key enzyme. COX-1 is a constitutive enzymatic isoform involved, at skeletal muscle level, in the regulation of microcirculation, basal turnover and in various stages of myogenesis [15
] and so it may also be involved in fibromyalgic pathogenesis. Furthermore, the decrease in mitochondrial mass and the rise in production of mitochondria derived radical oxygen species (ROS) have recently been proposed as relevant events in the fibromyalgia-related alterations [16
]. Many studies have noted a correlation between the increase of ROS and the reduction of endogenous antioxidant defenses, including superoxide dismutase (SOD), catalase (CAT) and glutathione, is strictly linked with the symptoms of FM [17
]. Recently, nucleotide oligomerization domain like receptor 3 (NLRP3) inflammasomes was found to be related to mitochondrial oxidative stress. ROS production is induced by many NLRP3 inflammasome stimulators and, at the same time, elevated ROS are essential for inflammasome activation [18
]. Interestingly, in fibromyalgia patients, after ROS-mediated activation, NLRP3 promotes the production of pro-inflammatory cytokines [19
The association of FM with inflammation and oxidative stress suggest that ant-inflammatory and/or antioxidant therapy might be important in FM management and modulation. The degree of improvement achieved by many drugs prescribed for FM is modest at best; in fact, 40–60% of patients do not respond to drug therapy [20
] and most fibromyalgia patients are sensitive to the side effects of these medications [21
]. Consequently, efforts to identify and promote new therapeutic strategies for fibromyalgia patients are still under consideration.
Recent evidence suggests that melatonin may be suitable and useful because of its multitasking properties as shown in several clinical studies [22
]. Melatonin is a small, highly conserved indoleamine with important chronobiological features [25
]. In addition to its chronobiological role, additional beneficial effects of melatonin have been reported including antioxidant, ant-inflammatory, antidepressant, sedative and analgesic activities [26
]. It is a pleiotropic agent that has been proven safe and remarkably well tolerated in human and animal models over a wide range of doses [31
Data supporting the claim that melatonin may have efficacy as a FM treatment are slight. Herein, we used a validated fibromyalgia animal model, the reserpine-induced myalgia rat (RIM), to obtain data related to this issue. We initially investigated motor activity, morphological, ultrastructural and oxidative stress and inflammatory changes at skeletal muscle level to document the basis of fibromyalgia etiopathogenesis and we studied in detail the involvement of NLRP3 inflammosome in the model of this disease. We also evaluated the effects of melatonin and its mechanism(s) of action to better understand the pathogenesis of this disorder.
The results provide evidence that melatonin, dose and duration of treatment dependently, reduced significantly the difficulties in spontaneous motor activity, alterations in musculoskeletal cytoarchitecture and induction of inflammatory and oxidative stress processes. Regarding the effect of melatonin on the inflammosome NLRP3, we report that its expression is significantly attenuated. Thus, we suggest that melatonin may be useful for minimizing the pathological processes related to FM disease.
Herein, we showed that the reserpine treated animals presented: (1) a significant decrease in number of spontaneous bouts and running distance traveled; (2) skeletal muscle atrophy due to the alteration of the gastrocnemius weight and diameter of myotubes and significant musculoskeletal ultrastructural alterations; (3) significant rise of TSH and, interestingly, also NLRP3 expressions in skeletal muscle levels; and (4) significant reduction of expression of endogenous antioxidant enzymes (SOD1 and CAT) and of constitutive molecules involved in inflammation, oxidative stress and myogenesis processes (COX-1 and SIRT3). Similar results have also been recorded in previous studies [1
] in which reserpine treatment causes a reduction in locomotor activity that could be related to long-lasting muscular mechanical hyperalgesia, tactile allodynia and tenderness, but also to depression characteristic of FM pathogenesis [34
]. Moreover, Blasco-Serra and colleagues [35
] documented the depressive-like symptoms in RIM group; they observed the rats had significant aversion to eating in a novel environment. In our study, rats treated with reserpine showed a clear decrease in food consumption and consequently in body weight. In addition, the atrophy of muscle fiber described by Bonaterra et al. [32
] is consistent with our results. According other authors [19
], the underlying mechanisms of these alterations could be inflammation and oxidative stress processes characteristic of FM and also in the RIM model. In particular, our data provide information on the possible mechanisms showing an increase in morphological damage caused by ROS and a reduction in endogenous antioxidants, including SOD and CAT, in gastrocnemius. We also evaluated COX-1, a constitutive marker of myogenesis in different muscles including cardiomyocytes [15
]. In particular, we showed that COX-1 was decreased in RIM animals, as already reported by De Almeida and colleagues [37
]. Therefore, we suggest that COX-1 has an important musculoskeletal protective role. Moreover, we studied SIRT3 expression and we demonstrated it to be highly expressed in RIM rats. Considering its role in redox regulation by deacetylating mitochondrial proteins like acetyl-coenzyme A synthetase 2, glutamate dehydrogenase and SOD [38
], we propose that it has important protective effects against oxidative stress also in FM syndrome [39
]. Related to this finding, we noted that the levels of SOD1 and SIRT3 of the control rats at two months were lower compared with control group at one month and, in agreement with other authors [40
], we considered this reduction to be a result of physiological aging and oxidative stress process.
After obtaining these data and, in particular, considering the morphological and ultrastructural alterations of mitochondria, we analyzed the expression of NLRP3, which is a prominent marker of mitochondrial ROS generation and inflammation processes [19
]. Interestingly, NLRP3 is increased in rats treated with reserpine but not in the control animals at two months, due to age-related alterations. It is known that NLRP3 promotes inflammation, oxidative stress and cell death through the activation of inflammatory caspase 1 and caspase 5 [41
]. Our findings suggest that NLRP3 is important for progression of fibromyalgic disease as well as in diabetic cardiopathy [42
] and renal dysfunction [43
However, in addition to an adequate fibromyalgic-animal model that is needed for the identification of markers related to pathogenesis, it is also very important to study appropriate treatments and pharmacological therapies against FM. Thus, the present study also evaluated the potential beneficial effects of melatonin administration against the alterations induced in the RIM model. In accordance with several previous studies conducted with fibromyalgic patients [4
], our results showed that melatonin reduces significantly the damage induce by FM pathogenic process. In fact, we observed that the treatment with melatonin improved the in volountary motor activity compared with the RIM experimental group and with the group treated with reserpine and then with only tap water. Probably, melatonin treatment causes an improvement in rat’s spontaneous running activity because of its antioxidants and ant-inflammatory mechanisms of action, well known melatonin properties [47
]. Although these beneficial effects are potentially related also to analgesic [30
] melatonin properties, experimental studies as well as clinical trials in humans have demonstrated that melatonin has an analgesic properties in chronic, acute, inflammatory and neuropathic pain conditions, including fibromyalgia [44
]. Importantly, these effects seem MT2 receptor-mediated, as shown by the group of Granados-Soto describing that selective MT2 receptor partial agonists have analgesic properties through modulation of brainstem descending anti-nociceptive pathways [28
]. Furthermore, in our study, we evaluated the effects of melatonin treatment on skeletal muscle cell atrophy induced by reserpine observing that melatonin causes a significant increment in muscle weight and in myotubes diameter compared with RIM experimental group and the animals treated with reserpine and then only with tap water. In addition previous studies reported that melatonin reduces skeletal muscle atrophy and oxidative stress [49
]. In order to confirm the antioxidant, ant-inflammatory and protective roles of melatonin, we evaluated the skeletal muscle expression of endogenous antioxidant and of constitutive molecules and we found that melatonin modulates muscoloskletal ultrastructure, increases expression of SOD1, CAT, COX-1 and SIRT3 and decreaes NLRP3 compared with RIM experimental group and also with the groups treated reserpine and then only with tap water.
In summary, we suggest that melatonin thought its important inhibiting effect against NLRP3 activation together with its known antioxidant, ant-inflammatory and analgesic properties may block the fibromyalgic pathological processes (Figure 8
However, further studies on this topic are mandatory to better assess the potential melatonin mechanism(s) of action.