Abstract: BACKGROUND: Degeneration of nucleus pulposus cells is a key component of intervertebral disc degeneration.  Ferroptosis, a novel form of programmed cell death, is closely associated with the onset and progression of intervertebral disc degeneration; however, its precise mechanisms remain unclear. 
OBJECTIVE: To establish an oxidative stress model in vitro by inducing ferroptosis in nucleus pulposus cells using tert-butyl hydroperoxide and to investigate the mechanisms of tert-butyl hydroperoxide-induced ferroptosis in nucleus pulposus cells, thereby elucidating the role of ferroptosis in the pathogenesis of intervertebral disc degeneration.
METHODS: Nucleus pulposus cells were treated with varying concentrations of tert-butyl hydroperoxide (0, 25, 50, 100, and 200 μmol/L), and cell morphology and viability were assessed using fluorescence microscopy and the cell counting kit-8 assay. Interventions with 100 μmol/L tert-butyl hydroperoxide, 10 μmol/L 
RSL3, or dimethylsulfoxide were applied to nucleus pulposus cells, and cell proliferation was evaluated using the EdU assay. The expression levels of ferroptosis-related proteins (glutathione peroxidase 4, ferritin heavy chain 1, PTGS2, and ACSL4) and intervertebral disc degeneration marker proteins (matrix metalloproteinase 13 and Col2A) were analyzed via western blot and immunofluorescence. Additionally, reactive oxygen species and lipid peroxidation levels were quantified using the reactive oxygen species detection kit and C11-BODIPY probe. Mitochondrial morphological changes were observed under transmission electron microscopy.
RESULTS AND CONCLUSION: (1) tert-Butyl hydroperoxide treatment significantly reduced the viability and proliferation of nucleus pulposus cells. (2) tert-Butyl hydroperoxide induced typical ferroptosis-related morphological changes in nucleus pulposus cells. (3) tert-Butyl hydroperoxide exposure led to a decrease in the expression of ferroptosis-suppressing proteins glutathione peroxidase 4 and ferritin heavy chain 1, while increasing the expression of ferroptosis-promoting factors ACSL4 and PTGS2. (4) tert-Butyl hydroperoxide elevated intracellular reactive oxygen species production and lipid peroxidation levels in nucleus pulposus cells. (5) Transmission electron microscopy revealed ferroptosis-specific mitochondrial changes in nucleus pulposus cells treated with tert-butyl hydroperoxide, including contraction, reduced cristae, and increased membrane density. (6) tert-Butyl hydroperoxide treatment also resulted in the increased expression of matrix metalloproteinase 13 and decreased expression of Col2A in nucleus pulposus cells. In conclusion, tert-butyl hydroperoxide induces ferroptosis in nucleus pulposus cells, contributing to the development of intervertebral disc degeneration. This process may represent a key pathological mechanism in intervertebral disc degeneration and offers potential targets for developing novel therapeutic strategies.

Key words: tert-butyl hydroperoxide, ferroptosis, nucleus pulposus cells, oxidative stress, intervertebral disc degeneration