Objective To investigate the molecular mechanisms by which ginsenoside Rb₃ (Rb₃) inhibits lipid accumulation in macrophages. Methods Oxidized low-density lipoprotein (ox-LDL) was used to induce lipid-associated macrophages (LAMs) model in murine RAW264.7 macrophages. After Rb₃ intervention, intracellular lipid accumulation was evaluated by oil red O staining and BODIPY-based neutral lipid fluorescence staining. Changes in lipid composition were analyzed using untargeted lipidomics. Transcriptomic and proteomic analyses were performed in parallel, followed by integrative multi-omics bioinformatic analysis. Molecular docking was conducted using Autodock to predict the binding potential between Rb₃ and candidate target molecules identified from multi-omics intersections. The expression levels of key genes and proteins were validated by qRT-PCR and Western blotting. Cell thermal shift assay (CETSA) was employed to evaluate the binding of Rb₃ to complement component 5a receptor 1 (C5ar1) and cluster of differentiation 36 antigen (Cd36) targets. Results A lipid-enriched LAMs model was successfully established. Within a non-cytotoxic concentration range, Rb₃ dose-dependently suppressed intracellular lipid accumulation (P < 0.05, 0.01, 0.001). Lipidomics revealed a marked elevation of triglycerides accompanied by a reduction in glycerophospholipids in model group, whereas Rb₃ treatment significantly reduced triglyceride levels and restored glycerophospholipids, with pathway enrichment mainly involving glycerophospholipid metabolism. Transcriptomic analysis identified 1 291 differentially expressed genes, which were significantly enriched in peroxisome proliferator-activated receptor (PPAR) signaling pathway. Proteomic analysis detected 254 differentially expressed proteins, with enrichment in pathways including amyotrophic lateral sclerosis-related signaling. Integrative multi-omics analysis identified six overlapping target molecules, including Cd36, C5ar1 and adenosine triphosphate binding cassette transporter G1 (Abcg1). Molecular docking predicted favorable binding affinities between Rb₃ and Cd36, C5ar1, Abcg1. CETSA results demonstrated that Rb₃ significantly protected Cd36 and C5ar1 proteins from temperature-dependent degradation, providing further evidence of direct binding between Rb₃ and these targets. qRT-PCR and Western blotting results showed that Rb₃ significantly down-regulated the expressions of Cd36 and C5ar1 in LAMs (P < 0.05, 0.01, 0.001), while up-regulated the expressions of PPARγ, Abca1 and Abcg1 (P < 0.05). Conclusion Rb₃ activates PPAR signaling pathway and regulates glycerophospholipid metabolism, targeting Cd36 and C5ar1 to inhibit their mediated lipid uptake and promote Abca1 and Abcg1 mediated lipid efflux, regulating the intracellular levels of triglycerides and glycerophospholipids, thereby exerting an inhibitory effect on lipid accumulation in macrophages.

lipid-associated macrophages  /  ginsenoside Rb₃  /  multi-omics analysis  /  PPAR signaling pathway  /  glycerophospholipid metabolism  /  Cd36  /  C5ar1