Melatonin Ameliorates Neuropathic Pain in Mice by Regulating Pyroptosis Through Gut Microbiota-Mediated Linoleic Acid

Neuropathic pain (NP) presents a significant challenge for patients with chronic diseases, characterized by spontaneous pain and hyperalgesia, with recent studies highlighting the involvement of the gut microbiota in its pathogenesis. Increasing evidence suggests that melatonin mitigates systemic inflammation and remodels gut microbiota. The present study further explored the mechanism by which oral melatonin ameliorates pain-related behavior following peripheral nerve injury. In this study, mice with chronic constriction injury (CCI) were orally administered melatonin (100 mg/kg) for 14 days. Pain behaviors were evaluated using mechanical allodynia and thermal hyperalgesia tests. Gut microbiota profiles were assessed via 16S rRNA sequencing, and microbiota-dependent effects were verified through antibiotic treatment (ABX) and fecal microbiota transplantation (FMT). Linoleic acid (LA) levels in serum and spinal cord were measured using liquid chromatography-mass spectrometry (LC-MS), and protein expression of NLRP3, caspase-1, and GSDMD in spinal cord tissue was examined by immunoblotting. Melatonin treatment significantly increased the mechanical pain threshold and prolonged thermal withdrawal latency in CCI mice. It also remodeled the dysbiotic gut microbiota, and these protective effects were abolished by antibiotic treatment but restored by FMT. Protein analysis revealed upregulation of NLRP3 and GSDMD in CCI mice, both of which were suppressed following melatonin administration. Moreover, melatonin-induced microbiota modulation elevated LA levels in serum and spinal cord. Supplementation with LA alone alleviated NP and inhibited NLRP3, caspase-1, and GSDMD activation. These findings suggest that oral melatonin treatment effectively ameliorates NP in CCI mice associated with the neuroinflammation and microbiota-metabolites-brain axis.