Enhanced systemic oxidative stress response in patients with idiopathic inflammatory myopathies

Abstract

Background

Idiopathic inflammatory myopathies (IIM) are characterized by chronic inflammation, endothelial dysfunction, and muscle tissue mitochondrial defect, leading to the local oxidative stress response. However, data on its systemic intensity and correlation with IIM clinical and laboratory characteristics remains scarce.

Methods

In clinically stable dermatomyositis (n = 18) and myositis (n = 38) patients and matched controls (n = 50), we measured global oxidative stress response in peripheral blood using a novel coumarin boronic acid (CBA) assay enabling real-time detection of protein hydroperoxides (HP) formed in serum.

Results

We documented 36% faster kinetics (p < 0.001) and a 68% increase in the cumulative (p = 0.003) fluorescent product generation in the IIM group compared to the control, which indicates higher HP formation associated with systemic oxidative stress. The dynamics of fluorescent product growth were similar in the dermatomyositis and myositis groups. Interestingly, myositis patients with a marked increase in HP formation were characterized by lower serum myoglobin levels (p = 0.038). There was also an inverse correlation between serum myoglobin and the kinetics of HP formation (e.g., for cumulative in-time generation r = –0.35, p = 0.03). The systemic oxidative stress response measures were not related to clinical characteristics of the disease and treatment, internal medicine comorbidities, smoking status, or autoantibody profile.

Conclusions

IIM are characterized by a global pro-oxidant imbalance reflected by enhanced HP generation in serum. Furthermore, muscle weakening without active signs of muscle damage may be related to the increased local and systemic oxidative stress response, suggesting non-inflammatory pathomechanism of the disease that our technically undemanding assay may evaluate.

Introduction

Idiopathic inflammatory myopathies (IIM) encompass a spectrum of rare autoimmune disorders primarily characterized by immune-mediated muscle injury [1]. Although classified into one group, their pathogenesis, clinical manifestation, and pattern of organ involvement vary [2, 3]. Adult IIM patients can be categorized into dermatomyositis (DM), myositis of an anti-synthetase syndrome, immune mediated-necrotizing myopathy (IMNM), inclusion body myositis (IBM), or undefined polymyositis, based on clinical symptoms and autoantibodies profile [4]. There is evidence of a correlation between specific types of myositis-specific antibodies (MSAs) and the clinical phenotype of the disease, including interstitial lung disease (ILD), dysphagia, polyarthritis, and constitutional symptoms, along with Raynaud’s phenomenon [1, 5, 6]. Nevertheless, MSAs are detected in about 50% of IIM patients [7], challenging the proper diagnosis of the disease. Despite such heterogeneity, most patients share fundamental characteristics, including proximal skeletal muscle weakness and signs of immune-mediated muscular injury [8].

A main barrier to advancements in IIM is a limited understanding of disease pathogenesis that results from a complex interplay of genetic predisposition and environmental triggers [9]. Moreover, IIM pathomechanisms have specific differences depending on the immunological basis. Among humoral factors, interferon (IFN) pathways have been identified as major contributors to certain disease subtypes, as evidenced, for example, by marked type I IFN (IFN-α, β, κ, ε, and w) signature in DM [10,11,12]. By contrast, anti-synthetase syndrome, IBM, and IMNM show relatively low activation of the I IFN pathway [10]. Still, smaller studies have documented type II IFN (IFN-γ) pathway engagement in those diseases [10, 13, 14], consistently with overexpression of major histocompatibility complex (MHC) class II [14, 15]. Elevated MHC classes I and II in muscle tissue have been reported as a hallmark of IIM, leading to T-cell and NK activation and muscle fiber damage [16]. That may be induced by IFNs themselves, but other pro-inflammatory cytokines that have been detected in the muscle tissues of myositis patients, such as interleukin(IL)−1α, IL-1β, tumor necrosis factor (TNF), and many chemokines may also contribute [17]. On the other hand, in anti-synthetase syndromes, disturbances in RNA processing seem to be critical for disease development, possibly implying the pathogenic roles of tRNA synthetic pathways [18]. However, other pro-inflammatory mechanisms have also been reported here. For example, histidyl-tRNA synthetase antigen (anti-Jo-1 antigen) can function as a chemokine acting through the CCR5 chemokine receptor. Hence, it could be involved in recruiting CD4 + and CD8 + T lymphocytes and antigen-presenting cells to the site of auto-antigen overexpression, e.g., in the lungs [19].

Besides, the pathology of IIM is also related to non-immune-mediated mechanisms, such as a disturbed microcirculation due to phenotype changes of endothelial cells or capillary loss [18, 20, 21]. Emerging data show that endothelial cells constitute a primary immune response target, leading to vasculopathy, followed by tissue hypoxia and metabolic alterations, at least in a subset of IIM patients. Consequently, a deficiency in energy sources for muscle contractions could cause muscle weakness and fatigue [18]. Another possible non-immune-mediated mechanism of muscle dysfunction in IIM is the so-called endoplasmatic reticulum (ER) stress response [22]. That is likely due to the MHC class I up-regulation in muscle fibers, which leads to the ER overload response and unfolded protein response [22]. It is postulated that ER stress might be a major non-immune mechanism responsible for skeletal muscle dysfunction without muscle fiber damage [22].

Both inflammation and hypoxia may lead to increased reactive oxygen species (ROS) generation by inflammatory and endothelial cells, driving oxidative stress response [23,24,25]. However, recent research points to the muscle tissue mitochondria and respiratory chain defects as the primary source of locally produced ROS, contributing to muscle weakness and fatigue in IIM patients, particularly those with IBM [16, 26,27,28]. Nevertheless, it remains unclear whether this phenomenon is local, limited to muscles, or results in global oxidative stress with its systemic implications. Data on measuring pro-oxidative balance in peripheral blood are scarce, and comprehensive studies have not been performed. Earlier studies focused primarily on the antioxidant status, including analysis of the levels of high-density lipoprotein [29], superoxide dismutase [30], serum bilirubin or uric acid [31].

Thus, in the current study, we sought to evaluate the global oxidative stress response in the peripheral blood of IIM patients based on a recently developed novel coumarin boronic acid (CBA) assay that measures the kinetics of amino acid and protein hydroperoxides (HP) formed in serum [32]. We analyzed their relationship with clinical disease manifestations and laboratory findings to gain insight into the IIM pathomechanism.

Patients and methods

Study population

IIM patients (n = 56) were enrolled at the Department of Allergy and Clinical Immunology and the Department of Dermatology of the University Hospital, Kraków, Poland, between 2016 and 2019. IIM diagnosis was based on the 1975 Bohan and Peter diagnostic criteria [33, 34]. Only patients who met the criteria for "definite" or "probable" IIM with clinically stable disease were included. In 2017, the European League Against Rheumatism/American College of Rheumatology (EULAR/ACR) published the new classification criteria for IIM [35]. According to these criteria, we distinguished IIM patients as having dermatomyositis (DM), which consisted of DM with signs of muscle injury (n = 9, 50%), amyopathic dermatomyositis (n = 2, 11%), and hypomyopathic dermatomyositis (n = 7, 39%) and a group of “other myositis”. The ‘other myositis’ group comprised patients diagnosed with IMNM (n = 6, 16%), antisynthetase-syndrome myositis (n = 19, 50%), and other polymyositis (n = 13, 34%). For simplicity, this group of different diagnoses is called "myositis" in the text and figures. We did not enroll patients with IBM due to its unique clinical and pathological features [28].

Disease stability was assessed during the six months preceding patient enrollment. Every two months, the physician analyzed patient- and physician-reported outcome measures using a visual analog scale and 0–10 points scoring of the Manual Muscle Testing 8 (MMT-8) sheet, as indicated by Disease Activity Core Set Measures (DACSM), according to the International Myositis Assessment and Clinical Studies Group (IMACS) recommendations [36, 37]. During each visit, we also assessed serum levels of muscle-associated markers, including creatine kinase and myoglobin. Only those with low variance and stable measures, that is, with no trend toward disease progression in scores or biomarkers during the previous 6 months, were involved in the study [38].

The control group (n = 50) comprised volunteers from the hospital personnel with no personal or family history of connective tissue disorders. Control subjects were matched with the IIM group regarding age, sex, body mass index (BMI), smoking habit, and internal medicine comorbidities.

The exclusion criteria for both groups consisted of the following: any acute illness within the last two months, a history of cardiovascular events, such as stroke and myocardial infarction, active malignancy (e.g., cancer diagnosis <5 years, metastatic or recurrent cancer), heart failure with ejection fraction below 40%, atrial fibrillation, ongoing anticoagulant therapy, liver injury (alanine aminotransferase [ALT] > twofold above the upper normal range), and kidney insufficiency (estimated glomerular filtration rate evaluated using Modification of Diet in Renal Disease formula <60 mL/min/1.73 m²), pregnancy, postpartum period and breastfeeding. Subjects with arterial hypertension, diabetes mellitus, or hypercholesterolemia, as well as individuals with a history or current smoking, were eligible to participate. IIM-associated interstitial lung disease (ILD) was characterized by typical radiological findings on chest computed tomography imaging. A high probability of pulmonary hypertension was defined as a pulmonary artery systolic pressure >45 mm Hg measured via transthoracic echocardiography.

The study was approved by the Bioethics Committee of the Jagiellonian University Medical College (No. 1072.6120.200.2021). All procedures adhered to the ethical principles outlined in the Declaration of Helsinki. Additionally, each participant received detailed information about the study’s methods and safety procedures and provided written informed consent before enrollment.

Laboratory investigations

Complete blood cell count, myoglobin and C-reactive protein (CRP) serum levels, the activity of alanine aminotransferase (ALT), aspartate transaminase (AST), creatine kinase, and other blood measurements were assessed with standard laboratory techniques. Anti-nuclear antibodies (ANA) were screened using an indirect immunofluorescence method. MSAs and myositis-associated autoantibodies were quantified with line-blot immunoassay (Autoimmune Inflammatory Myopathies 16 Ag #DL1530-4; Euroline, Lϋbeck, Germany). Serum aliquots were stored at −70 °C for further CBA measurements.

Coumarin boronic acid assay

To evaluate oxidative stress response in peripheral blood, we used a modification of the CBA assay that allows for real-time monitoring of protein and amino acid hydroperoxides (HP) formation. It has been described in detail in our previous publication [32]. In brief, 50 μL of serum sample was mixed with 150 μL phosphate buffer (pH 7.4) containing catalase (100 units/mL), DTPA (diethylenetriaminepentaacetic acid; 0.1 mM), and non-fluorescent CBA as a substrate (0.8 mM). The fluorescence (FL) intensity of the reaction product, 4-hydroxycoumarin (COH), was recorded for 20 h at 10-min intervals (Excitation/Emission: 360/465 nm; ClarioStar plate reader, BMG Labtech, Ortenberg, Germany). Background FL was subtracted, and the results were normalized to the total serum protein concentration. The FL intensity exhibited exponential growth, which was fitted to the logistic model. The resulting curve reflects the dynamics of fluorescent product formation; therefore, HP contents were characterized by three parameters: (1) the saturating concentration K, indicating the numerical upper limit of fluorescent product growth; (2) the rate factor R, describing the velocity of the fluorescent product growth; and (3) the area under the curve until the saturation level reached, representing the cumulative in time CBA fluorescent product generation.

Statistical methods

Statistical analysis was conducted using Statistica 13.3 software (TIBCO Software Inc., Palo Alto, CA, USA) and R (version 3.6.1). Categorical variables were presented as numbers (percentages), and group differences were calculated using the Chi² or exact Fisher test, as appropriate. The distribution of continuous data was assessed using the Shapiro–Wilk test. They were non-normally distributed, thus presented as the median with the Q1-Q3 range and compared by the Mann–Whitney test. The associations between continuous variables were analyzed using the Spearman rank correlation test. For the CBA assay variables, Box-Cox transformation was applied, and one-way covariance analysis (ANCOVA) was utilized to adjust for potential confounders, including age, sex, and BMI. Statistical significance was set at a p-value less than 0.05 for all analyses.

Results

Characteristics of the patients

We enrolled 56 IIM patients (median age 58.5 years; 66.1% women) and 50 control subjects (median age 54.0 years; 54.0% women). The studied groups (patients vs. controls) were similar according to demographic factors, including age, sex, BMI, and comorbidities (Table 1). However, in the subgroup analyses, the myositis group compared to controls was slightly older (p = 0.02), had more often hypercholesterolemia (p < 0.001) and comprised more patients who were smoking in the past (p = 0.03), whereas th DM group compared to controls was more often diagnosed with hypercholesterolemia (p = 0.03). As expected, IIM patients showed increased serum levels of CRP compared to the control group. They also had lower blood hemoglobin levels, decreased serum creatinine concentrations, increased serum activity of ALT and AST, and elevated fibrinogen in plasma (Table 1). Furthermore, IIM patients showed moderately increased levels of triglycerides, decreased HDL cholesterol, and a higher frequency of hypercholesterolemia. Based on clinical characteristics, IIM patients were stratified into DM (n = 18, 32.1%) and myositis (n = 38, 67.9%) subgroups (Table 2). There were no differences in the baseline laboratory tests if both subgroups were compared (Table 1).

Table 1 Demographic characteristics, internal medicine comorbidities, smoking habits, and basic laboratory test results in dermatomyositis and myositis patients and a control group

Table 2 Autoimmune disease-specific features in dermatomyositis and myositis patients

Clinical and laboratory characteristics of dermatomyositis and myositis patients

The median disease duration was 2 years (Table 2). Among the muscle-related clinical features, weakness in the lower limbs was the most common symptom (n = 37, 66.1%), followed by shoulder girdle weakness (n = 30, 53.6%). ILD was documented in more than half of the patients (n = 31, 55.4%), representing the most frequent internal organ involvement, more prevalent in the myositis group (68.4% compared to 27.8% in DM; p = 0.004). All DM patients had typical skin symptoms during the disease, with Gottron’s sign (n = 13, 72.2%), V-sign (n = 11, 61.1%), and heliotrope eruption (n = 8, 44.4%) being the most common.

Overall, anti-nuclear antibodies were detected in 94.6% of IIM patients, with no difference between DM and myositis subgroups regarding the titer (Table 2). Anti-Ro52 antibodies were the most prevalent (39.3% in all IIM) and detected more frequently in myositis compared to DM cases (50% vs. 16.7%; p = 0.017). Anti-Jo-1 antibodies were also more common in myositis than in DM (31.6% vs. 5.6%; p = 0.042).

We observed six cases of previous neoplasms (thyroid gland, bladder, cervix, ovarian cancer, endometrial cancer, and lymphoma) in the myositis group and only one case in the DM group (breast cancer); yet the occurrence of neoplasms was similar between groups (p = 0.65).

Enhanced oxidative stress response in dermatomyositis and myositis patients

The intensity of the fluorescent product generation in the CBA assay exhibited a rapid increase in both studied groups. However, IIM patients showed considerable heterogeneity in the course of fluorescence curves and the kinetics of fluorescent product growth (Fig. 1ab), independently of DM and myositis diagnosis (Table 3). Overall, there was a 1.36-fold increase in the velocity of fluorescence product growth (factor R) and a 1.38-fold increase in saturating concentration K in IIM patients compared to the control group (Table 3). Similarly, cumulative in-time fluorescent product generation was nearly 70% higher in the IIM group than in the control (Table 3). These results were also present in separate analyses of DM and myositis cases (Table 3) but not in comparing two patient subgroups (Fig. 1c, Table 3).

Fig. 1

Figure legend. a Fluorescent product curves in the real-time coumarin boronic acid (CBA) assay in control subjects and IIM patients. Bold lines indicate median values (for clarity, axes were truncated at 1600 FLU/mL and 10 h). b Summary plot showing the median FLU values with interquartile range (shaded area) in all IIM patients (n = 56) and controls (n = 50). c The saturating concentration (K), growth velocity (R factor), and cumulative fluorescent (FL) product generation in the real-time coumarin boronic acid (CBA) assay in dermatomyositis (DM) and polymyositis (PM) patients and control group. Data are presented as median with interquartile range. * p < 0.05, ** p < 0.01 (Mann–Whitney test). Abbreviations: IIM – idiopathic inflammatory myopathy; DM – dermatomyositis; FLU – fluorescence units, ns – non-significant

Table 3 Parameters of fluorescent product generation in the real-time coumarin boronic acid assay in idiopathic inflammatory myopathies patients

The dynamics of HP generation were similar concerning DM and myositis subsets, organ involvement, and smoking habits (Supplementary Tables S1-S3). Furthermore, disease duration and antibody profile were unrelated to significant changes (data not shown).

Oxidative stress response correlated inversely with myoglobin levels in myositis patients

Next, we determined if systemic HP formation was associated with IIM-related clinical and laboratory measures. First, we compared subgroups of myositis patients with signs of high vs. low/moderate systemic oxidative stress, using 0.75 quartile of cumulative fluorescent product generation as a cutoff value (Fig. 2ab). Only the myositis cohort was included in this analysis, as merely three DM patients showed markedly increased CBA assay kinetics. Myositis patients with signs of high oxidative stress (n = 10) did not differ in demographic and clinical variables compared to myositis cases with low/moderate HP formation (n = 28), including similar organ involvement and markers of inflammation (Table 4). Interestingly, myositis patients with markedly increased parameters of HP formation were characterized by a less pronounced muscle injury. For example, in this subgroup, we observed decreased serum myoglobin levels compared to the remaining patients (32.3 vs. 67.5 µg/L, p = 0.037; Fig. 2c), and also a trend toward decreased serum activity of creatine kinase (p = 0.08, Table 4). Moreover, in myositis patients characterized by high oxidative stress, we also documented a tendency (p = 0.1) toward lower reported (during the course of the disease) maximum activity of serum creatine kinase (Fig. 2d). Surprisingly, these patients also showed increased median ANA titers (1:10,240 vs. 1:1280 in low/moderate group, p = 0.014) and lower prevalence of anti-Ro-52 myositis-associated autoantibodies with no differences regarding MSAs as compared to myositis patients with low/moderate systemic oxidative stress response (Table 4).

Fig. 2

Figure legend. Marked systemic oxidative stress response is associated with decreased serum myoglobin levels in most myositis patients. a Graph showing cumulative generation of fluorescent (FL) product in CBA assay showing stratification of myositis patients into those with signs of high (≥ 0.75 quartile [Q], n = 10) or low/moderate (< 0.75 Q, n = 28) systemic stress response. b FL curves of real-time CBA assay in individual myositis patients stratified as in “a”. Bold lines indicate median values with shaded areas marking 0.25 to 0.75 Q ranges. For clarity, axes were truncated at 1600 FLU/mL and 10 h. c Serum myoglobin concentration in myositis patients with high or low/moderate serum protein hydroperoxides (HP) formation. Abbreviations: UL, an upper limit of the reference range. *p < 0.05 (Mann–Whitney test). d The highest reported (disease history) serum creatine kinase activity in the indicated subgroups of myositis. e Scatter plot of the correlation between cumulative FL product generation (FLU/mLxmin) and serum myoglobin levels (µg/L) in myositis patients. Abbreviations: r_S – Spearman coefficient

Table 4 Clinical and laboratory characteristics of myositis patients stratified into high vs. low/moderate systemic oxidative stress response

These results were also confirmed by association analysis that showed a weak negative correlation between systemic oxidative stress parameters and serum myoglobin (e.g., for R: r = –0.34, p = 0.034, and for cumulative fluorescent product generation: r = –0.35, p = 0.03) in the myositis group (Fig. 2e). However, there was no significant correlation with makers of inflammation (e.g., for CRP and cumulative fluorescent product generation, p = 0.28) or other laboratory measures (data not shown).

We did not document similar associations in separate analyses of the DM group; however, patients with DM who reported general muscle weakness (n = 10, 55.6%) showed 56% higher dynamics of HP formation compared to the remaining DM cases (for R: 63.9 [50.5–73.8] vs. 40.8 [33.5–43.9], respectively, p = 0.037).

Discussion

In the present study, we document increased global oxidative stress measured in the peripheral blood of IIM patients using a real-time CBA assay. The velocity of fluorescent product growth (factor R) and the saturating concentration (K) were nearly 40% higher, while cumulative in-time fluorescent product generation was approximately 70% higher in IIM patients compared to the control group. We also confirmed a significant variance in CBA results in the IIM group, with only one-fourth of myositis patients characterized by very high fluorescent product generation. That suggests a considerable heterogeneity of ROS production and subsequent protein HP formation in the IIM group. Finally, subgroup analysis revealed that myositis patients with signs of high systemic oxidative stress response showed decreased serum myoglobin levels and a tendency toward lower muscle injury markers in the disease history, yet with a similar disease course, organ involvement, and myositis-specific antibody profile.

Several potential sources of ROS have been suggested to contribute to the excessive local oxidative stress in muscle tissue of IIM patients [39]. One is related to the immunologic factors, such as ROS produced directly by inflammatory cells localized in muscle fibers or skin and stimulated by cytokines, e.g., IFNs and IL-1β [27, 40]. Another source refers to muscle mitochondrial dysfunction, whose causes are not fully elucidated but lead to cellular hypoxia and changes in calcium influx [27, 41]. Mitochondria and respiratory chain defects in muscle tissue contribute to muscle weakening and local ROS production, with further muscle fiber fatigue and aberrant ER function as consequences [26, 27, 40]. However, the most important reason for ER dysfunction is the ER stress pathway secondary to the abundant accumulation of unfolded or misfolded proteins, likely overproduced MHC class I complex. It may disrupt further pro-oxidative balance essential for proper ER function [26, 40]. ER stress may subsequently induce pro-inflammatory signaling, for example, via the NF-κB activation, enhancing further ROS production but also inducing the autophagy pathway [40, 42, 43]. All the abovementioned abnormalities lead to excessive local muscle ROS generation, cell hypoxia, and reduced ATP synthesis, ultimately contributing to muscle weakness and fatigue. Our data show that this local pro-oxidant balance likely draws muscle weakening but also increases global systemic oxidative stress, analyzed by HP formation in peripheral blood. This corresponds to our intriguing negative correlation between oxidative stress parameters and serum myoglobin levels in the myositis group and increased general weakness in DM patients with higher HP formation. That outcome seems to be one of the most essential in our results.

The current study’s oxidative stress parameters were unrelated to the disease clinics and the presence of specific MSAs. Previously, Huang et al. [30] suggested a potential connection between systemic oxidative stress, documented by reduced levels of superoxide dismutase (SOD), and the occurrence of interstitial lung disease (ILD) in individuals with DM who tested positive for anti-MDA5 antibodies. We did not confirm similar associations, which may be related to low sample size and considerable heterogeneity in the magnitude of systemic oxidative stress response identified in our cohort. Our data suggests that increased ROS production in IIM reflects non-inflammatory mechanisms unrelated to clinical presentation, such as lung involvement, specific autoantibody profile, and systemic inflammatory state. Thus, one might speculate that non-immunologic pathways, like mitochondrial dysfunction, ER stress, capillary damage, and metabolic defects, could be critical for them.

Interestingly, there were no differences in the dynamics of CBA assay parameters regarding sex, BMI, and internal medicine comorbidities or smoking status in the IIM group. Previously, we reported that systemic oxidative stress corresponding measures are also increased in other inflammatory diseases, such as asthma [44] and systemic sclerosis [45]. Nevertheless, compared to them, oxidative stress parameters in IIM were unrelated to inflammatory biomarkers. This observation suggests a different regulatory mechanism of pro-oxidant balance in IIM compared to asthma or systemic sclerosis, where inflammation and smoking were significant determinants of higher CBA assay parameters also analyzed in serum. Furthermore, that discrepancy again underlines that redox imbalance in IIM is likely linked to molecular mechanisms on the muscle cell level, corresponding to the disease pathogenesis and requiring further investigation.

Finally, the clinical significance of enhanced oxidative stress response in a subset of IIM is not yet fully understood, necessitating further research to uncover the specific mechanisms linking oxidative stress to the pathogenesis of these conditions. Nevertheless, promoting lifestyle modifications, such as a balanced diet and regular exercise to reduce oxidative stress, could complement standard treatments for managing IIM patients.

Study limitation

The general limitation of our study is the relatively small number of individuals, particularly in the subgroup analyses. We did not evaluate pro-inflammatory cytokines in peripheral blood, which could provide further insights into IIM pathogenesis. We also did not include IBM patients, for which high oxidative stress response is well documented [26, 27]. Here, we aimed to focus on other IIM subsets that are not well characterized in terms of oxidative stress response. Consequently, some relationships observed may be incidental rather than indicative of a cause-and-effect relationship. Nevertheless, considering the rarity of IIM, even a study on a small patient cohort is valuable. Our analysis focused solely on proteins and did not encompass other molecules like lipids and nucleic acids. Next, the CBA assay was conducted as a one-time assessment; thus, we cannot exclude the variability of HP formation over time.

Conclusions

In conclusion, the current study reveals that patients with IIM exhibit elevated systemic oxidative stress response, as indicated by increased amino acid and protein HP levels detected in the serum using real-time CBA assay. Considerable heterogeneity in the magnitude of HP formation in IIM patients and negative association with muscle injury biomarkers suggest a variable contribution of oxidative stress in the pathogenesis of the disease. Further investigation is necessary to elucidate the underlying mechanisms and potential therapeutic targets of oxidative stress-related pathways, paving the way for more effective treatment strategies and improved patient outcomes in IIM.