Daphnetin alleviates neuropathic pain in rats by modulating spinal microglial polarization via the Nrf2/NLRP3...

Neuropathic pain (NP) is a frequent sequel to peripheral nerve injury and maladaptive nervous system function. Daphnetin (DAP), a bioactive 7,8-dihydroxycoumarin derived from the traditional Chinese medical plant Daphne giraldii Nitsche, has potential anti-nociceptive effects and alleviated NP following intrathecal injection. However, the molecular target of DAP for this action remains unknown.

This study aims to investigate the underlying mechanisms of DAP against NP.

Chronic constriction injury (CCI) rat model and lipopolysaccharide (LPS)-treated HAPI microglial cells were established to evaluate the analgesic and anti-inflammatory effects and underlying mechanisms of DAP. Rat behavioral assessments included mechanical withdrawal threshold (MWT), thermal withdrawal latency (TWL), and gait analysis. The expression of mRNA was determined by qRT-PCR. Protein expression levels were detected by Western blot, immunofluorescence, and enzyme linked immunosorbent assay. Molecular docking, surface plasmon resonance, molecular dynamics simulation, and site-directed mutagenesis were employed to investigate the interaction between Kelch-like ECH-associated protein 1 (Keap1) and DAP.

DAP treatment significantly increased MWT and TWT in CCI rats. It also improved gait parameters and attenuated spinal inflammation, neuronal damage, and hyperexcitability in CCI rats. DAP treatment markedly increased the expression of Nrf2 and its nuclear translocation in the rat spinal cord and HAPI cells. DAP treatment inhibited the activation of NLRP3 inflammasome and microglial M1 phenotype polarization, while promoting M2 phenotype polarization. Furthermore, the NLRP3 inhibitor MCC950 also alleviated hyperalgesia in CCI rats, inhibited NLRP3 inflammasome activation, suppressed microglial M1 polarization, and promotes M2 polarization, whereas the Nrf2 inhibitor ML385 exerted the opposite effect. It reversed the regulatory effect of DAP on spinal microglial polarization in the CCI rat spinal cord and LPS-induced HAPI cells. Molecular interaction studies further demonstrated that DAP binds to Tyr334 and Tyr572 residues in the Kelch domain of Keap1, thereby blocking Keap1/Nrf2 interaction to activate Nrf2.

DAP treatment inhibits microglial M1 polarization and promotes M2 polarization via the Nrf2/NLRP3 inflammasome pathway, attenuates neuroinflammation and central sensitization, and finally achieves analgesic effects. These findings identify DAP as a novel Nrf2 activator with therapeutic potential for the management of NP.