Mechanism of ferrostatin-1 hydrogel in treatment of lumbar disc herniation

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, matrix metalloproteinase 3 were detected. (2) Ferrostatin-1-loaded poly(lactic-co-glycolic acid)-poly(ethylene glycol)-poly(lactic-co-glycolic acid) hydrogel (drug-loaded hydrogel) was prepared, and its microstructure and in vitro drug release were characterized. Eighty C57BL/6 mice were randomly divided into normal, model, free drug, and drug-loaded hydrogel groups (n=20 per group). Except for the normal group, the other three groups were established as L5/6 lumbar disc herniation models. The model, free drug, and drug-loaded hydrogel groups received perivertebral injections of PBS, ferrostatin-1 solution, and drug-loaded hydrogel, respectively. Mechanical and thermal pain thresholds were dynamically monitored. On day 7 after administration, nucleus pulposus tissues were harvested to detect inflammatory factors (tumor necrosis factor α and interleukin-1β), malondialdehyde, glutathione levels, and ferroptosis pathway-related genes glutathione peroxidase 4 and solute carrier family 7 member 11 mRNA expression. RESULTS AND CONCLUSION: (1) Compared with the control group, the model group showed increased intracellular malondialdehyde levels, iron ion accumulation, and matrix metalloproteinase 3 mRNA expression (P < 0.05), while glutathione levels and type II collagen and aggrecan mRNA expression decreased (P < 0.05). Compared with the model group, the ferrostatin-1 group showed decreased intracellular malondialdehyde levels, iron ion accumulation, and matrix metalloproteinase 3 mRNA expression (P < 0.05), while glutathione levels and type II collagen and aggrecan mRNA expression increased (P < 0.05). (2) Scanning electron microscopy showed that the drug-loaded hydrogel had a loose porous structure with vacuoles of varying sizes, and the hydrogel exhibited good sustained-release properties. Compared with the model group, both free drug and drug-loaded hydrogel groups showed pain relief, decreased inflammatory factors and malondialdehyde levels (P < 0.05), and increased glutathione levels and glutathione peroxidase 4 and solute carrier family 7 member 11 mRNA expression (P < 0.05), with the drug-loaded hydrogel showing stronger effects than the free drug. (3) These results indicate that ferrostatin-1 exerts a protective effect on nucleus pulposus cells by regulating oxidative stress and ferroptosis-related gene expression, thereby treating lumbar disc herniation in mice.

1. Introduction

Lumbar disc herniation is characterized by low back pain and radiating leg pain, severely affecting patients' quality of life [1-2]. Studies have found that the nucleus pulposus tissue of patients with lumbar disc herniation often exhibits significant oxidative stress, which is closely related to cell damage and ferroptosis [3-4]. Ferroptosis is a form of programmed cell death dependent on iron and triggered by accumulation of lipid peroxides, playing an important role in the progression of disc herniation [5-6]. During lumbar disc degeneration, nucleus pulposus cells are stimulated by mechanical stress, oxidative stress, and inflammatory responses, leading to iron metabolism imbalance and lipid peroxidation accumulation, thereby inducing ferroptosis [7].

Research has shown that in an oxidative stress model induced by tert-butyl hydroperoxide, changes in ferroptosis marker protein levels and increased lipid peroxidation lead to degeneration of disc cells, exacerbating lumbar disc herniation [8]. Iron overload promotes degeneration and calcification of the cartilaginous endplate by inducing oxidative stress and ferroptosis, significantly aggravating the progression of lumbar disc herniation. Therefore, reducing iron levels and inhibiting related pathological processes may become an effective strategy for preventing and treating lumbar disc herniation [9]. Thus, intervening in iron metabolism or inhibiting lipid peroxidation to regulate ferroptosis may provide new strategies and research directions for the treatment of lumbar disc herniation, with important clinical significance.

Hydrogels, due to their excellent biocompatibility and high water content, have been widely used in drug delivery and tissue engineering [10-11]. Given the special physiological structure of the intervertebral disc, compared with traditional administration methods, local hydrogel delivery offers advantages such as improved drug bioavailability, targeted and sustained release, reduced systemic toxicity, and improved local microenvironment [12-13]. Poly(lactic-co-glycolic acid) and polyethylene glycol are biocompatible polymers approved by the FDA for medical products. Their copolymer, poly(lactic-co-glycolic acid)-poly(ethylene glycol)-poly(lactic-co-glycolic acid), also has low toxicity and does not easily cause immune rejection in vivo, ensuring safety in applications such as drug carriers and tissue engineering [14]. Studies have proven that poly(lactic-co-glycolic acid)-poly(ethylene glycol)-poly(lactic-co-glycolic acid) copolymer... (truncated for brevity)