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

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.