Abstract

Dysregulated macrophage polarization is a critical pathological driver of sepsis-induced acute lung injury (ALI). However, the absence of novel therapeutic targets constitutes a significant translational barrier, underscoring the urgent need for advancements in precision medicine. In this study, we demonstrate that the natural product luteolin (LU) effectively promotes LPS-induced M1-to-M2 macrophage polarization by modulating the expression of pro-inflammatory and anti-inflammatory cytokines. Subsequently, thermal proteome profiling identifies receptor-type protein tyrosine phosphatase α (RPTPα) as a direct cellular target of LU in macrophages, which is validated by drug affinity responsive target stability, microscale thermophoresis, and surface plasmon resonance assays. Meanwhile, LU treatment inhibits RPTPα phosphatase activity. Molecular docking suggests that LU interacts with the 405 PFTP 408 motif, impairing substrate recognition and subsequently suppressing the enzymatic activity of RPTPα. Furthermore, transcriptomic profiling reveals that LU significantly dysregulates 1,402 genes. Integrated kyoto encyclopedia of genes and genomes (KEGG) and gene set enrichment analysis demonstrate that LU suppresses the tumor necrosis factor (TNF) signaling pathways, which is reversed upon RPTPα silencing. In vivo, LU exhibits potent anti-inflammatory effects in both BALB/c mice with sepsis-induced ALI and CuSO 4 -induced zebrafish inflammation models. Collectively, our study reveals that RPTPα is a potential therapeutic target for modulating macrophage polarization. Moreover, LU may serve as a lead compound targeting RPTPα for the treatment of sepsis-induced ALI. Graphical Utilizing the stable isotope labeling by amino acids in cell culture-thermal proteome profiling platform, we identified receptor-type protein tyrosine phosphatase α (RPTPα) as a direct target of the natural flavonoid luteolin (LU), inducing M1-to-M2 macrophage polarization and conferring anti-inflamma