Targeting Nox4 alleviates TMJ osteoarthritis by restoring redox homeostasis and suppressing ferroptosis in macrophages

Objectives: Temporomandibular osteoarthritis (TMJ OA) is a degenerative disease with pain, abnormal sound, and restricted mouth - opening. Oxidative stress and ferroptosis contribute to its progression, and inflammatory macrophages in the synovium are crucial. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (Nox4), a major Reactive Oxygen Species (ROS) source, is involved in joint disorders, but the mechanism of Nox4 regulating macrophage ferroptosis in TMJ OA is unclear. Method: RNA-sequencing was performed on synovial tissues obtained from patients or rats with or without TMJ OA to identify potential therapeutic targets for TMJ OA. Synovial tissues were examined histologically after staining with immunofluorescence. The impact of silencing Nox4 gene expression on inflammatory macrophages was explored. RT-qPCR and Western blotting were used to evaluate the expression of inflammatory and antioxidant genes. Intracellular and mitochondrial ROS were detected using DCFH-DA, DHE, or MitoSOX probes. Mitochondrial membrane potential was detected using the JC-1 probe. Lipid peroxidation and ferroptosis were measured using C11-BODIPY and FerroOrange. RNA-seq was used to analyze the anti-inflammatory mechanisms of the Nox4 inhibitor GLX351322 (40 μM) in LPS-stimulated macrophages. Results: In this study, we observed a marked upregulation of Nox4 expression in synovial macrophages from both TMJ OA patients and rats. Inhibition of Nox4 significantly attenuated inflammatory responses through activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. Upon inhibition of Nox4, ROS production was reduced, accompanied by a reduction in mitochondrial damage. Meanwhile, Nox4 inhibition decreased ferroptosis in inflammatory macrophages by mitigating oxidative stress-induced lipid peroxidation. Conclusion: In this study, we identified Nox4 as a potential therapeutic target in TMJ OA. Targeting Nox4 counld reduce macrophage inflammation by alleviating oxidative stess-induced ferroptosis.