METTL3 mediated m6A modification downregulates WWP2 and aggravates pulmonary ischaemia–reperfusion injury via ubiquitination of DDX3X

Pulmonary ischemia–reperfusion injury (PI-RI) is a serious and clinically prevalent complication that frequently occurs following lung transplantation, cardiopulmonary bypass, or thromboembolectomy, serving as a major determinant of adverse patient prognosis. Despite its profound clinical significance and well-recognized pathogenic role, safe and efficacious therapeutic interventions for PI-RI remain lacking, with no definitive targeted treatment available in clinical practice. The present study was designed to identify the core molecular regulators underlying PI-RI pathogenesis, elucidate the critical signaling pathways driving disease progression, and explore and optimize targeted therapeutic candidates for PI-RI. Specifically, this work focuses on dissecting the regulatory mechanisms of macrophage-mediated inflammatory responses, which are central to PI-RI development. Differentially expressed genes in lung tissues and infiltrating immune cells from PI-RI patients and mouse experimental models were initially screened and validated via quantitative real-time polymerase chain reaction (qRT-PCR). A series of functional validation experiments were subsequently performed, including lentivirus-mediated WWP2 genetic knockdown (sh-WWP2), in vitro macrophage pyroptosis functional assays, mitochondrial stress level profiling, and molecular docking analysis. The regulatory effect of m6A RNA methylation on target gene expression was verified by modulating METTL3 expression and conducting YTHDF1 dependency assays. Co-immunoprecipitation combined with protein structural modeling was utilized to characterize the protein–protein interaction (PPI) between key molecules. In addition, high-throughput drug screening was performed to identify abietic acid as a novel small-molecule modulator targeting WWP2. Significantly downregulated WWP2 expression was observed in lung tissues from clinical PI-RI patients and murine PI-RI models, and single-cell RNA sequencing (scRNA-seq) data further confirmed that WWP2 downregulation was predominantly enriched in pulmonary macrophages. In vivo functional experiments demonstrated that WWP2 knockdown markedly aggravated pulmonary pathological damage in PI-RI mice. Consistently, in vitro assays verified that WWP2 deficiency exacerbated mitochondrial oxidative stress and promoted macrophage pyroptosis. Mechanistically, METTL3-mediated m6A RNA methylation modification reduced the stability of WWP2 mRNA in a YTHDF1-dependent manner. WWP2 directly interacted with DDX3X and facilitated the ubiquitination and proteasomal degradation of DDX3X protein. Structural molecular modeling further revealed that multiple key amino acid residues of WWP2 (S584, S587, Y805, D887, T930, E929, K954, and E955) formed specific hydrogen bonds with DDX3X residues (D134, K138, R145, E149, R259, R296, Q328, K324, G374, and R590). Functional validation assays indicated that WWP2-mediated DDX3X suppression effectively inhibited NLRP3 inflammasome activation, and this protective regulatory effect was completely abrogated by pharmacological inhibition of NLRP3. Furthermore, abietic acid was identified to directly bind to WWP2, enhance the biological function of WWP2, and ultimately alleviate macrophage pyroptosis by repressing excessive inflammatory responses and mitochondrial oxidative stress during PI-RI pathogenesis. This study identifies a previously uncharacterized WWP2–DDX3X–NLRP3 signaling axis that modulates macrophage pyroptosis in PI-RI. Mechanistically, WWP2 restricts macrophage pyroptosis by promoting ubiquitination-dependent DDX3X protein degradation, thereby inhibiting aberrant NLRP3 inflammasome activation and subsequent inflammatory and oxidative damage. Targeting this novel regulatory pathway, particularly via WWP2-stabilizing small-molecule agents such as abietic acid, provides a promising and actionable therapeutic strategy for the clinical prevention and treatment PI-RI.