<p>Neuropathic pain is a chronic pain condition characterized by complex pathogenesis and poor prognosis. EB (Eupalinolide B), a highly bioactive sesquiterpene lactone derived from Eupatorium lindleyanum DC, has been demonstrated to possess multiple pharmacological activities, including antihistamine, antibacterial, and antioxidant effects. USP7 (ubiquitin-specific protease 7) is a crucial deubiquitinating enzyme in eukaryotes, while the Keap1, Nrf2, and HO-1 signaling pathways play pivotal roles in the development of neuropathic pain. Our study established a spared nerve injury model in mice and employed multiple molecular biology experiments to investigate the regulatory role of EB in the USP7/Keap1/Nrf2 pathway and its mechanisms in neuropathic pain. Results showed significantly elevated USP7 and Keap1 protein expression in the spinal cord of SNI mice, while Nrf2 and HO-1 levels were markedly reduced. EB treatment downregulated USP7 expression, promoted Keap1 ubiquitination and degradation, thereby elevating Nrf2/HO-1 protein levels. This inhibited microglial proliferation and M1 polarization, reduced the production of proinflammatory factors (TNF-α, IL-1β, IL-6), and significantly ameliorated mechanical and thermal hyperalgesia in SNI mice. Long-term intraperitoneal injection of EB did not cause any significant side effects in the heart, liver, or kidneys of SNI mice. In summary, EB exerts anti-inflammatory and analgesic effects by modulating the USP7/Keap1/Nrf2 signaling pathway, offering a potential novel therapeutic strategy for neuropathic pain.</p> Graphical Abstract <p>The mechanism by which EB regulates Keap1 levels through USP7 to relieve neuropathologic pain (Created with BioRender.com)</p> <p></p>