Differential expression of lysine-acetylated proteins in the anti-inflammatory effects of luteolin-gadolinium on macrophages

This study investigates the anti-inflammatory mechanisms of luteolin-gadolinium rare earth complexes (LutGdRCS) in LPS-activated macrophages, focusing on lysine-acetylated protein regulation. Transcriptional (qPCR), proteomic (4D-DIA tandem mass spectrometry), and functional analyses revealed that LutGdRCS significantly downregulated pro-inflammatory mediators (iNOS, IL-6, IL-1β) and nitric oxide (NO) levels. Quantitative acetylome profiling identified 1260 lysine-acetylation sites across 796 proteins, with 775 upregulated and 485 downregulated sites. Key proteins-Eef2, Rpl5, and Atp5c1-emerged as central hubs in protein-protein interaction networks, while Atp5f1cK_89 was identified as a critical deacetylation site linked to mitochondrial ATP synthase activity. Subcellular localization analysis showed cytoplasmic (34.7 %), nuclear (34.1 %), and mitochondrial (10.6 %) enrichment of modified proteins. Pathway mapping highlighted LutGdRCS-driven modulation of NF-κB, JAK-STAT, oxidative phosphorylation, and nitrogen metabolism pathways. Bioinformatic clustering further revealed altered ubiquitin ligase activity, antioxidant responses, and ribosomal functions. These findings demonstrate that LutGdRCS attenuates inflammation by dynamically regulating lysine acetylation, particularly through mitochondrial energy metabolism and immune signaling pathways. The study positions LutGdRCS as a novel rare earth-based therapeutic candidate for inflammatory disorders, offering mechanistic insights into its acetylome-level anti-inflammatory effects.