Role and mechanism of AOPPs-induced NOX4-mediated ferroptosis in intervertebral disc degeneration

Intervertebral disc degeneration (IVDD) is a major cause of low back pain, characterized by extracellular matrix breakdown, loss of hydration, and disc height. Ferroptosis, an iron-dependent cell death driven by lipid peroxidation, is implicated in IVDD pathogenesis, but the upstream signals in disc cells remain unclear. Advanced oxidation protein products (AOPPs), oxidative stress-derived protein modifications, accumulate in degenerating discs and activate NADPH oxidase 4 (NOX4), a critical regulator of reactive oxygen species (ROS) and ferroptosis. We hypothesize that AOPPs induce ferroptosis in disc cells via NOX4 activation. In our study, using a rat puncture-induced IVDD model, intradiscal administration of AOPPs exacerbated IVDD, as shown by decreased disc height, higher Pfirrmann grade, matrix degradation, and upregulation of NOX4, TXNIP, and FTH, alongside downregulation of GPX4. In vitro, AOPPs and the ferroptosis inducer Erastin induced ferroptotic changes in nucleus pulposus and annulus fibrosus cells, including elevated MDA, iron overload, GSH depletion, mitochondrial shrinkage, and altered ferroptosis-related proteins, which were reversed by Ferrostatin-1. Pharmacological inhibition or genetic knockdown of NOX4 attenuated AOPPs-induced ferroptosis in vitro and protected against IVDD in vivo. Our results highlight NOX4 as a central regulator in AOPPs-induced ferroptosis, promoting IVDD, and suggest that targeting NOX4 to inhibit ferroptosis may offer a novel therapeutic strategy against IVDD.