Attenuation of reserpine-induced fibromyalgia via ROS and serotonergic pathway modulation by fisetin, a plant flavonoid polyphenol

Abstract

Fibromyalgia (FM) is a chronic complex musculoskeletal disorder characterized by widespread musculoskeletal pain accompanied by fatigue, sleep disturbance, memory defects and mood changes. Fisetin, a plant flavonoid polyphenol, has been reported to possess potent antioxidant, antinociceptive and neuroprotective activities. The present study aimed to evaluate the efficacy of fisetin against reserpine-induced FM (RIF) in rats. RIF was induced in male Wistar rats (180-220 gm) using reserpine (1 mg/kg; subcutaneous; once daily for 3 consecutive days) and the rats were treated with fisetin (5, 10 and 25 mg/kg) for 21 days. Various behavioral, biochemical and molecular parameters were evaluated. Administration of reserpine induced allodynia, hyperalgesia and depression, which were significantly ameliorated (P<0.05) by fisetin (10 and 25 mg/kg), as reflected by an increase in paw and tail withdrawal latency, increased paw withdrawal threshold, and decreased immobility time. Reserpine led to decreased biogenic amine levels [5-hydroxytryptamine (5-HT), noradrenaline (NA) and dopamine (DA)] and increased the ratio to their metabolite 3,4-dihydroxyphenylacetic acid. 5-hydroxyindoleacetic acid in the spinal cord, thalamus and prefrontal cortex was significantly decreased (P<0.05) by fisetin. Immunohistological analysis of brain tissue revealed that fisetin significantly inhibited (P<0.05) reserpine-induced depletion of 5-HT. It also significantly inhibited (P<0.05) elevated oxido-nitrosative stress and reactive oxygen species (ROS) levels, as analyzed by flow cytometry in RIF rats. Fisetin exerts its antinociceptive and anti-depressive potential via modulation of decreased levels of biogenic amines (5-HT, NA and DA), elevated oxido-nitrosative stress and ROS to ameliorate allodynia, hyperalgesia, and depression in experimental RIF.

Keywords: 5-HT; FM; NA; ROS; dopamine; fisetin; immunohistochemistry; reserpine.

Figure 1.

Effect of fisetin on reserpine-induced alterations in (A) mechanical hyperalgesia in the paw pressure test, (B) mechanical allodynia in the von Frey hair test, (C) thermal allodynia in the Hargreaves test, and (D) thermal hyperalgesia in the tail flick test in reserpine-induced fibromyalgia rats. Data are expressed as the mean ± standard error of the mean (n=4) and were analyzed by two-way ANOVA followed by Tukey's multiple range test. *P<0.05 vs. vehicle control group, ^#P<0.05 vs. normal animals and ^$P<0.05 vs. 30 mg/kg pregabalin or 5, 10 and 25 mg/kg fisetin. Fisetin (5), 5 mg/kg fisetin for 21 days; fisetin (10), 10 mg/kg fisetin for 21 days; fisetin (25), 25 mg/kg fisetin for 21 days; pregabalin (30), 30 mg/kg pregabalin for 21 days.

Figure 2.

Effect of fisetin on reserpine-induced alterations in the duration of immobility in the (A) forced swim test and (B) tail suspension test in reserpine-induced fibromyalgia rats. Data are expressed as mean ± standard error of the mean (n=4) and analyzed by two-way ANOVA followed by Tukey's multiple range test. *P<0.05 vs. vehicle control group, ^#P<0.05 vs. normal animals and ^$P<0.05 vs. 30 mg/kg pregabalin or 5, 10 and 25 mg/kg fisetin. Fisetin (5), 5 mg/kg fisetin for 21 days; fisetin (10), 10 mg/kg fisetin for 21 days; fisetin (25), 25 mg/kg fisetin for 21 days; pregabalin (30), 30 mg/kg pregabalin for 21 days.

Figure 3.

Effect of fisetin on reserpine-induced alterations in neural ROS of RIF rats. Representative image of intracellular ROS production detected by flow cytometry using H₂DCFDA probe in (A) normal, (B) vehicle control, (C) Fisetin (10 mg/kg)-treated, (D) Fisetin (25 mg/kg)-treated and (E) pregabalin (30 mg/kg)-treateds (E). The quantitative analysis of the effect of fisetin on reserpine-induced alterations in percentage H₂DCFDA fluorescence (F) of RIF rats. Data are expressed as mean ± standard error of the mean (n=4) and analyzed by one-way ANOVA followed by Tukey's multiple range test. *P<0.05 vs. vehicle control group, ^#P<0.05 vs. normal animals and ^$P<0.05 vs. 30 mg/kg pregabalin or 5, 10 and 25 mg/kg fisetin. ROS, reactive oxygen species; RIF, reserpine-induced fibromyalgia; H₂DCFDA, dichlorodihydrofluorescein diacetate; F (5), 5 mg/kg fisetin for 21 days; F (10), 10 mg/kg fisetin for 21 days; F (25), 25 mg/kg fisetin for 21 days; P (30), 30 mg/kg pregabalin for 21 days.

Figure 4.

Effect of fisetin on reserpine-induced alterations in 5-HT expression in the brain thalamus of RIF rats. Representative images (at ×40 and ×100 magnifications) are presented of a section of brain thalamus of (A) normal, (B) vehicle control, (C) fisetin (25 mg/kg)-treated and (D) pregabalin (30 mg/kg)-treated rats. (E) Quantitative analysis of the effect of fisetin on reserpine-induced alterations in 5-HT expression in the brain thalamus of RIF rats. Data are expressed as mean ± standard error of the mean (n=4) and analyzed by one-way ANOVA followed by Tukey's multiple range test. *P<0.05 vs. vehicle control group, ^#P<0.05 vs. normal animals and ^$P<0.05 vs. 30 mg/kg pregabalin or 5, 10 and 25 mg/kg fisetin. RIF, reserpine-induced fibromyalgia; 5-HT, 5-hydroxytryptamine; F (5), 5 mg/kg fisetin for 21 days; F (10), 10 mg/kg fisetin for 21 days; F (25), 25 mg/kg fisetin for 21 days; P (30), 30 mg/kg pregabalin for 21 days.