Pre-eclampsia-related TLR2/4 signaling activates NOX2/4 to induce oxidative stress and ferroptosis in trophoblasts.

BACKGROUND: Oxidative stress and ferroptosis have been recognized as detrimental factors in trophoblast malfunction in pre-eclampsia (PE). Here, we investigated the roles of toll-like receptor (TLR) signaling in PE-induced ferroptosis in the placenta tissues and explored the therapeutic potential of targeting TLR signaling in the animal model of PE. METHODS: Placental tissues from 20 PE patients and 20 healthy controls were analyzed for TLR2/4 expression and MAPK signaling activation using Western blot and transmission electron microscopy. HTR-8/SVneo trophoblasts were subjected to hypoxia/reoxygenation (H/R) induction to mimic PE conditions. Cell proliferation (CCK-8 assay), migration (scratch assay), invasion (transwell assay), and angiogenesis (tube formation assay) were assessed. Ferroptosis markers were evaluated by Western blot, and oxidative stress parameters including reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), and labile iron were measured using commercial kits. In vivo, a reduced uterine perfusion pressure (RUPP) rat model of PE (n = 6 per group) was established on gestational day 14, and animals were treated with robinin (TLR2/4 inhibitor, 5 mg/kg/day) or VAS2870 (NADPH oxidase [NOX] inhibitor, 5 mg/kg/day) via intraperitoneal injection. Blood pressure, proteinuria, coagulation parameters, and placental ferroptosis markers were analyzed on gestational day 18. RESULTS: TLR2/4 upregulation and mitogen-activated protein kinase (MAPK) signaling over-activation were found in the placental tissues of PE patients and in H/R-induced trophoblasts. Inhibiting TLR2/4 reduced MAPK signaling and improved trophoblast proliferation, migration, invasion, and angiogenesis. TLR2/4-dependent NADPH oxidase 2 and 4 (NOX2 and NOX4) upregulation contributed to the oxidative stress and ferroptosis in trophoblasts upon H/R induction. In the rat model of PE, inhibiting TLR2/4 and NOX activity prevented ferroptotic events in the placental tissues and ameliorated PE syndromes, including elevated blood pressure and proteinuria. CONCLUSIONS: TLR2/4-dependent oxidative stress and ferroptosis may undermine trophoblast function in the PE placenta, and targeting TLR signaling-mediated ferroptosis could serve as an intervention strategy of PE.