Rhoifolin inhibits ferroptosis and alleviates pulmonary arterial hypertension via the TNF-α/TNF-R1/CASP8/CASP3 pathway.

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, 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.