Rheumatoid arthritis is a chronic autoimmune disease that primarily affects the joints. The disease may progress from painful synovitis to cartilage loss, bone erosion, functional impairment and systemic complications [1]. The pathogenesis results from a complex interaction between innate defenses, adaptive immune responses, stromal cell activation, autoantibody formation and cytokine amplification, ultimately leading to tissue damage and prolonging the disease's duration [2,3]. ACR/EULAR 2010 classification rules were put forward in order to hone early RA recognition, especially for people who show inflammatory arthritis before evidence erosive injury actually takes place [4]. In both clinical practice and research work, disease activity is often measured with combined scores like DAS28, which pulls together swollen joint counts plus inflammatory measures or a patient global assessment so that you get a workable sense of how heavy the disease burden is [5].

Interleukin-22 is now discussed more in relation to autoimmune inflammation and tissue-response mechanisms [6]. In the RA joint environment, IL-22 may support synovial fibroblast activation, drive RANKL upregulation and aid osteoclast-mediated bone breakdown, so in that sense it has a biological rationale for  being  followed as an  inflammatory  biomarker  [7]. Clinical research has found IL-22 included to be higher in RA patients than in healthy controls and there may be a connection to inflammatory patterns as well as serological readouts [6,8].

MicroRNAs is small, non-coding RNA that affect gene regulation after transcription. Therefore, they can steer immune-cell activation, cytokine pathways, inflammatory feedback loops and tissue responses too [9,10]. MiRNA-146a has attracted special concern in RA because it is reported to be increased in peripheral blood mononuclear cells, CD4+ T cells, Th17-related compartments and synovial tissue from affected patients [9,11,12].

Recent findings also support the diagnostic relevance of miRNA-146a as a biomarker. Some research has shown that miRNA-146a can help RA patients from controls and may also follow disease activity, so in a sense it becomes a benefit contender for a wider biomarker panel [13,14]. Circulating microRNA patterns have also been put forth as complementary aids, next to conventional serological tests, particularly when early detection or activity readout is needed [15,16].

Oxidative stress is another meaningful part of RA biology. Reactive oxygen species, lipid peroxidation and antioxidant defense systems overlap quite a lot with inflammation and together they may drive tissue damage, endothelial impairment and a steady, chronic immune arousal [17,18]. Even though several studies have described a reduced antioxidant capacity in RA, overall total antioxidant results can end up moving around depending on how active the disease is, treatment exposure, the dietary or nutritional status and the type of sample that gets used as well as the exact assay method [19,20].

On this basis, the current manuscript presents IL-22 as an inflammatory cytokine marker, miRNA-146a as an epigenetic immune-response marker and total antioxidant status as a redox-related marker. The purpose is to provide a clear and publishable interpretation of the available results while identifying the analyses that still require raw data for final validation.

Study Design

A case-control dataset including 40 RA patients and 40 controls was analyzed. The previously excluded immunological variables were removed from the analysis according to the revised study plan.IL-22 was detected by ELISA Technique, whereas miRNA was restricted by qPCR and spectrophotometer was used to estimate total antioxidant. Statistical analysis was performed in an SPSS-style framework using descriptive statistics, independent-samples comparisons, Spearman correlation and Kruskal-Wallis testing where appropriate.

This case-control study included 40 RA patients and 40 apparently healthy controls. The study collection was conducted from February 2023 to November 2023. All participants had been selected from Al-Forat Hospital and private labs in Najaf Governorate. The provided dataset included serum IL-22, total anti-oxidants and miRNA whole blood, by using ELISA, Spectrophotometer and qPCR respectively.

Biomarkers

Serum IL-22 was considered an inflammatory cytokine biomarker. MiRNA-146a was handled as a relative-expression marker, ideally measured by RT-qPCR and normalized to an endogenous reference gene using the 2^-ΔΔCt method when Ct values are available [14]. Total antioxidant status was treated as a biochemical indicator of global antioxidant capacity and redox balance.

Statistical Analysis Plan

The dataset was arranged for analysis using SPSS version 26. A full statistical workflow should include the Shapiro-Wilk test for normality, independent-samples t-test or Mann-Whitney U test for group comparison, Cohen’s d for effect-size interpretation, ROC analysis for diagnostic performance, chi-square or Fisher’s exact test for classification tables and Pearson or Spearman correlation according to the distribution of the data.

Descriptive Comparison of Biomarkers

All measured biomarkers were higher in the RA group than in the control group. IL-22 increased by 40.6%, miRNA-146a increased by 484.7% and total antioxidant status increased by 104.0%. These findings point toward a combined inflammatory, epigenetic and antioxidant alteration in the RA samples (Table 1).

Table 1: Descriptive Statistics for IL-22, miRNA-146a and total Antioxidant Status in RA Patients and Controls

Derived Effect-Size Summary

The derived statistics showed large effect sizes for the three biomarkers. The most pronounced effect was seen for miRNA-146a, which also showed the highest fold change. These values should be interpreted as approximate because they were calculated from the reported summary data rather than from participant-level measurements (Table 2, Figures 1-4).

Table 2: Derived Summary Statistics Calculated from Reported mean ± SD Values

Figure 1: Corrected mean ± SD Comparison for IL-22 between RA Patients and Controls

Figure 2: Corrected mean ± SD Comparison for miRNA-146a between RA Patients and Controls

Figure 3: Corrected mean ± SD Comparison for Total Antioxidant Status between RA Patients and Controls

Figure 4: Sequential Grouped Comparison of all Biomarkers. The Figure is Descriptive because the Biomarkers have different Measurement Units

ROC Curve Results and Diagnostic Performance

The reported ROC findings indicated excellent diagnostic performance for miRNA-146a and total antioxidant status. IL-22 showed only moderate to borderline discriminatory value. The perfect AUC reported for miRNA-146a should be interpreted carefully, because complete separation between patients and controls is uncommon in clinical biomarker research and should be confirmed in an independent cohort (Table 3, Figures 5-8).

Table 3: Reported ROC Cut-Off Values and Diagnostic Performance after Correction to RA Terminology

Figure 5: Combined ROC Curves based on the Reported AUC, Sensitivity and Specificity Values. Curves are Visual Reconstructions and not recalculated from Raw Data

Figure 6: ROC Curve Visual Reconstruction for IL-22

Figure 7: ROC Curve Visual Reconstruction for miRNA-146a

Figure 8: ROC Curve Visual Reconstruction for Total Antioxidant Status

Diagnostic Classification at Reported Cut-Off Values

The diagnostic classification table was rewritten using RA-appropriate terminology. The word “infected” was removed because RA is not classified as an infectious disease. The classification therefore refers to predicted RA positivity based on the reported cut-off values (Table 4).

Table 4: Diagnostic Classification after Correction to RA Terminology

Correlation Results

The correlation results showed a strong positive association between miRNA-146a and total antioxidant status and also between miRNA-146a and IL-22. By contrast, the relationship between IL-22 and total antioxidant status was weak and not statistically significant (Table 5, Figures 9-11).

Table 5: Reported Pearson Correlations Corrected to RA Biomarker Terminology

Figure 9: Scatter-Plot Visual Representation for miRNA-146a and IL-22 based on the Reported Correlation Coefficient, not on Raw Participant-Level Data

Figure 10: Scatter-Plot Visual Representation for miRNA-146a and Total Antioxidant Status based on the Reported Correlation Coefficient, not on Raw Participant-Level Data

Figure 11: Scatter-Plot Visual Representation for IL-22 and Total Antioxidant Status based on the Reported Correlation Coefficient, not on Raw Participant-Level Data

The present findings show that IL-22, miRNA-146a and total antioxidant status were all increased in patients with RA compared with healthy controls, which honestly looks kind of consistent overall. This pattern is biologically reasonable since RA really does involve immune activation, cytokine signaling, epigenetic regulation and oxidative stress related responses at the same time [3,18]. The rise in IL-22 is in line with earlier clinical observations in RA, where higher IL-22 levels were described in patients and were linked to inflammatory measures or serological markers [6]. Mechanistically speaking, IL-22 might help drive synovial inflammation by nudging fibroblast-like synoviocytes, boosting RANKL expression and also supporting osteoclastogenesis, so in the end these pathways can favor bone erosion and joint damage [7].

In the present dataset, however, the ROC performance of IL-22 was moderate and statistically borderline, so it kinda points to IL-22 being more useful as a supportive inflammatory marker rather than something that works well alone as a single diagnostic test.

MiRNA-146a showed the clearest difference between RA patients and controls. Its marked increase lines up with earlier studies, where miRNA-146a was reported as upregulated in PBMCs, CD4+ T cells, Th17-related cells and also in synovial tissue from RA patients [9-12]. The strong correlation between miRNA-146a and IL-22 in this dataset may suggest that miRNA-146a acts as a proxy for ongoing immune activation, kind of like an inflammatory feedback loop. This view also matches prior work that put forward miRNA-146a as a diagnostic marker, or an activity-linked biomarker in RA [13,14].

The reported perfect diagnostic performance of miRNA-146a should not be overstated. Although it is an encouraging result, perfect AUC values are uncommon in real-world biomarker studies. Such findings may occur when the sample is small, when the two groups are very clearly separated, or when no independent validation cohort is used. Therefore, miRNA-146a appears to be the strongest marker in the available data, but its diagnostic accuracy should be verified using raw data and an external group of patients.

Total antioxidant status was also significantly higher in RA patients and showed excellent reported ROC performance. Result warrants discussion because a few oxidative stress studies have reported that RA has reduced antioxidant capacity along with elevated oxidant markers such as MDA or TOS [19,20].

The higher total antioxidant status in this dataset might be like a compensatory response to chronic inflammation, you know. It may also be due to differences in treatment use, dietary antioxidant intake, disease stage, sample handling, or even the biochemical assay that was used [17,18].

Also, the strong positive correlation between miRNA-146a and total antioxidant status is an interesting outcome, honestly. It suggests that the epigenetic immune response and the redox-related response could be linked in RA. This possible connection should be investigated again in a larger dataset, including disease activity scores, inflammatory markers, treatment details and direct oxidative-stress markers such as MDA, TOS and oxidative stress index.

In summary, the available data indicate that IL-22, miRNA-146a and total antioxidant status are significantly altered in RA patients. Among the three markers, miRNA-146a showed the strongest difference and the best reported diagnostic profile. Total antioxidant status also provided strong diagnostic information, while IL-22 added important inflammatory context.

Informed Consent

Written informed consent was obtained from all participants before sample collection. 

Conflict of Interest

The authors declare no conflict of interest.

Funding

This research received no external founding. 

Data Availability

The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Ethical Approval

This study was approved by Ethics committee of Jaber Ibn Hayyan for Medical and Pharmaceutical sciences.

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