Zonisamide attenuates morphine tolerance in association with reduced spinal TLR4/p38 MAPK signaling.

Introduction: Morphine tolerance limits its long-term clinical utility, and neuroinflammation is a key underlying mechanism. This study investigated whether zonisamide, an antiepileptic drug with anti-inflammatory properties, attenuates morphine-induced neuroinflammation and morphine tolerance. Methods: In vitro, BV-2 microglial cells were treated with morphine (200 μM) with or without zonisamide (10 μM). Quantitative real-time PCR, enzyme-linked immunosorbent assay (ELISA), and Western blot analysis were used to assess inflammatory mediators and signaling pathways. In vivo, male C57BL/6 mice received repeated morphine injections (10 mg/kg, s.c., twice daily) for 9 days to induce tolerance, with or without zonisamide (30 mg/kg, i.p.). Behavioral tests (hot plate and tail flick) were performed to evaluate analgesic tolerance. Immunofluorescence was performed to assess microglial activation (Iba1), and Western blot analyses were conducted to evaluate TLR4 expression, p38 phosphorylation, and pro-inflammatory cytokine protein levels. Pharmacological inhibition of TLR4 with TAK-242 was used to verify the involvement of TLR4 signaling. Results: In vitro, zonisamide suppressed morphine-induced upregulation of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and Toll-like receptor 4 (TLR4) mRNA, reduced morphine-induced secretion of IL-1β, IL-6, and TNF-α at the protein level, and inhibited morphine-induced p38 mitogen-activated protein kinase (MAPK) phosphorylation. In vivo, zonisamide did not affect acute morphine analgesia but significantly attenuated the development of chronic morphine tolerance. Spinal cord analyses revealed that zonisamide reduced microglial activation (Iba1), TLR4 expression, p38 phosphorylation, and pro-inflammatory cytokine (IL-1β, TNF-α) protein levels. Pharmacological inhibition of TLR4 with TAK-242 produced a behavioral effect similar to that of zonisamide, and combined treatment did not provide additional benefit, suggesting overlapping signaling effects. Discussion: These findings indicate that zonisamide attenuates morphine tolerance in association with reduced spinal TLR4/p38 MAPK signaling and neuroinflammation. This study provides a mechanistic rationale for repurposing zonisamide as an adjunct therapy to improve long-term pain management and mitigate opioid-related adverse effects.