Endoplasmic reticulum stress and oxidative imbalance is the missing link in fibromyalgia pathophysiology

Fibromyalgia (FM), or fibromyalgia syndrome (FMS), is the most common musculoskeletal disorder after low back pain and osteoarthritis, and it is one of the most prevalent causes of chronic pain. Pain is the primary and distinguishing symptom of FMHowever, the condition is also characterized by a range of other symptoms, including fatigue, sleep disturbances, joint stiffness, cognitive impairment, chronic pain at multiple tender points, anxiety, and depression³. FM can develop at any age, including during childhood⁴. The average global prevalence is estimated at 2.7%, with a reported female-to-male ratio of approximately  Various factors—such as oxidative stress, mitochondrial dysfunction, and disruption of intracellular calcium (Ca²⁺) homeostasis—may play significant roles in the etiopathogenesis of FMS However, due to the lack of consensus on diagnostic and classification criteria, and particularly because of the uncertainty surrounding its underlying mechanisms, there is currently no consistent or universally effective treatment available for this condition.

Pathological conditions that induce endoplasmic reticulum (ER) stress can impair ER function by causing the accumulation of unfolded or misfolded proteins within the ER lumen. In response, the cell initiates a series of adaptive mechanisms known as the unfolded protein response (UPR), aimed at restoring cellular homeostasis. Recent research suggests that ER stress plays a key regulatory role by activating various molecular pathways that contribute to the development and persistence of chronic pain Glucose-regulated protein 78 (GRP78), a key ER chaperone from the heat shock protein 70 (HSP70) family, is a central regulator of ER stress signaling. It is involved in protein folding, quality control, and Ca²⁺ binding, and directs misfolded proteins toward degradation via the ER-associated degradation (ERAD) pathwayGRP78 expression is upregulated under ER stress conditions and is widely used as a marker of ER stress¹⁵. The UPR is primarily regulated by ER transmembrane proteins, including protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activating transcription factor 6 (ATF6), which transmit signals to the cytosol and nucleus to enhance protein-folding capacity. PERK activation reduces global protein synthesis and promotes the expression of genes involved in antioxidant defense and amino acid metabolism. It also regulates pro-apoptotic proteins, such as growth arrest and DNA damage-inducible protein 34 (GADD34) and C/EBP homologous protein (CHOP). During ER stress, ATF6 translocates to the Golgi apparatus, where it is cleaved by site-1 and site-2 proteases (S1P and S2P), releasing the cytosolic domain (ATF6f), which then activates the transcription of specific UPR target genes

To assess the oxidative-antioxidative balance in both clinical and experimental settings, Total Oxidant Status (TOS), Total Antioxidant Status (TAS), and the Oxidative Stress Index (OSI) are frequently used. Research has shown that oxidative stress plays a significant role in the pathophysiology of FMS Patients with FMS often exhibit elevated levels of free radicals and reduced antioxidant capacity compared to healthy individuals. Moreover, increased oxidative stress has been correlated with disease progression, while improvements in symptoms have been observed in patients treated with antioxidants, vitamins, and certain antidepressants. ER stress and oxidative stress are interconnected processes that influence a wide array of cellular signaling pathways. Recent studies suggest that targeting these pathways may open new therapeutic avenues for FMS managemen

The primary objective of this study is to elucidate the molecular pathophysiology of fibromyalgia syndrome (FMS) by examining both oxidative stress and ER stress parameters. To achieve this, we assessed key ER stress markers—GRP78, PERK, CHOP, and ATF6—while concurrently measuring total antioxidant status, total oxidant status, and the oxidative stress index, to provide a comprehensive evaluation of the oxidant-antioxidant balance.

Demographic characteristics

The demographic characteristics of the study population are presented in Table 1. There were no statistically significant differences between the FMS and control groups in terms of age and body mass index (BMI) (p > 0.05). However, a statistically significant difference was observed between the groups regarding pain duration (p = 0.001), with the FMS group reporting longer durations.

General laboratory test results

As shown in Table 2, statistically significant differences between the groups were observed in the levels of uric acid, total cholesterol, and platelet count (p < 0.05). No significant differences were found in other parameters, including glucose, albumin, triglycerides, and C-reactive protein (CRP) (p > 0.05).

FMS scale scores

Scores from fibromyalgia-related questionnaires and scales are illustrated in Fig. 1; Table 3. Participants diagnosed with FMS showed significantly higher scores across all scales and questionnaires compared to the control group (p < 0.001), indicating a greater symptom burden and functional impairment.

Serum ER stress and oxidative stress parameters

Figures 2 and 3display the serum levels of ER stress and oxidative stress markers for the control and FMS groups based on the data in Table 4. Patients in the FMS group exhibited significantly elevated serum levels of GRP78, PERK, CHOP, and ATF6 compared to controls (p = 0.001), suggesting increased ER stress.

Comparison of the endoplasmic reticulum (ER) stress parameters between control and fibromyalgia groups. All the ER stress parameters, namely GRP78 (A), ATF6 (B), PERK (C), and CHOP (D), were found higher in the fibromyalgia patients (***p = 0.001). Data expressed as mean ± S.D. GRP78, glucose-regulated protein 78; ATF6, activating transcription factor 6; PERK, PKR-like endoplasmic reticulum kinase; CHOP, C/EBP homologous protein; FMS, fibromyalgia syndrome group.

Comparison of the oxidative stress parameters between control and FMS groups. Total oxidant status (TOS) levels were found higher in FMS group whereas total antioxidant status (TAS) levels were lower. Depending on these results the oxidative stress index (OSI) was higher in the fibromyalgia patients (***p = 0.001). Data expressed as mean ± S.D. FMS, fibromyalgia syndrome group.

Additionally, oxidative stress markers demonstrated notable differences between the groups. As shown in Fig. 3, TOS (Total Oxidant Status) and OSI (Oxidative Stress Index) levels were significantly higher in the FMS group, while TAS (Total Antioxidant Status) levels were significantly lower compared to the control group (p = 0.001). These findings support the presence of both elevated oxidative stress and impaired antioxidant defense in FMS patients. An integrated summary of these findings is illustrated in Fig. 4, which presents a proposed mechanistic model highlighting the interplay between ER stress and oxidative stress in the pathogenesis of fibromyalgia.

Proposed mechanistic model illustrating the interplay between endoplasmic reticulum (ER) stress and oxidative imbalance in the pathophysiology of fibromyalgia. Chronic stress and genetic predisposition initiate ER stress, characterized by increased levels of GRP78, PERK, ATF6, and CHOP. This process promotes reactive oxygen species (ROS) production and oxidative stress (↑TOS, ↓TAS), which in turn exacerbates ER stress and central sensitization—creating a self-perpetuating loop. The resulting cellular dysfunction contributes to neuroinflammation, mitochondrial impairment, and central sensitization, ultimately manifesting as the core symptoms of fibromyalgia: pain, fatigue, sleep disturbance, anxiety, and depression.

In this study, we evaluated the involvement of endoplasmic reticulum (ER) stress in the pathophysiology of fibromyalgia syndrome (FMS). Our results demonstrated that serum levels of key ER stress markers—GRP78, PERK, CHOP, and ATF6—were significantly elevated in patients with FMS compared to healthy controls. In parallel, we assessed oxidative stress parameters, which, in conjunction with ER stress, are thought to contribute to the underlying mechanisms of FMS. Consistent with previous literature, our findings provide strong support for the significant role of oxidative stress in the disorder. Collectively, these results offer compelling evidence that ER stress, alongside oxidative imbalance, plays a critical role in the molecular pathogenesis of FMS.

The pathogenesis of FMS is believed to involve dysregulation of central pain processing mechanisms and increased sensitivity or hyperactivity of the central nervous system, which contribute to disease onset and chronicityAlthough various biological factors are implicated in the development of FMS, the potential involvement of ER stress has not been thoroughly investigated. ER stress arises from cellular conditions such as protein misfolding, inflammation, hypoxia, and nutrient deprivation—all of which are also associated with a range of human diseasesIt is therefore not surprising that ER stress is implicated in the pathophysiology of numerous conditions, including neurodegenerative diseases (e.g., Alzheimer’s and Parkinson’s), atherosclerosis, liver and kidney diseases, stroke, metabolic syndrome, and cancer

Inflammatory pathways are known to contribute to chronic pain states, and ER stress can induce cytokine synthesis even in the absence of physical injury²⁴. Chopra et al. demonstrated that ER stress and unfolded protein response (UPR) activation are associated with prostaglandin synthesis²⁵. Beyond the induction of cytokines and prostaglandins, ER stress also affects critical biological processes such as ion channel dysregulation, mitochondrial dysfunction, reactive oxygen species (ROS) generation, and apoptosis, all of which influence pain perception and responseGRP78, PERK, CHOP, and ATF6 are core UPR proteins expressed in nearly all nucleated cells, so their elevation in serum likely reflects systemic ER stress rather than secretion from a single tissueHowever, metabolically active organs such as the liver, CNS, and skeletal muscle are particularly susceptible to ER stressGiven the prominent muscle involvement and mitochondrial dysfunction reported in fibromyalgia, skeletal muscle may plausibly contribute to these circulating markers, although their precise origin remains to be clarified

ER stress has been implicated in a variety of peripheral disorders, including rheumatoid arthritis osteoarthritisneuropathic pain, and postherpetic neuralgia³³. In our study, analysis of ER stress markers (GRP78, PERK, CHOP, and ATF6) revealed significantly elevated levels in FMS patients compared to controls. These findings are consistent with those of Cordero et al., who reported increased ER stress markers in individuals with chronic pain conditions, including fibromyalgia Furthermore, our results confirm previous reports of increased oxidative stress in FMS patientsHowever, unlike earlier studies that primarily focused on markers such as lipid peroxidation or nitric oxide levels, our investigation provides direct quantitative evidence of ER stress through measurement of GRP78, PERK, and CHOP levels. Notably, previous animal model studies have shown increased expression of phosphorylated ERK (pERK) following electroacupuncture treatment in fibromyalgia models, which aligns with our finding of elevated PERK levels in FMS patients. This observation further supports the involvement of ER stress-related kinase pathways in central sensitization and chronic pain processingThese findings underscore the critical role of ER stress in the pathophysiology of FMS and highlight the potential therapeutic value of targeting ER stress pathways in the clinical management of this complex disorder. Future research should focus on further elucidating the interconnected roles of ER stress and oxidative stress in FMS, with the goal of developing targeted therapeutic strategies. Potential approaches may include antioxidant therapies, pharmacologic agents aimed at modulating ER stress responses, or combination treatments. Such strategies could offer more effective options for symptom relief and potentially halt or reverse disease progression.

ER stress has emerged as a key contributor to the pathophysiology of chronic pain, functioning beyond its classical role in protein homeostasis. Chronic activation of the unfolded protein response (UPR), particularly via GRP78, PERK, ATF6, and CHOP, has been consistently observed in preclinical models of bone cancer pain, diabetic neuropathy, and peripheral nerve injury, with elevated expression of these markers in dorsal root ganglia (DRG), trigeminal ganglia, and spinal dorsal horn neuronsThe PERK–eIF2α–ATF4–CHOP axis promotes inflammatory gene transcription, mitochondrial dysfunction, and neuronal apoptosis, while ATF6 contributes to neuroinflammation through upregulation of pro-inflammatory cytokines such as TNF-α and IL-6 GRP78 not only regulates UPR initiation but also acts as a molecular chaperone facilitating protein folding and ER-associated degradation; however, its sustained expression may reflect unresolved or pathological ER stress Notably, inhibition of ER stress through pharmacological agents such as 4-phenylbutyric acid (4-PBA) tauroursodeoxycholic acid (TUDCA) salubrinal, an eIF2α dephosphorylation inhibitor⁴⁰, or small-interfering RNA targeting ATF6has been shown to ER stress marker expression. Additionally, extracellular GRP78 may exert immunomodulatory effects, influencing peripheral inflammation and immune cell activation⁴². These findings collectively support the view that ER stress is not merely a marker but an active driver of chronic pain, making the UPR a promising therapeutic target.

It is important to note that all participants in this study were female. This decision was intentional and based on the well-established epidemiological pattern of FMS, which predominantly affects women, with a female-to-male ratio of approximately 3:1. By including only female participants, we aimed to minimize biological variability due to sex differences and ensure greater homogeneity within the study population. However, future studies should incorporate both sexes to explore potential sex-specific pathophysiological mechanisms and improve the generalizability of findings.

A major strength of this study lies in its integrative approach, which simultaneously evaluates ER stress and oxidative stress parameters—an area that remains underrepresented in the current literature. Unlike previous investigations that often assess these molecular mechanisms in isolation, our study provides evidence for a potentially interdependent relationship between the unfolded protein response and oxidative imbalance in the pathogenesis of FMS. By combining biochemical analysis with validated clinical scales, we offer a more holistic perspective that connects symptomatic presentation with underlying molecular dysfunction.

In conclusion, our findings offer new insights into the cellular stress responses associated with fibromyalgia and may pave the way for the development of biomarker-driven, targeted therapeutic strategies. These insights have the potential to improve clinical outcomes and quality of life for patients living with this challenging chronic pain disorder.

This study has several limitations that should be acknowledged. First, the cross-sectional design limits our ability to establish causal relationships between ER stress, oxidative stress, and the development or progression of fibromyalgia syndrome (FMS). While associations can be observed, longitudinal studies are necessary to track changes in these biomarkers over time and to determine their potential role in disease onset, symptom fluctuations, and progression.

Second, although the study included 44 FMS patients and 44 healthy controls, the relatively small sample size may affect the statistical power and generalizability of the findings. Larger, multicenter studies are needed to validate these results and to better assess the potential variability introduced by demographic and clinical factors such as age, ethnicity, and disease severity.

Third, although previous studies have reported no significant correlation between the menstrual cycle and fibromyalgia pain severity hormonal variations are known to modulate immune function, pain perception, and cellular stress responses, potentially introducing interindividual variability. We did not synchronize the menstrual phases of participants in our study. While blood samples were collected outside of active menstrual periods, the lack of standardization with respect to the premenstrual and postmenstrual phases introduces potential variability in hormone-related biochemical responses and thus remains a limitation.

Additionally, our study applied strict exclusion criteria to minimize the influence of confounding variables such as autoimmune diseases, metabolic disorders, and other chronic illnesses. While this approach strengthens internal validity, it may also limit the generalizability of the results to the broader FMS population, many of whom present with such comorbidities in real-world clinical settings.

Taken together, these limitations highlight the need for future research involving larger, more diverse populations and longitudinal designs to confirm our findings and further clarify the role of ER stress and oxidative stress in FMS pathogenesis.

Establishment of the FMS patient and control cohort

This study was conducted in accordance with the Declaration of Helsinki (1989) and received approval from the Ethics Committee of Malatya Turgut Özal University Training and Research Hospital (approval number: 2022/37; date: 18.08.2022). Written informed consent was obtained from all participants prior to enrollment. Individuals presenting to the Department of Physical Therapy and Rehabilitation at Malatya Turgut Özal University Training and Research Hospital were recruited. Socio-demographic data—including medical history, age, body mass index (BMI), duration of pain, occupation, marital status, and education level—were recorded at baseline.

Exclusion criteria

Participants were excluded if they had any of the following conditions:

• Inflammatory rheumatic diseases (e.g., rheumatoid arthritis, osteoarthritis).
• Metabolic and endocrine disorders (e.g., obesity, diabetes, metabolic syndrome, thyroid disorders).
• Malignancies.
• Autoimmune diseases.
• Cardiovascular diseases (e.g., hypertension, coronary artery disease).
• Neurological or psychiatric disorders.
• Acute or chronic kidney pathologies.
• Chronic obstructive pulmonary disease (COPD).
• Allergic or dermatological conditions.
• Acute infectious diseases within the past three weeks.
• Tobacco, alcohol, or illicit substance use.
• Use of any medication (including birth control pills) or antioxidant supplements.
• Use of pregabalin, gabapentin, or antidepressants for FMS treatment.
• Use of glucocorticoids, analgesics, or antidepressants.
• Pregnancy or lactation.
• Following a vegetarian diet.

Patient selection and clinical assessment

Because fibromyalgia syndrome (FMS) lacks specific laboratory tests, radiographic findings, or definitive biomarkers, diagnosis is based on clinical criteria. In this study, patients were selected according to the American College of Rheumatology (ACR) 1990 and 2016 diagnostic criteria All participants were informed about the study’s purpose and provided written informed consent in line with the Declaration of Helsinki. Clinical assessments and patient classification were conducted by a physical therapy and rehabilitation specialist. Forty-four women aged 18 years and older who met the ACR criteria for FMS were included in the patient cohort. To assess clinical status, the following validated questionnaires and scales were administered: Fibromyalgia Impact Questionnaire (FIQ), Visual Analog Scale (VAS)⁴⁹, Pittsburgh Sleep Quality Index (PSQI)⁵⁰, Hospital Anxiety Score (HAS), Hospital Depression Score (HDS), and Fatigue Severity Scale (FSS)

Control group selection

The same procedures regarding medical history, exclusion criteria, and adherence to the ACR 1990 and 2016 criteria were applied to recruit healthy volunteers for the control group. Accordingly, 44 healthy women aged 18 years and older who did not meet the diagnostic criteria for FMS were included as controls. The control participants were matched with the FMS patients by age and BMI. Although the control group was matched to the FMS group primarily based on age and BMI to minimize the influence of these confounding variables on biochemical outcomes, psychological variables such as anxiety and depression were not part of the matching criteria. Instead, these variables were evaluated post hoc using validated scales (e.g., HAS and HDS) to characterize psychological profiles and confirm expected differences between groups. This approach reflects a common practice in observational studies; however, we acknowledge that lack of matching on psychological parameters may introduce residual confounding.

Evaluation of ER stress and oxidative stress in serum samples

To reduce the influence of medications on ER stress parameters, FMS patients were instructed to discontinue any medications at least two weeks prior to blood sample collection. However, if pain persisted, patients were permitted to take up to 3,000 mg of acetaminophen orally per day. Blood samples from both FMS patients and healthy controls were collected in the morning after an overnight fast (from 8:00 PM the previous evening until 8:00 AM). Samples were drawn into gel separator tubes for serum isolation and centrifuged at 1200 rcf for 10 min. Serum aliquots were stored in micro-volume Eppendorf tubes at − 80 °C until biochemical analysis.

Serum levels of ER stress markers—GRP78, PERK, CHOP, and ATF6—were measured using commercially available ELISA kits (Cloud-Clone Corp, China; Cat. Nos: SEC343Hu, SEB393Hu, SEE975Hu, SEC260Hu). Total antioxidant status (TAS) and total oxidant status (TOS) were assessed using commercial colorimetric kits (Rel Assay, Gaziantep, Turkey; Cat. Nos: RL0017 and RL0024) following the manufacturers’ protocols.

Statistical analysis

Power analysis was conducted using G*Power software (version 3.1) to estimate the required sample size⁵³. The sample size was determined as 88 (44 in each group) with an effect size of 0.63 (Cohen’s d), a margin of error of 0.05, a confidence level of 0.95, and a representativeness of the universe of 0.90.

Statistical analyses were performed using SPSS version 25. The normality of data distribution was assessed by the Kolmogorov-Smirnov test with a significance threshold of 0.05. Since most variables deviated from normal distribution (p < 0.05), non-parametric tests were applied. Comparisons between two independent groups were conducted using the Mann-Whitney U test. Categorical data were analyzed using cross-tabulations and chi-square tests. The effect size interpretation was based on the following scales: 0.2 = small effect, 0.5 = medium effect, 0.8 = large effect, and > 1.2 = very large effect.