Dual TYK2/JAK1 Inhibition by Brepocitinib Suppresses TNF-α–Driven Oxidative Stress, PI3K/AKT/mTOR Activation and FAK-Dependent Migration in Rheumatoid Arthritis Synoviocytes

Background: Rheumatoid arthritis (RA) synoviocytes acquire pathogenic phenotype characterized by oxidative stress, sustained activation of pro-survival signaling, enhanced migration, and excessive production of inflammatory mediators. Brepocitinib, a dual TYK2/JAK1 inhibitor, has demonstrated immunomodulatory activity; however, its effects on TNF-α–driven redox imbalance, kinase signaling, and migratory behavior in RA synoviocytes remain incompletely defined. Methods: Human RA synoviocytes were stimulated with TNF-α (10 ng/mL) and treated with brepocitinib (1–5 µM). Intracellular reactive oxygen species (ROS) were quantified using DCFDA fluorescence. Nrf2–Keap1 antioxidant signaling was examined by nuclear and cytosolic fractionation followed by immunoblotting for Nrf2, Keap1, HO-1, and NQO1. Activation of PI3K–AKT–mTOR and focal adhesion kinase (FAK)–associated cytoskeletal signaling was assessed by immunoblotting for phosphorylated PI3K, AKT (Ser473), mTOR (Ser2448), FAK (Y397), and cofilin. Cell migration was evaluated using a scratch wound-healing assay. Inflammatory responses were analyzed by qRT-PCR and ELISA for cytokines, chemokines, and matrix metalloproteinases, and correlation analysis was performed between ROS and IL-6 expression. Results: TNF-α markedly increased intracellular ROS and disrupted antioxidant defenses by suppressing nuclear Nrf2, elevating Keap1, and downregulating HO-1 and NQO1. Brepocitinib dose-dependently reduced ROS accumulation and restored Nrf2-dependent antioxidant signaling. TNF-α–induced activation of PI3K–AKT–mTOR and FAK pathways and synoviocyte migration were significantly attenuated by brepocitinib. In parallel, brepocitinib suppressed TNF-α–induced expression and secretion of IL-6, TNF-α, chemokines, and matrix metalloproteinases. ROS levels showed a strong positive correlation with IL-6 expression. Conclusion: Brepocitinib reprograms TNF-α–activated RA synoviocytes by restoring antioxidant defenses, suppressing survival and migratory signaling, limiting inflammatory amplification and pathogenic synoviocyte behavior in RA.