Abstract

Background Pulmonary arterial hypertension (PAH) is a fatal disease driven by progressive vascular remodeling. Emerging evidence links ferroptosis, an iron-dependent form of regulated cell death, to PAH pathogenesis, suggesting that inhibiting ferroptosis is a promising therapeutic strategy. Rhoifolin (ROF), a natural flavonoid with multiple bioactivities, has not been studied in PAH. Purpose This study aimed to systematically investigate the therapeutic potential of ROF against PAH, focusing on its inhibition of ferroptosis and the underlying molecular mechanisms. Methods An integrative approach was employed. Network pharmacology predicted common targets of ROF, ferroptosis, and PAH. Molecular docking and dynamics simulations assessed binding stability. Predictions were rigorously validated in vitro using Erastin-stimulated rat pulmonary arterial smooth muscle cells (rPASMCs) and in vivo using a monocrotaline-induced PAH rat model. Results Bioinformatics analysis identified 60 common targets and highlighted the TNF-α/TNF-R1/CASP8/CASP3 axis within the lipid and atherosclerosis pathway as a key mechanism. In vitro, ROF directly rescued rPASMCs from Erastin-induced ferroptosis, confirming its cell-protective effect. In vivo, ROF treatment ameliorated hemodynamic and remodeling indices, reduced pulmonary ferroptosis markers (Fe 2+ , MDA), and restored anti-ferroptotic defenses (GSH, GPX4). Concurrently, it downregulated the protein levels of the TNF-α/TNF-R1/CASP8/CASP3 axis. Computational studies confirmed stable binding of ROF to key targets. Conclusions This study is the first to demonstrate that ROF alleviates PAH by inhibiting ferroptosis, a mechanism linked to modulation of the TNF-α/TNF-R1/CASP8/CASP3 signaling axis. Our findings position ROF as a novel multi-target candidate for PAH therapy.