Hypermethylation of the Nrf2 Promoter Induces Ferroptosis by Inhibiting the Nrf2-GPX4 Axis in COPD

Abstract

Background Nuclear factor E2-related factor 2 (Nrf2) is involved in oxidative stress and lung inflammation and regulates the etiology of chronic obstructive pulmonary disease (COPD). Ferroptosis is characterized by the accumulation of lipid reactive oxygen species (ROS) via ferrous ion-dependent Fenton reactions and is involved in COPD. However, the role of Nrf2 in ferroptosis and its epigenetic regulation in the pathogenesis of COPD remain unclear. Methods Ferroptosis was detected by 4-HNE, MDA, C11BODIPY, DCFH-DA, Peals’ staining and CCK-8 assays. qPCR and Western blotting were performed to examine the Nrf2 levels in peripheral lung tissues, primary epithelial cells collected from patients with COPD and subjects with normal pulmonary function (never-smoker [control-NS]; smoker [control-S]), and cigarette smoke extract (CSE)-treated human bronchial epithelial (HBE) cells. ELISA was used to quantify IL-8 and IL-1β levels. Methylation of the Nrf2 promoter was analyzed by bisulfite sequencing and pyrosequencing. Results Ferroptosis was involved in COPD and glutathione peroxidase 4 (GPX4) expression was downregulated in the COPD group. Reactive oxygen species (ROS), lipid peroxides and MDA were increased, but GPX4 and SOD were exhausted in CSE-treated HBE cells. The production of IL-1β and IL-8 was promoted in HBE cells in response to CSE but could be reversed by the ferroptosis inhibitor fer-1. The Nrf2 level was significantly decreased in the COPD group compared with the control-S and control-NS groups. Increased Nrf2 expression enhanced GPX4 and SOD levels and inhibited ferroptosis and proinflammatory cytokines in the supernatant. Inhibition of GPX4 reversed the effect of Nrf2 overexpression and promoted ferroptosis. Two specific CpG sites within the Nrf2 promoter were hypermethylated in the COPD group. Similarly, CSE-treated HBE cells exhibited hypermethylation of the Nrf2 gene. Conclusion Nrf2 expression was downregulated in the lungs of COPD patients due to hypermethylation of the Nrf2 promoter, inhibiting Nrf2/GPX4 and ferroptosis, which is related to the initiation and progression of COPD. Targeting Nrf2/GPX4 may inhibit ferroptosis, which could provide strategies to delay or treat COPD.

Relationship Between ACSL4-Mediated Ferroptosis and Chronic Obstructive Pulmonary Disease

“…Mice in the CSE+LV-shNC and CSE+LV-shACSL4 groups received intratracheal infusions of Lentiviral vector containing unrelated sequence (LV-shNC) (Fenghbio, BR004, Changsha, Hunan, China) and Lentiviral vector containing ACSL4 (LV-shACSL4) (10 8 TU/mL) (Fenghbio, BR004, Changsha, Hunan, China) on Day 14, respectively, while mice in the control and CSE groups received intratracheal transfusions of an equal volume of PBS on Day 14. 12 , 13 Each group of mice was sacrificed on Day 28, and lung tissue was collected for follow-up assessments. Before being sacrificed on Day 28, the respiratory frequency (f) and the enhanced pause (Penh) of the mice in each group were detected with a non-invasive pulmonary function testing system for small animals (analog interface-16, Emka, France).…”

“…The mean linear intercept (MLI) and destructiveness index (DI) were measured to assess emphysema. 12,13 https://doi.org/10.2147/COPD.S391129…”

The molecular mechanism of ferroptosis and its role in COPD

“…Recent studies reported that CpG hypermethylation in the promoter causes downregulation of Nrf2 expression in the lung tissue of patients with COPD (65). Zhang et al found that, in HBE cells treated with CSE, the expression levels of reactive oxygen species (ROS), lipid peroxides, and MDA are increased, and they also reported that the levels of IL-1β and IL-8 are increased but that the expression levels of GPX4 and SOD are decreased, indicating that CSE induces ferroptosis in HBE cells and increases the release of inflammatory factors (63). However, increasing the expression of Nrf2 enhances the expression of GPX4 and SOD but inhibits the expression of ferroptosis and related inflammatory factors in the supernatant.…”

Identification of Ferroptosis-Related Hub Genes and Their Association with Immune Infiltration in Chronic Obstructive Pulmonary Disease by Bioinformatics Analysis

“…Hypermethylation of the nuclear factor erythroid 2-related factor 2 (Nrf2) promoter could inhibit Nrf2/GPX4 axis, thus affecting ferroptosis in COPD. 10 Otherwise, many studies suggest that various immune cells play vital roles in chronic airway diseases such as COPD. Innate immune cells, which were enhanced in small airways, modulated airway inflammation and remodeling.…”