# Eupalinolide B Alleviates Oxidative Stress in LPS-Induced RAW264.7 Macrophages via Covalently Binding to PRDX4

## TL;DR

Eupalinolide B reduces oxidative stress in macrophages by covalently binding to PRDX4, a key antioxidant protein.

## Contribution

This study reveals a novel mechanism where a natural compound enhances antioxidant capacity by covalently stabilizing PRDX4.

## Key findings

- Eupalinolide B reduces ROS, NO, and MDA levels while increasing SOD activity and GSH/GSSG ratio in LPS-induced macrophages.
- Eupalinolide B covalently binds to Cys54 and Cys248 residues of PRDX4, stabilizing the protein and enhancing antioxidant effects.
- PRDX4 knockdown diminishes the antioxidant effects of Eupalinolide B, confirming its functional role.

## Abstract

Background/Objectives: Eupalinolide B (EB), a natural compound derived from Eupatorium lindleyanum DC, has demonstrated multiple pharmacological activities. However, its role in modulating oxidative stress remains incompletely understood. Methods: In this study, we investigated the antioxidant effect and underlying mechanism of EB in lipopolysaccharide (LPS)-induced RAW264.7 macrophages. Results: EB significantly attenuated LPS-induced oxidative stress as evidenced by reduced levels of intracellular reactive oxygen species (ROS), nitric oxide (NO), and malondialdehyde (MDA) alongside enhanced superoxide dismutase (SOD) activity and an increased reduced/oxidized glutathione (GSH/GSSG) ratio. Using activity-based protein profiling, we identified peroxiredoxin 4 (PRDX4) as a key binding target of EB. Direct interaction was confirmed through labeling and competitive binding assays with purified PRDX4 protein. High-resolution mass spectrometry revealed that EB covalently binds to Cys54 and Cys248 residues of PRDX4. Furthermore, EB treatment upregulated PRDX4 protein expression in LPS-stimulated RAW264.7 cells. siRNA-mediated knockdown of PRDX4 significantly blunted the antioxidant effects of EB, confirming the functional relevance of this target. Conclusions: Our findings demonstrate that EB alleviates LPS-induced oxidative stress in macrophages by covalently binding to and stabilizing PRDX4, thereby enhancing cellular antioxidant capacity. This study unveils a novel mechanism whereby a natural product enhances cellular antioxidant capacity by covalently stabilizing a key peroxidase, highlighting the potential of EB as a therapeutic agent and PRDX4 as a promising target for oxidative stress-related diseases.