# Aspirin Eugenol Ester Alleviates Vascular Endothelial Ferroptosis by Enhancing Antioxidant Ability and Inhibiting the JNK/c-Jun/NCOA4/FTH Signaling Pathway

## TL;DR

Aspirin eugenol ester (AEE) protects vascular endothelial cells from ferroptosis by boosting antioxidants and blocking harmful signaling pathways.

## Contribution

AEE's novel mechanism of inhibiting ferroptosis via the JNK/c-Jun/NCOA4/FTH pathway is identified.

## Key findings

- AEE reduces ferroptosis and oxidative stress in bovine endothelial cells.
- AEE suppresses JNK/c-Jun phosphorylation and ferritinophagy in endothelial damage models.
- Transcriptomic analysis confirms JNK as a key target of AEE in regulating ferroptosis.

## Abstract

Oxidative stress occurs within bovine when exposed to harmful stimuli, accompanied by substantial accumulation of reactive oxygen species. Without timely clearance, these reactive oxygen species attack vascular endothelial cells, concurrently inducing extensive production of lipid peroxides within the vascular endothelium, and thereby triggering ferroptosis. Aspirin eugenol ester (AEE) showed pharmacological activity against oxidative stress-induced vascular endothelial damage. However, whether it could alleviate vascular endothelial damage by inhibiting ferroptosis remains unclear. This study aimed to evaluate the effects of AEE on vascular endothelial ferroptosis and elucidate its underlying molecular mechanisms. This study established vascular endothelial damage models in vitro and in vivo to explore the ability of AEE to inhibit ferroptosis and oxidative stress by measuring ferroptosis- and oxidative stress-related biomarkers. Transcriptomic and network pharmacology analyses were performed to identify AEE-regulated pathways and key targets. Validation of the pathways were conducted using molecular docking, cellular thermal shift assay, and specific protein agonists/inhibitors. AEE inhibited oxidative stress and ferroptosis in bovine aortic endothelial cells induced by hydrogen peroxide (H2O2) or RSL3 via suppressing the upregulation of ferroptosis-related genes and enhancing the expression of antioxidant genes. Transcriptomic and network pharmacology analyses identified JNK as a core target of AEE in regulating ferroptosis. JNK agonists enhanced H2O2-induced ferritinophagy; on the contrary, JNK inhibitors alleviated it. AEE suppressed H2O2-induced phosphorylation of JNK/c-Jun and ferritinophagy. In a carrageenan-induced rat aortic vascular endothelial damage model, AEE alleviated vascular endothelial damage and ferroptosis-related gene changes, promoted antioxidant gene expression, and inhibited JNK/c-Jun phosphorylation and ferritinophagy. AEE inhibited vascular endothelial ferroptosis by enhancing antioxidant ability, blocking downstream ferritinophagy, and reducing ferrous ion release.