Digoxin inhibits HIF-1α to attenuate inflammation and fibrosis in thyroid eye disease.

Thyroid eye disease (TED) is characterized by orbital inflammation and fibrosis, with a hypoxic microenvironment implicated in disease pathogenesis. To determine whether hypoxia-inducible factor-1α (HIF-1α) mediates hypoxia-induced inflammation and fibrosis in TED, and whether the HIF-1α inhibitor digoxin suppresses these processes. Serum HIF-1α was measured by ELISA in TED patients (n = 12) and healthy controls (n = 7). Primary orbital fibroblasts (OFs) from TED patients were transduced with lentiviral HIF-1α or exposed to hypoxia (1% O2). Inflammatory and fibrotic markers were assessed by qRT-PCR, western blotting, and ELISA. STAT3 phosphorylation and GSDME-mediated pyroptosis were examined. Digoxin (1.5-6 nM) was tested for its ability to reverse HIF-1α-induced changes. Serum HIF-1α was significantly elevated in TED patients. HIF-1α overexpression recapitulated hypoxia-induced effects, upregulating IL-6, IL-8, CCL2, CCL8, CXCL1, CXCL2, IL-1β, TIMP1, α-SMA, and COL1A1, increasing STAT3 phosphorylation and activating GSDME-mediated pyroptosis. Digoxin dose-dependently suppressed HIF-1α-driven inflammatory and fibrotic responses, reduced STAT3 phosphorylation, and attenuated cleaved caspase-3 and GSDME-NT expression. HIF-1α is a critical downstream mediator of hypoxia-induced inflammation and fibrosis in TED, acting through STAT3 and GSDME-mediated pyroptosis. Pharmacological inhibition of HIF-1α by digoxin reverses these changes, positioning digoxin as a promising therapeutic candidate for TED.