Osteoarthritis (OA) is characterized by the activation of nucleotide-binding and oligomerization domain-like receptor protein 3 (NLRP3) by thioredoxin-interacting protein (TXNIP), which promotes inflammation and pyroptosis. This study investigates whether treadmill exercise (TE) enhances melatonin-mediated regulation of the TXNIP/NLRP3 pathway and attenuates OA progression by modulating pyroptosis in a destabilization of the medial meniscus (DMM) rat model. A total of 32 male Sprague–Dawley rats (6 weeks old; 220 ± 20 g) were randomly assigned to four groups: Sham, DMM, DMM + melatonin, and DMM + melatonin + TE. The intervention lasted for 8 weeks. Morphological staining, immunofluorescence (IF), microcomputed tomography with three-dimensional reconstruction, Western blot, quantitative real-time polymerase chain reaction, and enzyme-linked immunosorbent assay were used to assess protein and gene expression. Compared with the DMM + melatonin group, the DMM + melatonin + TE group showed greater reductions in cartilage–subchondral bone damage and OA progression through modulation of the pyroptosis pathway. IF staining revealed that TXNIP protein expression was significantly reduced in the DMM + melatonin + TE group. Both the DMM + melatonin and combination treatment groups significantly regulated TXNIP/NLRP3 signaling and inhibited OA progression through pyroptosis. Furthermore, combining TE with melatonin significantly reduced the expression of pyroptotic cytokines compared with the DMM + melatonin group. This study suggests a potential therapeutic approach for managing OA by combining melatonin treatment with moderate TE.

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