Multi-Omics Analysis Reveals Crosstalk between Signaling and Metabolic Reprogramming during Celastrol-Mediated Inflammatory Response

Celastrol (CEL), a bioactive compound derived from Tripterygium wilfordii, exerts potent anti-inflammatory and metabolic-regulating properties, though its underlying mechanisms remain incompletely elucidated. Herein, we combined phosphoproteomics, targeted lipidomics, and metabolomics to delineate how CEL reprograms macrophage metabolism to resolve inflammation. Temporal phosphoproteomic analysis in LPS-stimulated macrophages revealed that CEL dynamically suppressed phosphorylation events across inflammatory pathways, with the PI3K/AKT/mTOR axis emerging as a central pathway regulated by CEL. We successfully quantified 18 PIP2 and 14 PIP3 species by shotgun lipidomics and found that CEL significantly reduced PIP3/PIP2 ratios, inhibiting PI3K activity and attenuating mTOR-dependent phosphorylation of downstream effectors. We further found that the suppression of PI3K disrupted HIF-1α stabilization and nuclear translocation, reversing LPS-driven metabolic shifts: CEL dampened glycolysis while restoring oxidative phosphorylation (OXPHOS) and rectifying TCA cycle fragmentation, as evidenced by reduced succinate/α-ketoglutarate ratios. CEL skewed macrophages toward an anti-inflammatory M2 phenotype, marked by downregulated CD86 and upregulated CD163. Overall, we unveil HIF-1α as a critical mediator of CEL’s anti-inflammatory effect, bridging phosphoprotein signaling rewiring to metabolic reprogramming. This mechanistic insight supports the therapeutic potential of CEL for inflammatory diseases via metabolic modulation.