Novel anti-rheumatic potential of Eucalrobusone C: inhibition of rheumatoid arthritis fibroblast-like synoviocytes and metabolic reprogramming.

Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by synovial hyperplasia, inflammatory cell infiltration, and joint destruction. This study investigates the inhibitory effects and metabolic mechanisms of Eucalrobusone C (EC), a novel formyl-phloroglucinol meroterpenoid derivative isolated from Eucalyptus robusta, on Tumour Necrosis Factor-α (TNF-α)-induced rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs). EC was extracted and purified, with purity confirmed using 1H Nuclear Magnetic Resonance Spectrum (NMR) at 400 MHz. RA-FLSs were exposed to varying concentrations of EC, followed by comprehensive assessment including CCK8 assay for cell proliferation, flow cytometry for cell death, and Transwell assay for migration and invasion capacity. Metabolomic profiling employed Ultra-High Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (UHPLC-Q-TOF MS), integrated with multivariate statistical analysis and bioinformatics tools to identify metabolic alterations. Results indicated that EC suppressed RA-FLS proliferation in a time- and concentration-dependent manner, significantly enhanced apoptosis, and inhibited cell migration and invasion. Metabolomics analysis detected 898 metabolites, with 112 upregulated and 67 downregulated in EC-treated groups compared to TNF-α-induced controls. Key differentially expressed metabolites were enriched in pathways including ABC transporters, neuroactive ligand-receptor interactions, protein digestion and absorption, and cAMP signalling. These findings suggest that EC exerts anti-rheumatic effects by modulating these metabolic pathways, offering potential as a therapeutic agent for RA management.