Oral Supplementation of Melatonin Protects against Fibromyalgia-Related Skeletal Muscle Alterations in Reserpine-Induced Myalgia Rats

Abstract

Fibromyalgia is a chronic syndrome characterized by widespread musculoskeletal pain and an extensive array of other symptoms including disordered sleep, fatigue, depression and anxiety. Important factors involved in the pathogenic process of fibromyalgia are inflammation and oxidative stress, suggesting that ant-inflammatory and/or antioxidant supplementation might be effective in the management and modulation of this syndrome. Recent evidence suggests that melatonin may be suitable for this purpose due to its well known ant-inflammatory, antioxidant and analgesic effects. Thus, in the current study, the effects of the oral supplementation of melatonin against fibromyalgia-related skeletal muscle alterations were evaluated. In detail, 90 Sprague Dawley rats were randomly treated with reserpine, to reproduce the pathogenic process of fibromyalgia and thereafter they received melatonin. The animals treated with reserpine showed moderate alterations at hind limb skeletal muscles level and had difficulty in moving, together with significant morphological and ultrastructural alterations and expression of inflammatory and oxidative stress markers in the gastrocnemius muscle. Interestingly, melatonin, dose and/or time dependently, reduced the difficulties in spontaneous motor activity and the musculoskeletal morphostructural, inflammatory, and oxidative stress alterations. This study suggests that melatonin in vivo may be an effective tool in the management of fibromyalgia-related musculoskeletal morphofunctional damage.

Keywords: fibromyalgia; inflammation; melatonin; oxidative stress; skeletal muscle.

Figure 1

Voluntary locomotor activity. The graphs summarize: the number of spontaneous bouts (A); and the distance travelled expressed in meters (B) of all experimental groups, evaluated during 1 h of spontaneous locomotor activity. ANOVA, two-way analysis of variance; * p ≤ 0.05 vs. Reserpine four days; # p ≤ 0.05 vs. Reserpine plus tap water; + p ≤ 0.05 vs. Reserpine plus melatonin 5 mg/kg/day for 2 months and ° p ≤ 0.05 vs. Controls. H₂O: tap water; MEL: melatonin; Res: reserpine.

Figure 2

Skeletal muscle atrophy. The graphs summarize: the gastrocnemius muscle weight, normalized to body weight, expressed in grams (A); and the Feret’s myotube diameter of gastrocnemius skeletal muscle, expressed in µm (B). ANOVA, two-way analysis of variance; * p ≤ 0.05 vs. Reserpine four days; # p ≤ 0.05 vs. Reserpine plus tap water; + p ≤ 0.05 vs. Reserpine plus melatonin 5 mg/kg/day for 2 months and ° p ≤ 0.05 vs. Controls. H₂O: tap water; MEL: melatonin; Res: reserpine.

Figure 3

Skeletal muscle ultrastructure. Transmission electron microscopy photomicrographs of: reserpine-induced myalgia rats (A,B); rats treated with reserpine and then with only tap water for two months (C,D); and rats treated with reserpine and then with melatonin at the dose of 5 mg/kg/day for two months (E,F). Scale bar: 1 μm. (m) identifies the mitochondria.

Figure 4

Total thiols. The graph summarizes the gastrocnemius total thiol levels of all experimental groups, expressed as µmol/grams. ANOVA, two-way analysis of variance; * p ≤ 0.05 vs. Reserpine four days; # p ≤ 0.05 vs. Reserpine plus tap water; + p ≤ 0.05 vs. Reserpine plus melatonin 5 mg/kg/day for 2 months and ° p ≤ 0.05 vs. Controls. H₂O: tap water; MEL: melatonin; Res: reserpine; TSH: total thiol.

Figure 5

Skeletal muscle oxidative stress and constitutive markers expression. Immunofluorescence photomicrographs of: gastrocnem us skeletal muscle superoxide dismutase 1 (A–E); catalase (F–L); cyclooxygenase-1 (M–Q); and sirtuin 3 (R–V) expression of: reserpine-induced myalgia rats (A,F,M,R); controls (B,G,N,S); rats treated with reserpine for two months (C,H,O,T); rats treated with reserpine and then with melatonin at the dose of 2.5 mg/kg/day for two months (D,I,P,U); and rats treated with reserpine and then with melatonin at the dose of 5 mg/kg/day for two months (E,L,Q,V). Nuclei were stained with DAPI (blue). Scale Bar: 20 µm. The quantitative measurement of the immunopositivity of both antioxidant enzymes and constitutive molecules of all experimental animals are shown in Figure 6A–D.

Figure 6

Skeletal muscle oxidative stress and constitutive markers quantitative analyses. The graphs summarize the histomorphometrical analyses expressed in arbitrary units (AU) of: superoxide dismutase 1 (SOD1) (A); catalase (CAT) (B); cyclooxygenase-1 (COX-1) (C); and sirtuin 3 (SIRT3) (D) immunopositivity at gastrocnemius skeletal muscle level. ANOVA, two-way analysis of variance; * p ≤ 0.05 vs. Reserpine four days; # p ≤ 0.05 vs. Reserpine plus tap water; + p ≤ 0.05 vs. Reserpine plus melatonin 5 mg/kg/day for 2 months and ° p ≤ 0.05 vs. Controls. H₂O: tap water; MEL: melatonin; Res: reserpine.

Figure 7

Skeletal muscle inflammatory markers. Immunofluorescence photomicrographs of gastrocnemius muscle inflammosome NLRP3 expression of: reserpine-induced myalgia rats (A); controls (B); rats treated with reserpine for two months (C); rats treated with reserpine plus melatonin at the dose of 2.5 mg/kg/day for two months (D); and rats treated with reserpine and then with melatonin at the dose of 5 mg/kg/day for two months (E). Nuclei were stained with DAPI (blue). Scale Bar: 20 µm. The graph summarizes the histomorphometrical analyses, expressed in arbitrary units (AU), of inflammosomeNLRP3. ANOVA, two-way analysis of variance; * p ≤ 0.05 vs. Reserpine four days; # p ≤ 0.05 vs. Reserpine plus tap water; + p ≤ 0.05 vs. Reserpine plus melatonin 5 mg/kg/day for 2 months and ° p ≤ 0.05 vs. Controls. H₂O: tap water; MEL: melatonin; Res: reserpine.

Figure 8

Potential melatonin mechanism(s) of action. A proposed mechanism by which melatonin protects the gastrocnemius skeletal muscle against fibromyalgic alterations through the block of NLRP3 activation. Up arrow indicates an increase in the expression, whereas the down arrow a decrease.