Hibiscus sabdariffa protects against fibromyalgia by modulating the neuroinflammation–oxidative stress–microRNA axis

Abstract

Fibromyalgia (FM) is a chronic syndrome characterized by widespread pain, fatigue, cognitive dysfunction and neuropsychiatric disturbances, associated with dysregulated oxidative stress and neuroinflammation. Emerging evidence suggests that microRNAs guiding these pathways, including miR-145-5p and miR-451a, may play a central role in FM pathogenesis. However, currently available pharmacological therapies provide limited efficacy and are frequently associated with adverse effects. Therefore, the present study investigated whether the methanolic extract of Hibiscus sabdariffa (MeOH-Exs), a polyphenol rich extract with known antioxidant and anti-inflammatory properties, alleviates reserpine (Res)-induced FM in rats through modulation of the neuroinflammation–oxidative stress–microRNA axis.

FM was induced by Res (1 mg/kg, s.c., for three days), followed by 28-day treatment with MeOH-Exs (25 or 50 mg/kg, p.o.) compared with the standard drug Neurontin (Neu). Behavioral assessments, serum hydrogen peroxide, brain serotonin, peripheral expression of miR-145-5p and miR-451a, and spinal cord inflammatory and histopathological alterations were evaluated.

MeOH-Exs significantly improved hyperalgesia, depressive- and anxiety-like behaviors, locomotion, and cognition relative to the Res group. The 50 mg/kg dose exerted the most pronounced effects, restoring expression of miR-145-5p and miR-451a, reducing oxidative stress, and elevating brain serotonin levels. Histological and immunohistochemical analyses confirmed attenuation of spinal cord inflammation and neuronal degeneration, caspase 3 expression and TNF-α immunoreactivity with effects comparable to or greater than Neu.

Collectively, these findings indicate that Hibiscus sabdariffa may ameliorate FM – like menifistations by targeting an integrated biochemical pathway linking oxidative stress, neuroinflammation, apoptosis, microRNA dysregulation and spinal cord histopathological alterations. While the precise molecular mechanisms require further investigation, MeOH-Exs demonstrates a promising multi-target neuroprotective profile and may represent a potential complementary preclinical therapeutic candidate for FM.

Graphical abstract

1. Introduction

Fibromyalgia (FM) is a clinical syndrome characterized by widespread pain, chronic for at least 3 months as defined by the American College of Rheumatology (ACR)[1]. Studies have reported that people in industrial countries are more susceptible to FM, with an estimated prevalence of over 2% worldwide, affecting more women (61.4%) than men (38.6%), including children and adolescents [2,3]. FM is not only characterized by chronic pain but is also associated with various pathological symptoms, such as headaches, fatigue, joint stiffness, depression, anxiety, memory deficits, sleep disturbances, dizziness, neuropathic pain, and psychiatric disturbances [4].The pathophysiology of FM is complex, with various proposed effects, highlighting a main pathogenic axis concerning neuroinflammation, oxidative stress, and dysregulated microRNAs (miRNAs), which collectively drive central sensitization[5]. Raised inflammatory cytokines, particularly TNF-α, trigger glial cells and boost nociceptive transmission, whereas excessive ROS, remarkably H₂O₂, contribute to neuronal dysfunction and mitochondrial destruction. Reduced expression of regulatory miRNAs such as miR-145-5p and miR-451a further worsens inflammation, oxidative damage, and serotonin imbalance[6].

Recent evidence further supports the involvement of miRNAs in fibromyalgia pathophysiology and chronic pain-associated neuronal dysfunction. Notably, altered expression of miR-145-5p has been identified in cerebrospinal fluid samples from FM patients and was associated with pain and fatigue severity [7]. Moreover, bioinformatics and genetic studies have highlighted the potential contribution of miRNA-related regulatory pathways to FM pathogenesis [8,9]. Therefore, miR-145-5p and miR-451a were selected in the current study based on their reported association with neuroinflammatory and oxidative stress-related signaling pathways relevant to FM. Reserpine (Res) is widely used to induce fibromyalgia-like alterations in experimental animals, including nociceptive hypersensitivity, depressive-like behavior, depletion of central monoamines such as serotonin, and increased oxidative stress [10]. Despite the current medications approved by the FDA for FM, such as gabapentin (Neurontin®), that primarily target pain, they are often associated with various side effects [11]. Therefore, there is a need for alternative treatments that can address the multifaceted nature of FM and its associated symptoms.

Hibiscus sabdariffa L (Hs), is a polyphenol plant known as roselle or karkadeh native to Malaysia and India, and belonging to the Malvaceae family [12]. Phytochemical studies have shown that Hs calyces contain several antioxidant constituents, including anthocyanins (ANs) and flavonoids, which contribute to its multi-target effects, including anti-inflammatory activity [13]. In addition, Hs has antiapoptotic, anti-depressant, and neuroprotective effects [14]. Suggestion also proposes that plant polyphenols can influence miRNA expression, induce hippocampal neurogenesis, improve memory and spatial learning, attenuate neuroinflammation [15,16], and reduce oxidative stress [17], all of which are implicated in fibromyalgia pathogenesis.

Methanolic extract was selected because methanol efficiently extracts polyphenolic and anthocyanin-rich fractions with potent biological activities [18]. Moreover, the extraction solvent was completely evaporated prior to administration, ensuring that the observed biological effects were attributable to the plant constituents rather than residual methanol. Although aqueous preparations of H. sabdariffa are more commonly used in traditional consumption, methanolic extraction is widely employed experimentally to enrich bioactive compounds and facilitate mechanistic investigations of their pharmacological effects [19].

Although the antioxidant and anti-inflammatory properties of Hibiscus sabdariffa and its polyphenol-rich extracts have been documented in several experimental models, their potential role in modulating the interconnected pathways of oxidative stress, neuroinflammation, apoptosis, and microRNA dysregulation in fibromyalgia remains insufficiently explored. Moreover, limited information is available regarding the effects of Hibiscus sabdariffa on miR-145-5p and miR-451a expression in the context of fibromyalgia-associated neuronal dysfunction. Therefore, the present study aimed to evaluate the therapeutic effects of a chemically characterized methanolic extract of H. sabdariffa (MeOH-Exs) in a reserpine-induced fibromyalgia model. We hypothesized that MeOH-Exs would ameliorate fibromyalgia-like manifestations through coordinated modulation of oxidative stress, neuroinflammatory and apoptotic responses, and miR-145-5p and miR-451a expression.

2. Materials and methods

2.1. Drugs and chemicals

Reserpine was obtained from Sigma-Aldrich (St. Louis, MO, USA); and Neurontin® (Gabapentin) 300 mg capsules was purchased from (Pfizer, Egypt). Powdered chemicals: Ferric chloride, Anhydrous Citric acid, and Aluminum Chloride, ALFA Chemika, India; CuSO₄, Sigma- Aldrich, St. Louis, MO, USA; Lead (II) acetate trihydrate, ALFA CHEMICAL Group Giza, Egypt; and Anhydrous Sodium hydroxide, was purchased from Fisher Scientific, U.K.

2.2. Methods

2.2.1. Collection and preparation

From the garden of Faculty of Pharmacy, Delta University for Science and Technology, Egypt, the air-dry flowers of Hs were collected and then grinned. The extraction of powdered air-dried (300g) was concerned according to formerly designated research[20].

2.2.2. Preparation and purification of Hibiscus methanolic extract (MeOH-Exs)

Dried Hibiscus sabdariffa calyces were extracted using 80% methanol containing 0.1% citric acid by sonication at 60 °C for 45 min (three cycles). The combined extract was filtered, concentrated, lyophilized, and purified following reported procedures using solvent partitioning [21,22] and silica-gel column chromatography[23]. The purified MeOH-Exs was used for all biological experiments. Briefly, for purification and phytochemical characterization, the extract was processed according to the method of [22] using sequential solvent partitioning with petroleum ether, was collected, filtered, and concentrated under reduced pressure at 65 °C until dryness. It should be noted that these procedures were performed for purification and characterization purposes, whereas the pharmacological experiments described in the present study were conducted using the methanolic extract of Hibiscus sabdariffa (MeOH-Exs). The extract was tested for major metabolite classes using standard qualitative assays as provided in Supplementary Methods S1.

2.2.3. GC-MS coupled system for association compounds in the MeOH-Exs of Hs

The chemical constituents of MeOH-Exs were characterized by GC–MS using an Agilent 7890A system equipped with an HP-5ms column. Helium was used as the carrier gas and the oven temperature was programmed from 55 °C to 280 °C at 11 °C/min[24]. Compounds were identified by comparison of retention times and mass fragmentation patterns with the NIST/WILEY libraries. The full chromatogram and the complete list of identified compounds are provided in Supplementary File S1 (Fig. S1–1).

2.3. Animals

The animal experiments conducted in this study complied with the ARRIVE guidelines and were approved by the National Center for the Replacement, Refinement, and Reduction of Animals in Research (NC3Rs)[25]. Furthermore, all procedures agreed with the Institutional animal care and accepted at the Pharmacy Collage, Delta University for Science and Technology, Gamasa, Egypt, with approval number (FPDU12/2022). Female Wistar rats weighing 215-255g were under a 12-h light/dark cycle at room temperature (23 ± 2 °c) and provided with food and water ad libitum. The doses of reserpine (1 mg/kg), MeOH-Exs (25 and 50 mg/kg), and Neurontin (30 mg/kg) used in the study were based on previous literature reports [10,[26], [27], [28]];. Specifically, the selected reserpine dose has been widely used to induce fibromyalgia-like symptoms in experimental animals, whereas the MeOH-Exs doses were adopted from previous studies demonstrating pharmacological efficacy and safety of Hibiscus sabdariffa extracts. Two MeOH-Exs dose levels (25 and 50 mg/kg) were employed to assess the presence of a dose-dependent therapeutic response. Neurontin (30 mg/kg) was selected as a reference treatment based on its established efficacy in experimental pain and fibromyalgia-related models.

2.3.1. Experimental design

Female Wistar rats were classified into five groups. Animals were randomly allocated to experimental groups using a simple randomization procedure prior to treatment initiation. Fifteen animals were initially assigned to each group to account for potential unforeseen losses during the experimental period. Based on previous studies employing the reserpine-induced fibromyalgia model and similar outcome measures [10], a final sample size of 12 animals per group was considered sufficient for the behavioral, biochemical, molecular, and histopathological analyses and was used for statistical evaluation. The reserpine-induced model was selected because it reproduces key fibromyalgia-like features, including persistent hyperalgesia and behavioral disturbances, thereby allowing evaluation of both nociceptive and non-nociceptive treatment effects [10]. Fig. 1.

• 1.Control:

Fig. 1.

Experimental protocol for Res-induced FM model. Fibromyalgia is induced by subcutaneous administration of Res (1 mg/kg) for three consecutive days. After that, rats were treated with MeOH-Exs (25, 50 mg/kg p.o), and Neu (30 mg/kg) for 28 days. Behavioral assessments were conducted between days 26 and 30. On day 31, all rats were sacrificed, and blood as well as tissue samples were collected. MeOH-Exs, methanolic extract sample of Hs; Neu, neurontin; and Res: reserpine.

Received 1% DMSO/saline (10 mL/kg, s.c.) for 3 days, followed by distilled water (p.o.) for 28 days.

• 2.Reserpine (Res):

Received Res (1 mg/kg, s.c.) dispersed in 1% DMSO/saline daily for consecutive 3 days, then given distilled water (p.o.) for another 28 days.

• 3.MeOH-Exs 25 mg/kg:

Received Res for 3 days, then MeOH-Exs (25 mg/kg, p.o.) daily for another 28 days.

• 4.MeOH-Exs 50 mg/kg:

Received Res for 3 days, then MeOH-Exs (50 mg/kg, p.o.) daily for another 28 days.

• 5.Neurontin (Neu) 30 mg/kg:

Received Res for 3 days, then Neu (30 mg/kg, p.o.) daily for another 28 days. Neurontin was included as a reference treatment to provide a pharmacological comparator for the antinociceptive effects of MeOH-Exs.

Whenever feasible, behavioral assessments and histopathological evaluations were performed by investigators blinded to treatment allocation. Behavioral assessments were done on day 26, except for the MWM, which included training on days 26–30 and a probe test on day 30. At termination, rats were sacrificed under thiopental anesthesia (40 mg/kg, i.p.)[29], and blood was collected for PBMC expression of miR-145-5p and miR-451a and serum H₂O₂ analysis. Brains were rapidly frozen at −80 °C for serotonin measurement by ELISA. Spinal cords were fixed in 10% formalin for histopathology.

Caspase-3 and TNF-α expression were estimated by immunohistochemistry.

2.4. Behavioral assessments

Behavioral assessments were conducted by an investigator blinded to treatment groups. Tests included: the Tail Suspension Test (TST; immobility time; [30], Forced Swim Test (FST; mobility/immobility over 2 min; [31]), Hot Plate test (thermal nociception at 50–55 °C; latency to hind-paw licking; [32]), Rotarod test (motor coordination; latency to fall over 3 min; [33]), Open Field Test (OFT; locomotion, rearing, and latency to first move in a 30 × 30 × 30 cm arena; [34], Elevated Plus Maze (EPM; % time and entries in open vs. closed arms over 3 min; [35], and spatial learning and memory were assessed using the Morris Water Maze (MWM) test; escape latency during 5-day acquisition; [36]. All tests were recorded by video for later analysis. Full procedural details, including apparatus specifications, trial structure, timing, habituation, exclusion criteria, and representative videos, are provided in Supplementary Methods S2.

2.5. Biochemical estimations

2.5.1. Quantification of miR-145-5p, and miR-451a expression using qRT-PCR

Total RNA was isolated from PBMCs and subsequently reverse-transcribed using the miRCURY® LNA® RT Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. qPCR was carried out in triplicate using the miRCURY® LNA® SYBR Green PCR Kit, with U6 small nuclear RNA as the internal control. The amplification protocol conditions included an initial denaturation step at 95 °C for 15 min, followed by 35 cycles consisting of denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s, and extension at 72 °C for 15 s. Specificity of amplification was verified through melting curve analysis. Relative expression levels were calculated based on cycle threshold (Ct) values using the comparative Ct (2⁻ΔΔCt) method of [37], where ΔCt was determined by normalizing target miRNA Ct values to the endogenous control (U6 small nuclear RNA), and ΔΔCt values were calculated relative to the control group. Expression levels were subsequently reported as fold changes relative to controls. The expression analysis of miR-145-5p and miR-451a was conducted in accordance with the method reported by Ref. [38].

2.5.2. Estimation of H₂O₂ levels in the serum

From Biodiagnostic Company (GIZA, Egypt), a hydrogen peroxide (H₂O₂) kit was supplied. The H₂O₂ levels were measured via a spectrophotometer at 510 nm following the manufacturer's data kit. H₂O₂ concentration (mM/L) = A _Sample/A _Standard x 0.5.

2.5.3. Estimation of serotonin in the brain tissue

The ELISA kit for Serotonin was supplied by Biovision Company and relied on a competitive ELISA detection method. After collection of the brain from all groups, they homogenized for 5 min at 1077 x g using pre-cooling phosphate buffer saline (0.01MPBS, pH = 7.4) to obtain 10% tissue homogenates, then centrifuged at (5000 x g) at 4 °Cfor 30 min, and finally the supernatant was taken. Absorbance was measured at 450 nm and then revealed from the standard curve.

2.6. Histopathological assessment of spinal cord tissues

Spinal cord specimens were collected from all experimental groups and fixed in 10% neutral buffered formalin for 24 h. Following fixation, samples were dehydrated through a graded series of ethanol, cleared in xylene, and embedded in paraffin wax. Paraffin blocks were sectioned into 5–6 μm thick slices using a microtome. The obtained sections were stained with hematoxylin and eosin (H&E) for histological evaluation. Microscopic examination was performed using a light microscope (Olympus, Japan).

2.7. Immunohistochemical analysis of caspase-3 and TNF- α

Paraffin-embedded spinal cord sections were deparaffinized and rehydrated, followed by antigen retrieval via EDTA buffer (pH 8.0). Endogenous peroxidase activity was opposed using 0.3% hydrogen peroxide, as previously described[39]. Sections were then incubated with blocking solution for 30 min prior to overnight incubation with primary antibodies against caspase-3 and TNF-α (1:100; R&D Systems).

Subsequently, slides were preserved with horseradish peroxidase (HRP)-conjugated rabbit IgG secondary antibodies (EnVision System, Dako). Immunoreactivity was visualized using diaminobenzidine (DAB) as the chromogen, followed by counterstaining with Mayer's hematoxylin.

The expression levels of caspase-3 and TNF-α were quantified by calculating the percentage of positively stained neuronal cells among 1000 examined cells across ten high-power fields (400× magnification).

2.8. Statistical analysis

Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA) with GraphPad Prism software (version 8; San Diego, CA, USA). Comparisons were conducted between the experimental (diseased) and control (normal) groups. Differences were considered statistically significant at p < 0.05. Unless otherwise stated, data are presented from n = 12 animals per experimental group. For qRT-PCR analyses, technical triplicates were averaged to obtain a single value for each biological sample before statistical analysis.

3. Results

3.1. Phytochemical characterization of MeOH-Exs

The GC-MS analysis identified a total of 38 compounds in the MeOH-Exs, comprising various types of polyphenols and flavonoids. Flavonoids were the most abundant class, accounting for 57.44% of the total composition. Specifically, the MeOH-Exs contained 28 different flavonoid compounds, including a significant concentration of anthocyanins (10.22%). The major anthocyanins identified were Malvidin-3,5-diglucoside (1.27%), Delphinidin-3-galactoside (1.64%), Cyanidin-3-O-sambubioside (2.14%), Cyanidin-3-rutinoside (2.48%), and Petunidin-3-glucoside, which was the most abundant at 2.69%. In addition, the MeOH-Exs also contained terpenes, which represented 9.45% of the total composition, with linalool and terpinene-4-ol being the major terpene compounds (6.61% and 1.56%, respectively). Other notable compounds identified in the MeOH-Exs included coumarins (10.33%), phenols (17.17%), limonene (5.62%), and luteolin and its derivatives (4.66%). Overall, the GC-MS analysis revealed the rich and diverse phytochemical profile of the MeOH-Exs, with a predominance of polyphenols and flavonoids, particularly anthocyanins, terpenes, and other bioactive compounds. The full GC–MS chromatogram and complete compound list (retention times, peak areas, and molecular ions) are presented in Supplementary File S1.

3.2. Effect of MeOH-Exs (25, 50) of H.s and Neu 30 on depression and duration of mobility in FST and immobility time in TST and FST in Res-induced FM rats

Reserpine (1 mg/kg) markedly increased immobility in both the TST and FST compared to controls (p < 0.0001). Treatment with MeOH-Exs (25 or 50 mg/kg) and Neu (30 mg/kg) significantly attenuated this depressive-like behavior, reducing immobility and increasing mobility times versus the Res group (p < 0.0001) Fig. 2(A–C). The 50 mg/kg MeOH-Exs dose restored FST immobility and mobility to control levels, whereas the 25 mg/kg dose produced only a moderate effect, showing higher immobility and lower mobility than controls (p < 0.05). Notably, MeOH-Exs 50 mg/kg exerted the strongest antidepressant-like activity, producing significantly lower immobility in both tests than Neu (p < 0.01) and higher FST mobility compared with MeOH-Exs 25 mg/kg and Neu (p < 0.05, p < 0.01).

Fig. 2.

Effects of methanolic extract of Hibiscus sabdariffa (MeOH-Exs; 25 and 50 mg/kg) and neurontin (Neu; 30 mg/kg) on immobility time and mobility time in the forced swim test (FST) (Panels A and B), and immobility time in the tail suspension test (TST) (Panel C) in reserpine-induced fibromyalgia rats. Data are presented as mean ± SD, n = 12. Statistical analysis was performed using one-way ANOVA. Significance is indicated as follows: *^, **^, **** Significant compared to control group at p < 0.05, 0.01, 0.0001 respectively, ^#### Significant compared to Res group at p < 0.0001, ^$, ^$$ Significant compared to MeOH-Exs (25) group at p < 0.05, 0.01 respectively, ^aa significant compared to MeOH-Exs (50) group at p < 0.01. MeOH-Exs, methanolic extract of Hibiscus sabdariffa; Neu, Neurontin; and Res, Reserpine.

3.3. Effect of MeOH-Exs (25, 50) and Neu 30 on alterations in allodynia and thermal hyperalgesia in Res-induced FM rats

Reserpine significantly reduced pain latency versus control (p < 0.0001), indicating thermal hyperalgesia. MeOH-Exs (25 and 50 mg/kg) and Neu markedly increased latency compared with the Res group (p < 0.0001). The 50 mg/kg MeOH-Exs dose produced the strongest analgesic effect, fully normalizing latency with no significant difference from controls and outperforming both the 25 mg/kg and Neu groups (p < 0.05) (Fig. 3, Fig. 4).

Fig. 3.

Effects of methanolic extract of Hibiscus sabdariffa (MeOH-Exs; 25 and 50 mg/kg) and neurontin (Neu; 30 mg/kg) on mechanical allodynia and thermal hyperalgesia in reserpine-induced fibromyalgia rats. Data are presented as mean ± SD, n = 12. Statistical analysis was performed using one-way ANOVA. Significance is indicated as follows: ***, **** Significant compared to control group at p < 0.001, < 0.0001 respectively, ^#### Significant compared to Res group at p < 0.0001, ^$, ^$$ Significant compared to MeOH-Exs (25) group at p < 0.05, 0.01 respectively. MeOH-Exs, methanolic extract of Hibiscus sabdariffa; Neu, neurontin; Res, reserpine.

3.4. Effect of MeOH-Exs (25, 50) of H.s and Neu 30 on alteration in locomotor activity and strength of rats in (Rota-Rod test) and Open field test (OFT) in Res-induced FM rats

Reserpine caused a significant decline in motor performance (p < 0.0001). All treatments improved latency to fall versus the Res group (p < 0.0001). The (25 and 50 mg/kg) MeOH-Exs groups showed maximal improvement, restoring performance to control levels and exceeding Neu (p < 0.0001), MeOH-Exs 50 had higher effect than the low dose (25 mg/kg), but the difference was not statistically significant Fig. 4(A). Reserpine significantly increased latency to first movement, decreased rearing, and number of crossed square (p < 0.0001). MeOH-Exs (25 and 50 mg/kg) and Neu significantly reversed these deficits versus the Res group (p < 0.0001). MeOH-Exs 50 mg/kg exhibited the greatest effect, showing higher locomotion and exploration than Neu group (p < 0.0001), with values approaching controls Fig. 4(A–C).

Fig. 4.1.

A Effects of methanolic extract of Hibiscus sabdariffa (MeOH-Exs; 25 and 50 mg/kg) and neurontin (Neu; 30 mg/kg) on locomotor activity and motor strength in the rotarod test in reserpine-induced fibromyalgia rats. Data are presented as mean ± SD, n = 12. Statistical analysis was conducted using one-way ANOVA. Significance is indicated as follows: **** Significant compared to control group at p < 0.0001, ^#### Significant compared to Res group at p < 0.0001, ^$$$$ Significant compared to MeOH-Exs (25) group at p < 0.0001, ^aaaa significant compared to MeOH-Exs (50) group at p < 0.0001. MeOH-Exs, methanolic extract of Hibiscus sabdariffa; Neu, neurontin; Res, reserpine.

Fig. 4.2.

A-C Effects of methanolic extract of Hibiscus sabdariffa (MeOH-Exs; 25 and 50 mg/kg) and neurontin (Neu; 30 mg/kg) on locomotor activity in the open field test (OFT) in reserpine-induced fibromyalgia rats. Data are presented as mean ± SD, n = 12. Statistical analysis was performed using one-way ANOVA. *^, **, **** Significant compared to control group at p < 0.05, 0.01, < 0.0001 respectively, ^#### Significant compared to Res group at p < 0.0001, ^$$$$ Significant compared to MeOH-Exs (25) group at p < 0.0001, ^aaaa Significant compared to MeOH-Exs (50) group at p < 0.0001. MeOH-Exs, methanolic extract of Hibiscus sabdariffa; Neu, neurontin; Res, reserpine.

3.5. Effect of MeOH-Exs (25, 50) of H.s and Neu 30 on anxiety in Elevated Plus Maze test (EPM) in Res-induced FM rats

Reserpine markedly reduced open-arm time and entries (p < 0.0001). All treatments increased open-arm exploration versus Res (p < 0.001). MeOH-Exs 50 mg/kg demonstrated the strongest anxiolytic effect, significantly exceeding both MeOH-Exs 25 mg/kg and Neu significantly exceeding both MeOH-Exs 25 mg/kg (p < 0.05), and Neu 30 (p < 0.0001) in time spent in open arms % and approaching control levels in number of entries open arms Fig. 5 (A, B, C, D).

Fig. 5.

Effects of MeOH-Exs (25 and 50 mg/kg) and Neu (30 mg/kg) on anxiety-like behavior in the elevated plus maze (EPM) test in reserpine-induced fibromyalgia rats. Data are presented as mean ± SD, n = 12. Statistical comparisons were made using one-way ANOVA. Significance: **, **** Significant compared to control group at p < 0.01, < 0.0001 respectively, ^#### Significant compared to Res group at p < 0.0001, ^$$$$ Significant compared to MeOH-Exs (25) group at p < 0.0001, ^aa, ^aaaa Significant compared to MeOH-Exs (50) group at p < 0.01, < 0.0001 respectively. MeOH-Exs, methanolic extract of Hibiscus sabdariffa; Neu, neurontin; Res, reserpine.

3.6. Effect of MeOH-Exs (25, 50) of H.s and Neu 30 on memory cognitive and spatial learning in Morris Water Maze test (MWM) in Res-induced FM rats

Reserpine significantly increased escape latency during training and reduced target-quadrant time in the probe test (p < 0.0001). MeOH-Exs (25 and 50 mg/kg) and Neu significantly improved learning and memory indices versus Res (p < 0.0001). The 50 mg/kg MeOH-Exs dose produced the greatest cognitive enhancement, showing shorter escape latencies and higher target-quadrant time than both 25 mg/kg and Neu groups (p < 0.0001) (Fig. 6).

Fig. 6.

Effects of MeOH-Exs (25 and 50 mg/kg) and Neu (30 mg/kg) on cognitive and spatial learning in the Morris Water Maze (MWM) test in reserpine-induced fibromyalgia rats. Data are expressed as mean ± SD, n = 12. One-way ANOVA was used for statistical analysis. Significance: **, **** Significant compared to control group at p < 0.01, < 0.0001 respectively, ^#### Significant compared to Res group at p < 0.0001, ^$$$$ Significant compared to MeOH-Exs (25) group at p < 0.0001, ^aaaa Significant compared to MeOH-Exs (50) group at p < 0.0001. MeOH-Exs, methanolic extract of Hibiscus sabdariffa; Neu, neurontin; Res, reserpine.

3.7. Effect of MeOH-Exs (25, 50) of H.s and Neu 30 on alterations in both miRNA-145-5p and miRNA-451a expression in Res-induced FM rats

Reserpine significantly downregulated miR-145-5p and miR-451a (p < 0.0001). MeOH-Exs (25 and 50 mg/kg) and Neu significantly restored their expression (p < 0.001). MeOH-Exs 50 mg/kg induced the strongest upregulation, showing higher expression levels than both 25 mg/kg and Neu (p < 0.0001), consistent with its superior molecular effects (Fig. 7A and B).

Fig. 7.

Effects of MeOH-Exs (25 and 50 mg/kg) and Neu (30 mg/kg) on miR-145-5p and miR-451a expression in reserpine-induced fibromyalgia rats. Data are presented as mean ± SD, n = 12. One-way ANOVA was applied for statistical analysis. Significance: *, **** Significant compared to control group at p < 0.05, P < 0.0001 respectively, ^#### Significant compared to Res group at p < 0.0001, ^$$$, ^$$$$ Significant compared to MeOH-Exs (25) group at p < 0.001, P < 0.0001 respectively, ^aaaa Significant compared to MeOH-Exs (50) group at p < 0.0001. MeOH-Exs, methanolic extract of Hibiscus sabdariffa; Neu, Neurontin; and Res, Reserpine.

3.8. Effect of MeOH-Exs (25, 50) of H.s and Neu 30 on alterations in serum H₂O₂ levels in Res-induced FM rats

Reserpine produced a marked elevation in serum H₂O₂ (p < 0.0001). MeOH-Exs (25 and 50 mg/kg) and Neu significantly reduced oxidative stress versus Res (p < 0.0001). MeOH-Exs 50 mg/kg showed the greatest antioxidant effect, lowering H₂O₂ to near-control levels and performing better than the 25 mg/kg (p < 0.05), with values approaching Neu group (Fig. 8).

Fig. 8.

Effects of MeOH-Exs (25 and 50 mg/kg) and Neu (30 mg/kg) on serum H₂O₂ levels in reserpine-induced fibromyalgia rats. Data are expressed as mean ± SD, n = 12. Statistical comparisons were performed using one-way ANOVA. Significance: ***^, **** Significant compared to control group at p < 0.001, < 0.0001 respectively, ^#### Significant compared to Res group at p < 0.0001, ^$, ^$$ Significant compared to MeOH-Exs (25) group at p < 0.05, p < 0.01 respectively. MeOH-Exs, methanolic extract of Hibiscus sabdariffa; Neu, Neurontin; and Res, Reserpine.

3.9. Effect of MeOH-Exs (25, 50) of H.s and Neu 30 on neurotransmitter serotonin in the brain in Res-induced FM rats

Reserpine caused a profound decrease in brain serotonin (p < 0.0001). Both MeOH-Exs doses and Neu significantly increased serotonin versus Res (p < 0.0001). The 50 mg/kg MeOH-Exs dose produced the highest serotonin restoration, with levels significantly greater than MeOH-Exs 25 mg/kg and Neu (p < 0.01) (Fig. 9).

Fig. 9.

Effects of MeOH-Exs (25 and 50 mg/kg) and Neu (30 mg/kg) on brain serotonin levels in reserpine-induced fibromyalgia rats. Data are presented as mean ± SD, n = 12. One-way ANOVA was used for statistical analysis. Significance: **, ****: Significant compared to control group at p < 0.01, p < 0.0001 respectively, ^####Significant compared to Res group at p < 0.0001, ^$$ Significant compared to MeOH-Exs (25) group at p < 0.01, ^aa Significant compared MeOH-Exs (50) at p < 0.01. MeOH-Exs, methanolic extract of Hibiscus sabdariffa; Neu, Neurontin; and Res, Reserpine.

3.10. Effect of MeOH-Exs (25, 50) of H.s and Neu 30 on neuronal cells of the spinal cords in Res-induced FM rats

Reserpine caused pronounced neuronal degeneration, vacuolization, and structural disruption The control group exhibited normal neurons (black arrows), neuropil, and glial cells (black arrowheads). In contrast, the rats treated with Res showed severe degeneration and vacuolation of neurons (black arrows), as well as glial cell appearance (black arrowheads), neuronal necrosis (blue arrows), congestion (red arrows), and hemorrhage (red arrowheads). The rats treated with the low dose of MeOH-Exs (25 mg) demonstrated decreased vacuolation and degeneration of neurons (black arrows), as well as reduced congestion (black arrows) and hemorrhage (red arrowheads). The high dose of MeOH-Exs (50 mg) showed few degenerated neurons (black arrowheads) and mild congestion (red arrows), with some sections appearing normal. Furthermore, the spinal cords of rats treated with Neu 30 mg revealed satellitosis around a few degenerated neurons (black arrows) in some sections, while the other sections appeared normal.

All treatments improved spinal architecture, with MeOH-Exs 50 mg/kg showing the most preserved neuronal morphology and minimal degenerative changes, comparable to controls and superior to Neu (Fig. 10).

Fig. 10.

Effects of MeOH-Exs (25 and 50 mg/kg) and Neu (30 mg/kg) on neuronal morphology in spinal cord sections of reserpine-induced fibromyalgia rats (H&E staining, ×400, scale bar 50 μm). Control sections exhibited normal neuronal architecture, (black arrows) whereas the Res group showed marked neuronal degeneration, vacuolization, and disruption of tissue organization (black arrowheads). Treatment with MeOH-Exs 25 mg/kg partially improved these histopathological alterations, while MeOH-Exs 50 mg/kg demonstrated greater preservation of neuronal morphology and spinal cord architecture. Neu treatment also improved tissue morphology but to a lesser extent than MeOH-Exs 50 mg/kg. Groups: Res (reserpine), MeOH-Exs 25 mg/kg, MeOH-Exs 50 mg/kg, Neu 30 mg/kg Abbreviations: MeOH-Exs, methanolic extract of Hibiscus sabdariffa; Neu, neurontin; Res, reserpine.

3.11. Effect of MeOH-Exs (25, 50) of H.s and Neu 30 on Caspase 3 and TNF-α within the positive brown neuronal cells in spinal cords in Res-induced FM rats

Reserpine markedly increased neurodegenerative cells, caspase-3, and TNF-α expression (p < 0.0001) (Black arrows point to positive expression) (Fig. 11.1A, Fig. 11.1B), and (Fig. 11.2A, Fig. 11.2B) respectively. MeOH-Exs (25 and 50 mg/kg) and Neu significantly reduced these inflammatory and apoptotic markers versus Res (p < 0.0001). The 50 mg/kg MeOH-Exs dose demonstrated the strongest suppression, showing significantly lower expression than the 25 mg/kg (p < 0.001, p < 0.01) in caspase-3, and TNF-α respectively, as evidenced by the intense brown immunopositive staining observed in spinal cord neurons compared with the control group. Moreover, the reduced density and intensity of immunopositive neurons in the MeOH-Exs 50 mg/kg group corroborated the quantitative analyses and histopathological findings, indicating a greater attenuation of neuroinflammation and apoptosis.

Fig. 11.1A.

Effects of MeOH-Exs (25 and 50 mg/kg) and Neu (30 mg/kg) on caspase-3-positive neurons in spinal cord sections (IHC, Mayer's hematoxylin counterstain, ×400, scale bar 50 μm) in reserpine-induced fibromyalgia rats. The Res group exhibited intense caspase-3 immunoreactivity (black arrows), indicating enhanced apoptotic activity, whereas treatment with MeOH-Exs and Neu reduced the number and intensity of caspase-3-positive neurons. The greatest reduction was observed in the MeOH-Exs 50 mg/kg group. Groups: Res (reserpine), MeOH-Exs 25 mg/kg, MeOH-Exs 50 mg/kg, Neu 30 mg/kg. Abbreviations: MeOH-Exs, methanolic extract of Hibiscus sabdariffa; Neu, neurontin; Res, reserpine.

Fig. 11.1B.

Quantitative analysis of caspase-3-positive neurons in spinal cord sections of reserpine-induced fibromyalgia rats treated with MeOH-Exs (25 and 50 mg/kg) or Neu (30 mg/kg). Data are expressed as mean ± SD. One-way ANOVA was used. Significance: ****Significant compared to control group at p < 0.0001, ^####Significant compared to Res group at p < 0.0001, ^$$$, ^$$$$: Significant compared to MeOH-Exs (25) group at p < 0.001, < 0.0001 respectively. MeOH-Exs, extract of Hibiscus sabdariffa; Neu, Neurontin; and Res, Reserpine.

Fig. 11.2A.

Immunohistochemical detection of TNF-α-positive neurons in spinal cord sections of reserpine-induced fibromyalgia rats treated with MeOH-Exs (25 and 50 mg/kg) or Neu (30 mg/kg) (IHC, Mayer's hematoxylin, ×400, scale bar 50 μm). Black arrows indicate TNF-α-positive neurons. Groups: Res, MeOH-Exs 25 mg/kg, MeOH-Exs 50 mg/kg, Neu 30 mg/kg. MeOH-Exs, methanolic extract of Hibiscus sabdariffa; Neu, neurontin; Res, reserpine.

Fig. 11.2B.

Quantitative analysis of TNF-positive neurons in spinal cord sections of reserpine-induced fibromyalgia rats treated with MeOH-Exs (25 and 50 mg/kg) or Neu (30 mg/kg). Data are expressed as mean ± SD. One-way ANOVA was used. Significance: **, ****: Significant compared to control group at p < 0.01, 0.0001 respectively, ^#### Significant compared to Res group at p < 0.0001, ^$$, ^$$$: Significant compared to MeOH-Exs 25 group at p < 0.01, p < 0.001 respectively. MeOH-Exs, methanolic extract of Hibiscus sabdariffa; Neu, Neurontin; and Res, Reserpine.

To enhance transparency and reproducibility, the individual animal data used to generate all figures have been included in Supplementary File S3.

4. Discussion

The current study demonstrates that methanolic extract of Hibiscus sabdariffa (MeOH-Exs) exerts noticeable therapeutic effects in the Res-induced model of fibromyalgia (FM), restoring behavioral, biochemical, and neurohistological parameters towards normal levels. These improvements may be attributed to the modulation of the neuroinflammation–oxidative stress–microRNA axis, a central cascade behind FM pathology.

The observed reductions in oxidative stress, inflammatory mediators, and apoptotic signaling following MeOH-Exs treatment are consistent with previous reports demonstrating that suppression of oxidative and inflammatory signaling pathways contributes to tissue protection and functional recovery in experimental disease models [40]. reported that attenuation of oxidative stress together with inhibition of NF-κB and MAPK signaling significantly reduced pathological tissue injury and inflammatory responses. Similarly [41], demonstrated that the natural polyphenol resveratrol ameliorated neuroinflammatory alterations through modulation of inflammatory mediators and chemokine-related signaling pathways. Although these molecular signaling pathways were not directly investigated in the present study, the observed reductions in TNF-α, H₂O₂, and caspase-3, together with restoration of miR-145-5p and miR-451a expression, suggest that the neuroprotective effects of MeOH-Exs may involve broader regulation of oxidative stress- and inflammation-associated signaling networks. Further mechanistic studies are warranted to clarify the contribution of these pathways to the anti-fibromyalgia effects of Hibiscus sabdariffa.

According to the results of behavioral and biochemical tests, reserpine administration produced the expected FM-like characteristics, including mechanical and thermal hyperalgesia, reduced locomotion, depressive- and anxiety-like performances, weakened motor coordination, and memory deficits. This collection of symptoms aligns with clinical FM characteristics and supports the model's strength. At the molecular level, Res significantly elevated H₂O₂ levels and TNF-α expression, reduced serotonin, and suppressed circulating miR-145-5p and miR-451a— supporting the concept that FM is associated with integrated oxidative and inflammatory pathways with epigenetic involvement. Otherwise, Neurontin treatment improved nociceptive thresholds in the model but had limited impact on oxidative stress, miRNA deficits, or overall neuroinflammation. This is consistent with its role as a calcium channel modulator rather than a neuroimmune regulator[42].

MeOH-Exs markedly improved behavioral performance across all groups, with the 50 mg/kg dose showing the strongest effect. The extract ameliorated hyperalgesia, enhanced locomotor and motor functions, memory enhancement, and reversed depressive- and anxiety-like behaviors, suggesting restoration of central neurotransmission and nociceptive balance. These behavioral benefits likely stem from MeOH-Exs' ability to modulate key biochemical pathways. The therapeutic relevance of using a methanolic extract in the current study should also be considered. Methanolic extraction enables efficient recovery of polyphenols, anthocyanins, flavonoids, and other antioxidant constituents that may not be fully extracted using aqueous preparations alone [19]. These phytoconstituents are believed to contribute substantially to the observed neuroprotective and anti-inflammatory effects [43]. Importantly, the methanol solvent was fully removed during extract preparation before animal administration. Nevertheless, we acknowledge that traditional human consumption of H. sabdariffa commonly involves aqueous preparations rather than purified methanolic extracts. Therefore, while the present findings support the mechanistic and preclinical therapeutic potential of H. sabdariffa bioactive compounds, further translational studies using clinically relevant formulations and pharmacokinetic characterization are still required.

The behavioral improvements observed in the present study are consistent with previous reports describing analgesic, antidepressant-like, and anti-fatigue effects associated with H. sabdariffa and its bioactive constituents. The analgesic activity has been linked to anthocyanins, flavonoids, and terpenes with antinociceptive properties [44,45], whereas the antidepressant-like effects have been associated with polyphenol-mediated modulation of neurotransmitter signaling [46]. Previous evidence has also linked the anti-fatigue effects of H. sabdariffa to improved serotonin availability, antioxidant defenses, and neurotrophic signaling [47]. The present findings are consistent with these reports and further show that the behavioral improvements in the reserpine-induced fibromyalgia model occurred alongside restoration of serotonin levels and attenuation of oxidative stress, neuroinflammatory, and histopathological alterations.

One fundamental biochemical finding was the potent antioxidant activity of the extract. Elevated H₂O₂ levels in FM reflect enhanced ROS production and impaired antioxidant defenses[48]. In the present study, MeOH-Exs significantly reduced serum H₂O₂ levels, particularly at the higher dose, indicating strong redox-restoring activity. This finding is consistent with previous experimental evidence demonstrating that H. sabdariffa attenuates oxidative stress and exerts neuroprotective effects in the brain [49]. Oxidative stress is particularly relevant to FM inflammatory pathways [50,51]

Parallel to oxidative changes, neuroinflammation plays a central role in FM. Res increased TNF-α expression within spinal cord neurons, confirming inflammation-driven neurodegeneration[52]. MeOH-Exs reduced TNF-α expression in a dose-dependent manner and attenuated neuronal damage, as supported by histopathological examination showing reduced vacuolization, spongiosis, and necrosis. This suggests that Hibiscus’ anti-inflammatory polyphenols, including naringenin and quercetin, delphinidin, cyanidin derivatives, and protocatechuic acid, suppressed inflammatory signaling and protected neuronal integrity [53]. Polyphenols also stimulated the secretion of neurotrophic and anti-inflammatory factors, ultimately reducing the inflammatory response by activating peroxisome proliferator-activated receptor-γ (PPARγ)[54]. MeOH-Exs administration revealed a decline in caspase-3 expression and the number of neurodegenerative cells in the spinal cord, suggesting antiapoptotic activity[55]. This anti-apoptotic effect may be attributed to the antioxidant properties of Hs. Caspase-3 is also associated with the development of FM symptoms, such as depression and anxiety, brain cell damage, impaired memory and cognitive processes, as well as neuropathic pain [56,57] ; .

Collectively, these findings extend previous evidence on the antioxidant, anti-inflammatory, and neuroprotective properties of H. sabdariffa to a reserpine-induced fibromyalgia model, in which these effects were accompanied by broad behavioral improvement and attenuation of spinal cord histopathological alterations. Importantly, the present study further integrates these responses with the assessment of miR-145-5p and miR-451a expression, providing an additional molecular dimension to the observed protective effects.

In this context, MeOH-Exs significantly modulated the expression of miR-145-5p and miR-451a. The selected miRNAs were investigated based on previous experimental and profiling studies demonstrating their involvement in neuroinflammatory and oxidative stress-related pathways associated with neuronal dysfunction and chronic pain conditions [7,58]. Their reported regulatory roles in inflammatory signaling, apoptosis, and cellular oxidative responses make them biologically relevant targets in fibromyalgia-associated neurodegeneration and behavioral impairment. These miRNAs set neuronal survival[59], oxidative stress responses[60], and inflammatory pathways[61]. Their downregulation in the Res group aligns with previous studies connecting miRNA suppression to neurodegeneration, mood disorders, and enhanced nociception [62,63]. MeOH-Exs significantly reinstated their levels, suggesting a potential association between miRNA modulation and the observed biochemical and behavioral improvements. Reestablishment of these miRNAs may also be linked to the improved serotonin levels observed, given their roles in neurotransmitter regulation.

From a scientific perspective, the coordinated behavioral, biochemical, histopathological, and miRNA-related responses observed in the present study support the relevance of investigating multiple interrelated processes in experimental fibromyalgia rather than focusing on a single pathological component. These findings provide a preclinical basis for further investigation of H. sabdariffa as a source of multi-target bioactive compounds. However, translation of these findings into potential clinical application will require further mechanistic, pharmacokinetic, formulation, and clinical evaluation.

Several limitations of the present study should be acknowledged. First, the findings were obtained using a reserpine-induced rat model and therefore require validation in other experimental models and, ultimately, in clinical studies. Second, the use of a methanolic extract and the absence of detailed pharmacokinetic and pharmacodynamic characterization of the extract and its identified bioactive constituents, including their bioavailability, metabolism, blood–brain barrier penetration, and dose–response relationships, limit direct translational extrapolation of the findings. Third, neuroinflammation was evaluated primarily through TNF-α expression; assessment of additional inflammatory mediators and glial activation markers would provide a more comprehensive characterization of the neuroinflammatory response. Finally, although changes in miR-145-5p and miR-451a expression occurred alongside improvements in oxidative stress, inflammatory, and apoptotic responses, the present findings demonstrate coordinated associations rather than direct causal interactions among these molecular processes. Further mechanistic studies investigating upstream regulatory pathways, including NF-κB, Nrf2, and related signaling networks, together with pharmacokinetic and translational investigations, are therefore warranted.

5. Conclusion

The present study demonstrates that MeOH-Exs attenuated Res-induced fibromyalgia-like alterations in a dose-dependent manner. Treatment improved behavioral outcomes and serotonin levels, reduced oxidative stress, TNF-α and caspase-3 immunoreactivity, ameliorated spinal cord histopathological alterations, and restored miR-145-5p and miR-451a expression. Collectively, these findings indicate a multi-target protective effect of MeOH-Exs involving coordinated modulation of oxidative stress, neuroinflammatory and apoptotic responses, and miRNA expression.

These coordinated changes support the involvement of the neuroinflammation–oxidative stress–microRNA axis in the protective effects of MeOH-Exs in the reserpine-induced fibromyalgia model. However, the present findings demonstrate associations among these processes rather than establishing direct causal interactions between the individual components of the proposed axis. Thus, the findings support the preclinical therapeutic potential of Hibiscus sabdariffa and provide a basis for further mechanistic investigation. Additional studies are warranted to define the upstream molecular and transcriptional mechanisms, characterize the pharmacokinetic and pharmacodynamic properties of the extract and its bioactive constituents, and evaluate the translational relevance of these findings.

Author contributions

Heba Mostafa: Conceptualization of the research idea, methodology development, experimental work, data collection, investigation, validation, project administration, provision of resources, statistical analysis, interpretation of results, and writing – review & editing. Lamia Barakat: Conceptualization of the research idea, performing experiments, literature review, interpretation of results, and writing – review & editing.

Rania M. Khalil: Conceptualization of the research idea, experimental work, methodology development, data collection, interpretation of results, and writing – review & editing. Shymaa Abdullah Mohamed: Methodology and experiments related to miR-145-5p and miR-451a expression analysis.

Ethics declaration

This study was conducted in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. This study was approved by the Delta University for Science and Technology. (Approval No. FPDU12/2022)

Consent for publication

Not applicable.

Ethics declaration

The animal experiments conducted in this study complied with the ARRIVE guidelines and were approved by the National Center for the Replacement, Refinement, and Reduction of Animals in Research (NC3Rs). Furthermore, all procedures were in agreement with the Institutional animal care and submitted at the Faculty of Pharmacy, Delta University for Science and Technology, Gamasa, Egypt, approval number (FPDU12/2022).

Declaration of generative AI and AI-assisted technologies in the manuscript preparation process

During the revision of this work, the authors used ChatGPT (OpenAI) to assist with language refinement and improvement of clarity in responding to reviewer comments and revising manuscript text. Following the use of this tool, the authors critically reviewed, verified, and edited all content and take full responsibility for the content of the manuscript.

Funding

This study did not receive any specific funding from public, commercial, or non-profit organizations.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Data availability

Data will be made available on request.