Neferine alleviates intervertebral disc degeneration through KEAP1/NRF2/GPX4 and NF-κB signaling pathways

Objective·To investigate the therapeutic effects of neferine (Nef) on intervertebral disc degeneration (IDD) and the underlying regulatory pathways.Methods·The effects of Nef on the viability and proliferation of nucleus pulposus cells were assessed using the cell counting kit-8 (CCK-8) assay. Molecular docking software was employed to analyze the potential binding sites of Nef within the Kelch domain of kelch-like ECH-associated protein 1 (KEAP1). Tumor necrosis factor-α (TNF-α) was used to induce ferroptosis and inflammation in nucleus pulposus cells. Western blotting was performed to detect the expression levels of nuclear factor erythroid 2-related factor 2/glutathione peroxidase 4 (NRF2/GPX4) pathway- and nuclear factor-κB (NF-κB) pathway-related proteins under TNF-α stimulation with or without Nef. The effect of Nef on the metabolism of extracellular matrix in nucleus pulposus cells was evaluated using high-density cell culture. A needle puncture-induced IDD rat model was established, and 5 µL of 1.5 μmol/L Nef was injected twice into the intervertebral disc at the Co3/4 level (IDD+Nef group), while an equivalent volume of PBS was injected into the Co2/3 disc (IDD group). After 4 weeks, the intervertebral space height was detected by X-ray, disc degeneration was detected by magnetic resonance imaging, and disc structure was evaluated by histological staining.Results·The CCK-8 assay revealed that Nef at concentrations of 1.5 μmol/L and below did not inhibit the viability and proliferation of nucleus pulposus cells. Molecular docking results suggested that Nef might activate NRF2 by directly binding to the KEAP1 Kelch domain, thereby reducing the interaction between KEAP1 and NRF2. Western blotting indicated that Nef significantly increased the expression of the key ferroptosis-inhibiting proteins NRF2 and GPX4, while decreasing the expression of the phospho-P65 protein in the NF-κB pathway (all P<0.05). The high-density culture of nucleus pulposus cells demonstrated that Nef mitigated the TNF-α- induced degradation of the extracellular matrix (P<0.05). Animal study results showed that compared to the IDD group, the IDD+Nef group exhibited a greater intervertebral disc space height, a lower Pfirrmann grade (both P<0.05), and a reduced degree of histological degeneration.Conclusion·Nef may inhibit TNF-α-induced ferroptosis in nucleus pulposus cells by activating the KEAP1/NRF2/GPX4 pathway and reduce TNF-α-induced inflammation and extracellular matrix degradation by suppressing the NF-κB pathway, thereby alleviating IDD in rats.