Melatonin Activates KEAP1/NRF2/PTGS2 Pathway to Attenuate Hyperoxia-Driven Ferroptosis in Bronchopulmonary Dysplasia

Abstract

Background and purposes: Ferroptosis, a type of regulated cell death, has been confirmed to play a role in the pathogenesis of bronchopulmonary dysplasia (BPD). This study aimed to test the hypothesis that melatonin mitigates hyperoxia-induced BPD by inhibiting ferroptosis in alveolar epithelial cells, specifically through modulation of the KEAP1/NRF2/PTGS2 signaling pathway.

Methods: Hyperoxia-induced MLE-12 cells and neonatal mice were used to establish BPD models. The effects of melatonin on hyperoxia-induced ferroptosis in MLE-12 cells were assessed by administering melatonin and ferroptosis inducer erastin to these cells. Key target genes involved in melatonin's ameliorative effects on BPD were identified using bioinformatics analysis. To confirm the regulatory relationship between melatonin and the KEAP1/NRF2/PTGS2 pathway, MLE-12 cells were treated with the NRF2 inhibitor ML385 under hyperoxic conditions. Additionally, molecular docking was performed to predict interactions between melatonin and KEAP1.

Results: Melatonin (MT) treatment up-regulated the expression of glutathione peroxidase 4 (GPX4) and xCT in hyperoxia-treated alveolar epithelial cells. The anti-ferroptosis effect of MT on these cells was significantly reduced by ML385, confirming the role of the KEAP1/NRF2 pathway in MT's mechanism of action. In vivo experiments demonstrated that MT up-regulated NRF2, GPX4, and xCT levels and down-regulated KEAP1 and PTGS2 levels in hyperoxia-induced BPD models.

Conclusion: Melatonin exerts a protective effect against hyperoxia-induced BPD by inhibiting ferroptosis in alveolar epithelial cells, and this effect is mediated, at least in part, through the KEAP1/NRF2/PTGS2 axis.

Keywords: KEAP1; NRF2; PTGS2; bronchopulmonary dysplasia; ferroptosis; melatonin.

Figure 1

Melatonin attenuates hyperoxia-induced lung injury. (A) Molecular structure of melatonin (B) HE staining of lung tissue in mice (C) Body weight of mice (D) Radial alveolar count of lung tissue in mice (E) Mean linear intercept of lung tissue in mice (F–H) mRNA levels of IL-1β, IL-6 and TNF-α in lung tissue of mice. Data are expressed as mean ± SD (n = 5), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 indicate significant differences from each group.

Figure 2

Melatonin inhibits ferroptosis in BPD. (A–D) mRNA levels of ferroptosis marker proteins GPX4, xCT, NOX1 and ACSL4 in lung tissue of mice. (E) protein levels of GPX4 and xCT in lung tissue of mice and their quantitative analysis. (F–I) detection of GSH, MDA, Fe²⁺ and lipid ROS kits in lung tissue of mice. (J) immunohistochemical detection and quantitative analysis of lung tissue of mice. *p < 0.05, **p < 0.01; ***p < 0.001, ****p < 0.0001 indicate significant differences from each group.

Figure 3

Erastin abolished the effect of melatonin on MLE-12 cells under hyperoxia condition. (A–C) mRNA levels of ferroptosis marker genes GPX4, xCTand PTGS2 in MLE-12 cells. (D) protein levels of GPX4 and xCT in MLE-12 cells and their quantitative analysis. (E) Reactive oxygen species detection and fluorescence intensity analysis. *p < 0.05, **p < 0.01; ***p < 0.001, ****p < 0.0001 indicate significant differences from each group.

Figure 4

Melatonin can improve BPD by inhibiting ferroptosis by activating KEAP1/NRF2/PTGS2 signaling pathway. (A) Molecular docking prediction of Melatonin and KEAP1. (B)STRING analysis of the related targets of BPD and melatonin. (C) Interactions between KEAP1, PTGS2, and NFE2L2. (D and E) protein levels of KEAP1, NRF2 and PTGS2 in lung tissue of mice and their quantitative analysis. (F and G) Immunofluorescence and fluorescence intensity analysis of NRF2 and PTGS2 in lung tissues of mice. *p < 0.05, **p < 0.01; ***p < 0.001, ****p < 0.0001 indicate significant differences from each group.

Figure 5

ML385 abolished the effect of melatonin on MLE-12 cells under hyperoxia. (A–D) mRNA levels of NRF2, PTGS2, xCT and GPX4 in MLE-12 cells. (E) protein levels of NRF2, KEAP1, PTGS2, GPX4 and xCT in MLE-12 cells and their quantitative analysis. (F) mRNA levels of IL-1β,IL-6 and TNF-αin MLE-12 cells.(G) Reactive oxygen species detection and fluorescence intensity analysis. *p < 0.05, **p < 0.01; ***p < 0.001, ****p < 0.0001 indicate significant differences from each group.