Thonningianin a mitigates intervertebral disc degeneration by inhibiting ferroptosis via chelating Fe2+ and regulating the adenosine monophosphate-activated protein kinase (AMPK)/ nuclear factor erythroid 2-related factor 2 (Nrf2)/ glutathione peroxidase 4 (GPX4) pathway.

Background

Oxidative stress and ferroptosis play critical roles in the pathogenesis of intervertebral disc degeneration (IVDD). Thonningianin A (TA) is a complex polyphenolic ellagitannin found in plants like Thonningia sanguinea, and it exhibits free radical scavenging, anti-superoxide anion generation and metal-chelating activities. However, whether it can inhibit oxidative stress, ferroptosis and the progression of IVDD remains unclear.

Purpose

This study aimed to elucidate the molecular mechanism by which TA alleviates IVDD, specifically regarding oxidative stress and ferroptosis, and to validate these mechanisms in both cellular and animal models.

Methods

An RSL3-induced nucleus pulposus cells (NPCs) ferroptosis model was established, with rescue using Fer-1, DFO and various concentrations of TA along with siRNA validation. Ferroptosis, oxidative stress and extracellular matrix (ECM) related proteins were assessed by western blot, immunofluorescence, fluorescent probes, molecular docking, molecular dynamics simulations and DFT binding energy analysis. In vivo, a rat tail puncture IVDD model was treated with TA or Fer-1, and evaluated by X-ray, MRI, Safranin-O/fast green (S-O) staining, Hematoxylin and Eosin (HE) staining and immunohistochemistry.

Results

Mechanistic studies revealed that TA activates adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and promotes the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and subsequently upregulates the expression of glutathione peroxidase 4 (GPX4), thereby inhibiting oxidative stress, ECM degradation and ferroptosis. Additionally, TA modulates intracellular iron homeostasis by chelating Fe²⁺, thereby further attenuating ferroptosis. In a rat model, local intradiscal injection and intraperitoneal administration of TA effectively inhibited the progression of IVDD.

Conclusion

TA inhibits oxidative stress, ECM degradation and ferroptosis in NPCs by activating the AMPK/Nrf2/GPX4 axis and chelating Fe²⁺, thereby suppressing the progression of IVDD.