Abstract: BACKGROUND: Targeting the molecular mechanisms of ferrostatin, intervening in iron metabolism or inhibiting lipid peroxidation is expected to be a new strategy for the treatment of lumbar disc herniation, providing a new research direction for disease prevention and treatment.
OBJECTIVE: To investigate the mechanism of action of the ferroptosis inhibitor ferrostatin-1 on lumbar disc herniation through in vitro cell experiments and in vivo animal studies using poly(lactic-co-glycolic acid)-poly(ethylene glycol)-poly(lactic-co-glycolic acid) hydrogel as a carrier.
METHODS: (1) Third-generation mouse nucleus pulposus cells were divided into three treatment groups: the control group received no treatment; the model group received 10 ng/mL interleukin-1β, and the ferrostatin-1 group received 10 ng/mL interleukin-1β plus 25 μmol/L ferrostatin-1. Intracellular malondialdehyde levels, glutathione levels, iron ion content, and the mRNA expression of extracellular matrix-related genes type II collagen, aggrecan, and matrix metalloproteinase-3 were measured. (2) A poly(lactic-co-glycolic acid)-poly(ethylene glycol)-poly(lactic-co-glycolic acid) hydrogel loaded with ferrostatin 1 (drug-loaded hydrogel) was prepared and characterized for its micromorphology and in vitro drug release. Eighty C57BL/6 mice were randomly divided into a normal group, a model group, a free drug group, and a drug-loaded hydrogel group, with 20 mice in each group. A L5/6 lumbar disc herniation model was established in the remaining three groups, except the normal group. PBS, ferrostatin 1 solution, and drug-loaded hydrogel were injected into the paravertebral tissue of the model group, free drug group, and drug-loaded hydrogel group, respectively. Mechanical and thermal pain thresholds of the mice in each group were dynamically monitored. On the seventh day of drug administration, nucleus pulposus tissue was extracted and levels of inflammatory factors (tumor necrosis factor α and interleukin 1β), malondialdehyde, and glutathione were measured, as well as the mRNA expression of ferroptosis pathway-related genes, glutathione peroxidase 4 and solute carrier family 7 member 11.
RESULTS AND CONCLUSION: (1) Compared with the control group, the model group showed increased intracellular malondialdehyde levels, intracellular iron accumulation, and matrix metalloproteinase-3 mRNA expression (P < 0.05), while decreased glutathione levels and mRNA expression of type II collagen and aggrecan (P < 0.05). Compared with the model group, the ferrostatin 1 group showed decreased intracellular malondialdehyde levels, intracellular iron accumulation, and mRNA expression of matrix metalloproteinase-3 (P < 0.05), while increased glutathione levels and mRNA expression of type II collagen and aggrecan (P < 0.05). (2) Scanning electron microscopy revealed a loose, porous structure containing vacuoles of varying sizes, indicating that the hydrogel exhibited good sustained-release drug properties. Compared with the model group, both the free drug group and the drug-loaded hydrogel group showed pain relief, decreased levels of inflammatory factors and malondialdehyde (P < 0.05), and increased glutathione levels and mRNA expressions of glutathione peroxidase 4 and solute carrier family 7 member 11 (P < 0.05). The drug-loaded hydrogel exhibited a stronger effect than the free drug. (3) The results suggest that ferrostatin 1 protects nucleus pulposus cells by regulating the expression of genes related to oxidative stress and ferroptosis, treating lumbar disc herniation in mice.

Key words: nucleus pulposus cells, lumbar disc herniation, oxidative stress, ferroptosis, hydrogel, ferrostatin 1, biomaterials