Nrf2 protects against seawater drowning-induced acute lung injury via inhibiting ferroptosis

Abstract

Background: Ferroptosis is a new type of nonapoptotic cell death model that was closely related to reactive oxygen species (ROS) accumulation. Seawater drowning-induced acute lung injury (ALI) which is caused by severe oxidative stress injury, has been a major cause of accidental death worldwide. The latest evidences indicate nuclear factor (erythroid-derived 2)-like 2 (Nrf2) suppress ferroptosis and maintain cellular redox balance. Here, we test the hypothesis that activation of Nrf2 pathway attenuates seawater drowning-induced ALI via inhibiting ferroptosis.

Methods: we performed studies using Nrf2-specific agonist (dimethyl fumarate), Nrf2 inhibitor (ML385), Nrf2-knockout mice and ferroptosis inhibitor (Ferrostatin-1) to investigate the potential roles of Nrf2 on seawater drowning-induced ALI and the underlying mechanisms.

Results: Our data shows that Nrf2 activator dimethyl fumarate could increase cell viability, reduced the levels of intracellular ROS and lipid ROS, prevented glutathione depletion and lipid peroxide accumulation, increased FTH1 and GPX4 mRNA expression, and maintained mitochondrial membrane potential in MLE-12 cells. However, ML385 promoted cell death and lipid ROS production in MLE-12 cells. Furthermore, the lung injury became more aggravated in the Nrf2-knockout mice than that in WT mice after seawater drowning.

Conclusions: These results suggested that Nrf2 can inhibit ferroptosis and therefore alleviate ALI induced by seawater drowning. The effectiveness of ferroptosis inhibition by Nrf2 provides a novel therapeutic target for seawater drowning-induced ALI.

Keywords: Acute lung injury; Drowning; Ferroptosis; Nrf2; Seawater.

Fig. 1

Seawater exposure induced MLE-12 cell damage. MLE-12 cells were exposed to 25% seawater for different times (a-d). (a) Cell morphology images (Original magnification × 200. Bar = 50 μm). (b) CCK-8 assay for the determination of cell viability (n = 4). (c) Cell death was analyzed using Annexin V-FITC/PI staining with flow cytometry (n=3). (d) Quantitative results of the percentage of cell death (n = 3). (e) The expression of Ptgs2 mRNA in MLE-12 cells was measured by qPCR after 6 h of seawater exposure (n = 4). The level of Nrf2 expression was determined by Western blot (f) and qPCR (g) (n = 3). Data were presented as the mean ± SD. ^⁎P < 0.05 vs. Control group

Fig. 2

Inhibition of ferroptosis alleviated seawater-induced MLE-12 cell damage. (a) and (b). CCK-8 assay for the determination of cell viability (n = 4). (d) Cell death was analyzed using Annexin V-FITC/PI staining with flow cytometry (n = 3). (c) Quantitative results of the percentage of cell death (n = 3). (e) Intracellular ROS was monitored using the DCFH-DA fluorescent probes (n = 4). (f) Images of intracellular ROS levels in MLE-12 cells stained by DHE (10 μM) fluorescent probes. Scale bars: 50 μm. (g) lipid ROS was analyzed using 2 μM BODIPY® 581/591 C11 with flow cytometry (n = 3). (h) Quantitative results of lipid ROS (n = 3). Kit detected GSH (i), MDA (j) and SOD (k) levels in MLE-12 cells (n = 4). Data were presented as the mean ± SD. ^⁎P < 0.05 vs. Control group, ^#P < 0.05 vs. SW group

Fig. 3

Nrf2 attenuated seawater-induced MLE-12 cell damage. Images (a) and quantification (b) of Nrf2 expression levels were analyzed by Western blot (n = 3). (c) Nrf2 was detected by immunofluorescence (Red: Nrf2, Blue: nucleus, Original magnification × 400. Bar = 20 μm). (d) Cell death was analyzed using Annexin V-FITC/PI staining with flow cytometry (n = 4). (e) Quantitative results of the percentage of cell death (n = 4). (f) CCK-8 assay for the determination of cell viability (n = 4). Data were presented as the mean ± SD. ^⁎P < 0.05 vs. Control group, ^#P < 0.05 vs. SW group

Fig. 4

Nrf2 alleviated GSH depletion, lipid peroxidation and accumulation of ROS induced by seawater stimulation in MLE-12 cells. The content of GSH (a), MDA (b) and SOD (c) in MLE-12 cells (n = 4). (d) Intracellular ROS was monitored using the DCFH-DA fluorescent probes (n = 4). (e) Images of intracellular ROS levels in MLE-12 cells stained by DHE (10 μM) fluorescent probes. Original magnification × 200. Bar = 50 μm. (f) lipid ROS was analyzed using 2 μM BODIPY® 581/591 C11 with flow cytometry (n = 3). (g) Quantitative results of lipid ROS (n = 3). Data were presented as the mean ± SD. ^⁎P < 0.05 vs. Control group, ^#P < 0.05 vs. SW group

Fig. 5

Nrf2 protected mitochondria and regulated ferroptosis related gene expression. (a) Fluorescence images of 5 μM JC-1 stained MLE-12 cells. Scale bars: 50 μm. The ratio of red and green fluorescence reflected changes of mitochondrial membrane potential (n = 3). Fluorescence images (b) and relative quantification (d) of MitoSox Red (5 μM) stained MLE-12 cells. Original magnification × 200. Bar = 50 μm. (c) The mitochondrial membrane potential stained with rhodamine 123 (5 μM) was detected by a microplate reader (n = 4). Relative mRNA expression of ferroptosis-related genes GPX4 (e), FTH1 (f) and Ptgs2 (g) in MLE-12 cells (n = 4). (H) The protein–protein interaction (PPI) network of Nrf2, GPX4, Ptgs2 and FTH1 (String). Data were presented as the mean ± SD. ^⁎P < 0.05 vs. Control group, ^#P < 0.05 vs. SW group

Fig. 6

Inhibition of ferroptosis ameliorated lung injury in mice induced by seawater drowning. (a) Gross morphology of mice lung. H&E stained pathological tissue images (b) and ALI scores (d) (n = 4). Original magnification × 200 and × 400. Bar = 50 μm. (c) Lung wet/dry weight ratio (n = 4). (e) Micro CT images of mouse lungs. (f) Lung volume quantification was analyzed by Analyze 12.0 software based on the Micro CT data (n = 4). Kit detected GSH (g), MDA (H) and SOD (i) levels in mouse lung tissue (n = 4). Data were presented as the mean ± SD. Values are mean ± SD of four experiments. ^⁎P < 0.05 vs. Control group, ^#P < 0.05 vs. SW group

Fig. 7

Nrf2 agonist DMF inhibited ferroptosis and improved ALI in mice induced by seawater drowning. Images (a) and quantification (b) of Nrf2 expression level in lung were analyzed by Western blot (n = 3). (c) Immunofluorescence images were used to detect the expression of Nrf2 (red) in mouse lung tissue (n = 3). Nuclei were counterstained with DAPI (blue). Original magnification × 200. Bar = 50 μm. (d) Lung wet/dry weight ratio (n = 4). (f) Gross morphology of mice lung. H&E stained pathological tissue images (g) and lung injury scores (e) (n = 4). Original magnification × 200 and × 400. Bar = 50 μm. (h) Micro CT images of mouse lungs. (i) Lung volume quantification was analyzed by Analyze 12.0 software based on the Micro CT data (n = 3). Kit detected GSH (j) and MDA (k) contents in mouse lung tissue (n = 4). (l) qPCR was used to detect the expression of ferroptosis-related gene Ptgs2 mRNA (n = 4). Data were presented as the mean ± SD. ^⁎P < 0.05 vs. Control group, ^#P < 0.05 vs. SW group

Fig. 8

Nrf2 deficiency aggravated ALI and ferroptosis in mice induced by seawater drowning. Images (a) and quantification (b) of Nrf2 expression levels in lung were analyzed by Western blot (n = 3). (c) Immunofluorescence images were used to detect the expression of Nrf2 (red) in mouse lung tissue. Nuclei were counterstained with DAPI (blue). Original magnification × 200. Bar = 50 μm. (d) Lung wet/dry weight ratio (n = 4). (f) Gross morphology of mice lung. H&E stained pathological tissue images (g) and lung injury scores (e) (n = 4). Original magnification × 200 and × 400. Scale bars: 50 μm. (h) Micro CT images of mouse lungs. (i) Lung volume quantification was analyzed by Analyze 12.0 software based on the Micro CT data (n = 4). Kit detected GSH (j) and MDA (k) contents in mouse lung tissue (n = 4). (l) qPCR was used to detect the expression of ferroptosis-related gene Ptgs2 mRNA (n = 4). Data were presented as the mean ± SD. ^⁎P < 0.05 vs. Control group, ^#P < 0.05 vs. SW group

Fig. 9

A schematic model for Nrf2 activation attenuating seawater drowning-induced ALI in vitro and in vivo