RBX1 mitigates ferroptosis by inhibiting NCOA4-mediated ferritinophagy and contributes to the attenuation of intervertebral disc degeneration

Loss of nucleus pulposus (NP) cells is as one of the primary factors initiating intervertebral disc (IVD) degeneration (IVDD); however, the intrinsic physiological mechanisms of endogenous NP-derived stem cell (NPSC)-based therapy in IVDD remain poorly understood. Disturbed iron homeostasis is commonly observed in degenerative diseases, and an acidic microenvironment has been considered a crucial factor in IVDD. The molecular mechanism of ferroptosis in acidic microenvironments during IVDD has not been reported. Herein, we intended to investigate whether acidic conditions can induce ferroptosis in NPSCs and explore the mechanism of IVDD progression through NCOA4-mediated ferritinophagy, which is a type of selective autophagy mediating ferroptosis. The role of ring-box 1 (RBX1) in NCOA4-mediated ferritinophagy in NPSC ferroptosis and IVDD pathogenesis was also explored. First, clinical epidemiology research revealed that a reduction in serum ferritin level was an independent risk factor for IVDD. We then demonstrated that ferroptosis progressively increased in human NP tissues as IVDD advanced and the acidic conditions induced ferroptosis-associated decline in cell viability, reactive oxygen species accumulation, and extracellular matrix degradation in human NPSCs. In an acidic microenvironment, ferroptosis is promoted due to enhanced NCOA4-mediated ferritinophagy in NPSCs. A mechanistic study demonstrated that RBX1-mediated ubiquitination modulated NCOA4 expression and the inhibition of RBX1 promoted ferroptosis through NCOA4-mediated ferritinophagy in the human NPSCs. Our in vivo study further illustrated that RBX1 overexpression ameliorated ferroptotic effects on IVDD progression by suppressing NCOA4-mediated ferritinophagy. Results demonstrated the modulating role of RBX1 in NCOA4-mediated ferritinophagy and NPSC ferroptosis, providing valuable insights into the potential application of endogenous stem cell-based IVD self-repair and self-regeneration for IVDD treatment.