Ferroptosis, NF-κB expression, and antioxidant imbalance in chorioamnionitis-associated preterm birth

Abstract

Ferroptosis is a regulated form of cell death driven by iron-dependent lipid peroxidation and characterized by antioxidant imbalance, including reduced glutathione peroxidase 4 (GPX4) and an impaired nuclear factor erythroid 2–related factor 2 (NRF2)–heme oxygenase-1 (HO-1) axis. Infection-induced chorioamnionitis is a major risk factor for preterm birth (PTB). Emerging evidence indicates that infections can trigger ferroptosis, and nuclear factor kappa B (NF-κB) signaling may be involved. In this study, flow cytometry quantified plasma cytokines (IL-6, IL-1β, TNF-α) in normal controls (NC), non-chorioamnionitis-associated preterm birth (NC-PTB), and chorioamnionitis-associated preterm birth (CA-PTB) groups. ELISA measured malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and superoxide dismutase (SOD) in placental tissues; total iron (reported as Fe²⁺ equivalents) was quantified with a ferrozine-based assay after reducing ferric iron (Fe³⁺) to ferrous iron (Fe²⁺). Immunohistochemistry assessed NF-κB, GPX4, NRF2, and HO-1 in fetal membranes. The CA-PTB group showed higher inflammatory cytokines (IL-6, IL-1β, TNF-α) and NF-κB expression, alongside reduced antioxidant defenses (GPX4, NRF2, HO-1, SOD). Lipid peroxidation products (MDA, 4-HNE) and iron load (Fe²⁺) were also increased, indicating ferroptosis-related features. NF-κB correlated positively with inflammatory cytokines, lipid peroxidation products (MDA, 4-HNE) and iron load (Fe²⁺), and negatively with antioxidant indices (NRF2,SOD,HO-1); GPX4 showed no significant correlation with NF-κB. Antioxidant indices were inversely related to lipid peroxidation products and iron load. Taken together, our data indicate elevated ferroptosis-related features in CA-PTB alongside NF-κB expression and reduced antioxidant defenses. Whether infection-related NF-κB contributes to ferroptosis requires mechanistic confirmation.