Endothelial-derived FABP4 promotes EGFR activation in fibroblast-like synoviocytes during osteoarthritis synovial ferroptosis

Ferroptosis contributes to osteoarthritis (OA) synovial pathology, but upstream regulatory networks and druggable targets remain unclear. Huashi Dingtong Decoction (HSDT) was used as a pharmacological probe to identify candidate networks and active components inhibiting OA synovial ferroptosis. HSDT efficacy was assessed in a monosodium iodoacetate (MIA)-induced OA rat model and a TNF-α-stimulated fibroblast-like synoviocyte (FLS) model. Serum metabolomics, network pharmacology, machine learning, and single-cell transcriptomics prioritized candidate targets. Mechanistic validation used an RSL3-induced FLS ferroptosis model, siRNA silencing, the FABP4 inhibitor BMS309403, co-immunoprecipitation (Co-IP), and HUVEC–FLS paracrine co-culture. Rescue experiments used ferrostatin-1 (Fer-1) and deferoxamine (DFO); in vivo rescue used a DMM-induced OA rat model with AAV-mediated FABP4 overexpression. HSDT ameliorated synovitis and cartilage degeneration in MIA rats, reduced serum IL-1β and IL-6, and reversed metabolic disturbances. HSDT reversed ferroptosis phenotypes, including elevated Fe 2 ⁺, MDA, and 4-HNE and decreased GPX4, SOD, and GSH/GSSG. Integrated analysis prioritized a candidate FABP4/EGFR/HIF-1α/VEGFA network. FABP4 knockdown inhibited EGFR phosphorylation, restored GPX4, and reduced lipid peroxidation and intracellular Fe 2 ⁺, whereas additional EGFR knockdown produced no further effect. BMS309403 enhanced the anti-ferroptotic effect of HSDT. Co-IP suggested that HSDT reduced EGFR-associated FABP4 in parallel with reduced total FABP4. Paracrine co-culture supported a model in which endothelial-derived FABP4 activates EGFR phosphorylation in FLS. In DMM rats, AAV-FABP4 overexpression partially attenuated the ameliorative effects of HSDT. HSDT may inhibit endothelial-derived FABP4 expression and secretion, thereby attenuating endothelial–FLS paracrine activation. This was accompanied by reduced total FABP4 abundance and reduced EGFR-co-immunoprecipitated/co-localized FABP4 signal, along with decreased EGFR/HIF-1α/VEGFA network-associated activity and ferroptosis-related phenotypes. These findings are hypothesis-generating and require direct molecular validation. Scutellarein reproduced part of the HSDT effect in vitro and may serve as a candidate active component, although its in vivo efficacy, pharmacokinetics, and target engagement remain to be established. Together, these data suggest a candidate targetable multicellular network in OA synovial ferroptosis, but the causal hierarchy and direct molecular interactions remain to be established.