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

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-1ß 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-1ß 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.

Dynamic evolution of emphysema and airway remodeling in two mouse models of COPD.

BACKGROUND: Establishment of a mouse model is important for investigating the mechanism of chronic obstructive pulmonary disease (COPD). In this study, we observed and compared the evolution of the pathology in two mouse models of COPD induced by cigarette smoke (CS) exposure alone or in combination with lipopolysaccharide (LPS). METHODS: One hundred eight wild-type C57BL/6 mice were equally divided into three groups: the (1) control group, (2) CS-exposed group (CS group), and (3) CS + LPS-exposed group (CS + LPS group). The body weight of the mice was recorded, and noninvasive lung function tests were performed monthly. Inflammation was evaluated by counting the number of inflammatory cells in bronchoalveolar lavage fluid and measuring the expression of the IL-6 mRNA in mouse lung tissue. Changes in pathology were assessed by performing hematoxylin and eosin and Masson staining of lung tissue sections. RESULTS: The two treatments induced emphysema and airway remodeling and decreased lung function. Emphysema was induced after 1 month of exposure to CS or CS + LPS, while airway remodeling was induced after 2 months of exposure to CS + LPS and 3 months of exposure to CS. Moreover, the mice in the CS + LPS group exhibited more severe inflammation and airway remodeling than the mice in the CS group, but the two treatments induced similar levels of emphysema. CONCLUSION: Compared with the single CS exposure method, the CS + LPS exposure method is a more suitable model of COPD in airway remodeling research. Conversely, the CS exposure method is a more suitable model of COPD for emphysema research due to its simple operation.

Clinical Utility of Central and Peripheral Airway Nitric Oxide in Aging Patients with Stable and Acute Exacerbated Chronic Obstructive Pulmonary Disease.

PURPOSE: Exhaled nitric oxide has been used as a marker of airway inflammation. The NO concentration in the central and peripheral airway/alveolar can be measured by a slow and fast exhalation flow rate to evaluate inflammation in different divisions within the respiratory tract. We hypothesized that FeNO200 (exhaled NO at a flow rate of 200mL/s) could be used as an evaluation tool for peripheral airway/alveolar inflammation and corticosteroid therapy in chronic obstructive pulmonary disease (COPD) patients. METHODS: We recruited 171 subjects into the study: 73 healthy controls, 59 stable COPD patients, and 39 acute exacerbations of COPD (AECOPD) patients. Exhaled nitric oxide (FeNO50 (exhaled NO at a flow rate of 50mL/s)), FeNO200 and CaNO (peripheral concentration of NO/alveolar NO) and clinical variables including pulmonary function, COPD Assessment Test (CAT), C-reactive protein concentration (CRP) and circulating eosinophil count were measured among the recruited participants. FeNO50, FeNO200 and CaNO were repeatedly evaluated in 39 AECOPD patients after corticosteroid treatment. RESULTS: FeNO200 was significantly higher in stable COPD and AECOPD patients than in healthy controls. Nevertheless, CaNO could not differentiate COPD from healthy controls. No correlation was found between circulating eosinophil counts or FEV1 and exhaled nitric oxide (FeNO50, FeNO200, CaNO) in COPD patients. For AECOPD patients, 64% of patients had eosinophil counts >100 cells/µL; 59% of patients had FeNO200 >10 ppb; only 31% of patients had FeNO50 > 25 ppb. Among AECOPD patients, the high FeNO50 and FeNO200 groups' levels were significantly lower than their baseline levels, and significant improvements in CAT were seen in the two groups after corticosteroid treatment. These implied a good corticosteroid response in AECOPD patients with FeNO200>10ppb. CONCLUSION: FeNO200 is a straightforward and feasible method to evaluate the peripheral NO concentration in COPD. FeNO200 can be a type 2 inflammation biomarker and a useful tool for predicting corticosteroid therapy in COPD.

Let-7 mediated airway remodelling in chronic obstructive pulmonary disease via the regulation of IL-6.

BACKGROUND: Myofibroblast differentiation and extracellular matrix (ECM) deposition are observed in chronic obstructive pulmonary disease (COPD). However, the mechanisms of regulation of myofibroblast differentiation remain unclear. MATERIALS AND METHODS: We detected let-7 levels in peripheral lung tissues, serum and primary bronchial epithelial cells of COPD patients and cigarette smoke (CS)-exposed mice. IL-6 mRNA was explored in lung tissues of COPD patients and CS-exposed mice. IL-6 protein was detected in cell supernatant from primary epithelial cells by ELISA. We confirmed the regulatory effect of let-7 on IL-6 by luciferase reporter assay. Western blotting assay was used to determine the expression of α-SMA, E-cadherin and collagen I. In vitro, cell study was performed to demonstrate the role of let-7 in myofibroblast differentiation and ECM deposition. RESULTS: Low expression of let-7 was observed in COPD patients, CS-exposed mice and CS extract (CSE)-treated human bronchial epithelial (HBE) cells. Increased IL-6 was found in COPD patients, CS-exposed mice and CSE-treated HBE cells. Let-7 targets and silences IL-6 protein coding genes through binding to 3' untranslated region (UTR) of IL-6. Normal or CSE-treated HBE cells were co-cultured with human embryonic lung fibroblasts (MRC-5 cells). Reduction of let-7 in HBE cells caused myofibroblast differentiation and ECM deposition, while increase of let-7 mimics decreased myofibroblast differentiation phenotype and ECM deposition. CONCLUSION: We demonstrate that CS reduced let-7 expression in COPD and, further, identify let-7 as a regulator of myofibroblast differentiation through the regulation of IL-6, which has potential value for diagnosis and treatment of COPD.