NETosis-Associated Biomarkers and Pro-Inflammatory Cytokines in Collagen-Induced Arthritis: Therapeutic Modulation by Myricetin, RL6 (1,8-Naphthalimide), and CI-Amidine in an Albino Mouse Model

Abstract

Background: Neutrophil extracellular trap (NET) formation — NETosis — plays a central role in rheumatoid arthritis (RA) pathogenesis by generating citrullinated autoantigens that drive autoimmunity and perpetuating synovial inflammation through pro-inflammatory cytokine release. Despite growing evidence, integrated therapeutic targeting of the NET-cytokine-autoantibody axis remains incompletely explored.
Objectives: This study investigated NETosis-associated biomarkers and inflammatory mediators in a collagen-induced arthritis (CIA) mouse model and evaluated the therapeutic efficacy of three mechanistically distinct agents: Myricetin (natural flavonol; MPO and PADI4 inhibitor), RL6 (1,8-Naphthalimide; MPO inhibitor), and CI-Amidine (selective PAD4 inhibitor).
Methods: One hundred and eighty male albino mice were allocated into six groups (n=30 per group): negative control (G1), CIA-induced (G2), CIA untreated chronic (G3), CIA + Myricetin (G4), CIA + RL6 (G5), and CIA + CI-Amidine (G6). Seven biomarkers were quantified by ELISA: MPO-DNA, NE-DNA, IL-6, IL-1β, TNF-α, Cit H3-NE, and ACPA. NET formation was confirmed by fluorescence microscopy. One-way ANOVA with Games-Howell post-hoc test was applied.
Results: CIA induction produced dramatic elevations across all biomarkers, with Cit H3-NE showing the most striking increase (23.96-fold above control; P<0.0001). Progressive ACPA elevation from G2 (74.57±19.60 ng/mL) to G3 (81.87±6.17 ng/mL) demonstrated the self-amplifying autoimmune loop. All three therapeutic agents completely normalized Cit H3-NE to control levels. Myricetin demonstrated superior cytokine suppression (88.4% TNF-α reduction). Pearson correlation revealed the strongest IL-6–ACPA association in chronic CIA (r=0.74, P<0.001).
Conclusion: This study provides comprehensive evidence that NETosis drives RA pathogenesis through an integrated NET-cytokine-autoantibody axis. Myricetin, RL6, and CI-Amidine demonstrated significant anti-NETotic efficacy through distinct but convergent mechanisms, positioning NETosis as a viable multi-target therapeutic strategy in RA.