Ferulic acid ameliorates TLR4-mediated macrophage activation by irreversibly binding to peroxiredoxin 1 to inhibit its dimerization and secretion

Background: In the innate immune system, damage-associated molecular patterns (DAMPs) released by damaged cells, such as peroxiredoxin 1 (PRDX1), interact with Toll-like Receptor 4 (TLR4), exacerbating inflammation and tissue injury. Ferulic acid (FA), a dietary and herbal phenolic compound, exhibits notable anti-inflammatory properties. However, the precise anti-inflammatory mechanisms of FA are not fully understood.

Purpose: The objective of this study was to elucidate mechanisms underlying the anti-inflammatory effects of FA.

Methods: Lipopolysaccharide (LPS) was intratracheally instilled to establish a mouse model of LPS-induced pneumonia. Different doses of FA were injected intraperitoneally, and their anti-inflammatory effects were evaluated. An alkynyl-modified FA probe was used in conjunction with various chemobiological strategies to explore the localization, capture, and identification of FA targets. The pharmacophore of FA and its mechanisms of interaction with its target or pathway were further validated using rigorous biochemical assays and comprehensive transcriptomic profiling. The proposed mechanism of FA against systemic inflammation was validated in mice administered LPS intraperitoneally.

Results: FA alleviated LPS-induced pulmonary inflammation in mice by selectively targeting macrophages. Subsequently, PRDX1 was identified as an irreversible FA-binding target. Mechanistic investigations revealed that α,β-unsaturated ketone in FA serves as a critical pharmacophore that covalently binds to the Cys173 residue of PRDX1. This covalent binding event effectively suppressed PRDX1 dimerization, resulting in reduced PRDX1 secretion. The co-localization assay demonstrated that FA reduced TLR4-binding PRDX1 in LPS-treated RAW264.7 cells. Transcriptomic analysis indicated that NF-κB and TNF signaling pathways, downstream of TLR4 signaling, were involved in the FA-mediated anti-inflammatory effects. Reduction of TLR4 activation caused by FA treatment decreased levels of downstream inflammatory cytokines in LPS-treated RAW264.7 cells. Finally, reduced co-localization of TLR4 and PRDX1 by FA was confirmed in the lung slices of mice with LPS-induced intratracheal inflammation. FA also reduced PRDX1 dimerization and mitigated inflammation in mice administered LPS intraperitoneally.

Conclusion: Our study elucidated a novel mechanism in which FA covalently binds to the Cys173 residue of PRDX1, suppressing its dimerization and secretion and thereby alleviating inflammation by modulating the PRDX1/TLR4 signaling pathway. Our findings redefine plant phenylacrylic acids as covalent modulators of DAMPs, with therapeutic potential for ameliorating inflammation beyond conventional Antioxidants.

Anti-inflammation; Covalent inhibitors; Ferulic acid; PRDX1; TLR4.