Pirfenidone Sensitizes Hepatic Stellate Cells to Ferroptosis by Reprogramming Glutamine and Serine Metabolism for GSH Depletion

Pirfenidone (PFD) shows therapeutic potential for liver fibrosis, but its molecular mechanisms are not fully elucidated. Activation of hepatic stellate cells (HSCs) is central to liver fibrosis, making their targeted elimination a prime therapeutic strategy. Since amino acid metabolism governs both HSC activation and ferroptosis, we investigated whether PFD acts by reprogramming these metabolic pathways. Analysis of primary rat HSCs revealed that their in vitro activation induced fibrotic markers, including collagen type I and α-smooth muscle actin, as well as key metabolic enzymes. Specifically, we observed upregulation of glutaminase 1, initiating glutaminolysis to produce glutamate; serine hydroxymethyltransferase 2, which generates glycine from serine; and pyrroline-5-carboxylate synthase, the rate-limiting enzyme for de novo proline synthesis. Treatment with PFD suppressed HSC activation by reducing protein levels of these enzymes, an effect consistent with PFD's inhibition of activating transcription factor 4 nuclear accumulation. This created a dual metabolic vulnerability, limiting amino acid precursors for both collagen synthesis and the master antioxidant glutathione (GSH). Consequently, while PFD alone was not cytotoxic, GSH depletion sensitized activated HSCs to ferroptosis. Co-treatment with the ferroptosis inducer erastin triggered a synergistic increase in reactive oxygen species, labile iron, and lipid peroxidation, culminating in cell death. This synergistic lethality was abrogated by the ferroptosis inhibitor ferrostatin-1 and the antioxidant N-acetylcysteine, confirming ferroptosis as the specific cell death modality. Our study uncovers a dual anti-fibrotic mechanism for PFD: PFD inhibits collagen synthesis by limiting key amino acid precursors and depletes GSH. This compromises antioxidant defenses, creating vulnerability to ferroptosis. Our findings establish a rationale for using PFD in combination therapies designed to eliminate activated HSCs.