DOI: https://doi.org/10.21203/rs.3.rs-34579/v1
Drowning is one of the main causes of accidental injury and death (1, 2). It is estimated that more than 360,000 people die each year from drowning worldwide (3). Moreover, it is worth noting that acute lung injury (ALI) is one of the most common complications of drowning and can develop ultimately into acute respiratory distress syndrome (4, 5). Drowning can cause damage to alveolar epithelial cells, causing hypoxia, hemorrhage and oxidative stress, and pneumonia (6, 7). Oxidative stress plays an important role in drowning-induced ALI (4, 8). Our previous studies have shown that heme oxygenase-1 alleviated drowning-induced ALI (9). However, the molecular mechanisms involved in HO-1 induction in seawater-induced ALI was still unknown. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) has a key role in regulating cellular antioxidant stress and eliminating reactive oxygen species (ROS) (10). Recently research found that Nrf2 prevents oxidative stress-induced pulmonary diseases (11, 12). However, whether and how Nrf2 protects against drowning-induced ALI remain unknown. The main features of Ferroptosis are overwhelming cellular ROS production and iron-based lipid peroxide accumulation (13, 14). The latest research shows that inhibiting ferroptosis can alleviate the acute lung injury induced by lipopolysaccharide(15). However, the role of ferroptosis in ALI induced by seawater drowning is unclear. In this study, we employed Nrf2-specific agonist (dimethyl fumarate), Nrf2 inhibitor (ML385), Nrf2-knockout mice and ferroptosis inhibitor (Ferrostatin-1) to test the hypothesis that activation of Nrf2 attenuates drowning-induced ALI by inhibiting ferroptosis both in vivo and in vitro.
Artificial seawater (pH 8.2, osmolality 1300 mmol/L, specially weight 1.05, NaCl 26.518 g/L, MgSO4 3.305 g/L, MgCl2 2.447 g/L, CaCl2 1.141 g/L, KCl 0.725 g/L, NaHCO3 0.202 g/L, and NaBr 0.083 g/L) was prepared according to the main components of seawater in the East China Sea provided by the China Oceanic Administration (16). Dimethyl sulfoxide (DMSO) and dihydroethidium (DHE) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell Counting Kit-8 (CCK-8) was purchased from Biosharp Life Sciences (Biosharp, China). Dimethyl fumarate (DMF, Cat# HY-17363), ML385 (Cat# HY-100523) and Ferrostatin-1 (Fer-1, Cat# HY-100579) were obtained from Med Chem Express (MCE, USA). Primary antibodies for anti-GAPDH (Cat# ab181602) and anti-Nrf2 (Cat# ab62352) were obtained from Abcam (Abcam, USA).
MLE-12 cells (mice lung epithelial cell line, obtained from ATCC, USA) were maintained in DMEM (GIBCO, USA) medium supplemented with 10% fetal bovine serum (FBS, GIBCO, USA) and 1% penicillin-streptomycin solution (GIBCO, USA) and placed in a cell culture chamber containing 5% CO2 at 37 °C.
Cells exposed to 25% seawater (0.25 ml per 1 mL total volume) for 6 hours were used as the SW group (9). The other drug treatment groups were pretreated with final concentrations of Fer-1 (10 µM), DMF (20 µM) and ML385 (20 µM) for 2 hours before seawater exposure (17–19).
Cells viability was measured by using Cell Counting Kit-8 (CCK-8) kits. Cells were seeded at a density of 1 × 104/well in 96-well plates. After washing the cells with phosphate buffered saline (PBS), the CCK-8 solution was added to the medium at a dilution of 1:10 and incubated at 37 °C for 2 hours. Absorbance was measured at 450 nm using a microplate reader (BioTek, Winooski, USA).
The superoxide dismutase (SOD) activity, glutathione (GSH) and malondialdehyde (MDA) contents in cells and lung tissues were measured by a commercial assay kit (Nanjing Jiancheng Bio Co., Ltd., China) according to the manufacturer's instructions.
Reactive oxygen species (ROS) level was measured using a fluorometric intracellular ROS kit (Nanjing Jiancheng Bio Co., Ltd., China). MLE-12 was incubated with 10 µM 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) in the dark for one hour and then washed with PBS. The fluorescence intensity was measured by a fluorescence spectrophotometer (wavelength was 485 nm and the emission wavelength was 530 nm).
MLE-12 cells were treated with 10 µM dihydroethidium (DHE) in the dark for 30 minutes and then washed with PBS. Images were observed and collected by a Nikon TE-2000 fluorescence microscope (Nikon, Tokyo, Japan).
Mitochondrial membrane potential (MMP) of MLE-12 cells were detected using fluorescent probe JC-1 and rhodamine 123 (Sigma-Aldrich, St Louis, MO). Briefly, cells were incubated for 30 minutes at 37℃ in the dark with JC-1 (5 µM) or rhodamine 123 (5 µM). After washing with PBS, cell fluorescence images by JC-1 staining were observed and obtained using a Nikon TE-2000 fluorescence microscope (Nikon, Tokyo, Japan). The fluorescence intensity of rhodamine 123 staining was measured with a microplate reader using a 485 nm excitation and a 529 nm emission filter setup.
MLE-12 cells were treated with 5 µM MitoSox Red (Invitrogen) in the dark for 30 minutes and then washed with PBS. Images were observed and collected by a Nikon TE-2000 fluorescence microscope (Nikon, Tokyo, Japan).
Cell death was measured by flow cytometry (BD Biosciences, C6 Plus, USA) using Annexin V-FITC/PI Apoptosis Detection Kit (Beijing Cowin Biotech Co., Ltd., China) according to the manufacturer's instructions (20). The cell lipid ROS assay was performed by incubating the cells for 1 hour at a final concentration of 2 µM of BODIPY 581/591 C11 (Invitrogen) at 37 ℃. The cells were then washed twice with PBS, resuspended in PBS, and analyzed by flow cytometry (BD Biosciences, C6 Plus, USA) (20, 21).
Eight-week-old Nrf2-knockout (Nrf2−/−) and wild-type (WT) littermate male mice on a C57BL/6J background (obtained from Model Animal Research Center, MARC, Nanjing, project no. XM002783) were used to conduct the in vivo experiments. For the mice drowning model, mice were placed in a porous container and immersed in a water bath containing 6 cm deep 25 ± 2 ℃ for 35 seconds (9, 22). All experiments were conducted in accordance with established guidelines and approved by the Animal Care and Use Committee of Jiangnan University (JN. No 20180615b0841230). Wild-type and Nrf2-knockout mice were randomly assigned to the corresponding groups. DMF group and SW + DMF group were given DMF (80 mg/kg) by gavage at 3 h, 24 h and 48 h after drowning (23). Fer-1 and SW + Fer-1 groups were given Fer-1 (5 mg/kg) by intraperitoneal injection at 3 hours, 24 hours and 48 hours after drowning (24). All groups were sacrificed on the third day after drowning and lung tissue samples were collected for evaluation.
2.11 Tissue collection, wet-to-dry ratio analysis and histological analysis
Right lungs were collected for determined the lung wet-to-dry ratio, western blot and biochemical analysis. After the lung tissue were weighed, they were dried in an oven at 60 ℃ for 72 hours to constant weight, and then the wet-to-dry ratio was calculated to evaluate tissue edema. Left lung tissue samples were embedded in paraffin after fixed in 4% paraformaldehyde. Tissue pieces were cut into 4 µm sections and stained with hematoxylin and eosin (HE) for lung injury score (25).
2.12 Micro-computed tomographic analysis
Mice were anesthetized with isoflurane continuously delivered through a nose cone and scanned using a Quantum FX micro-CT Imaging System (PerkinElmer, USA). Each mouse was scanned for 4 minutes under the parameters of 70 kV, 88 µA, 36 mm FOV. The data acquired by the scan was analyzed and 3D reconstructed using the Analyze 12.0 software at the same level setting.
2.13 Immunofluorescence Staining
To observe Nrf2 localization of MLE-12 cells. Cells were then fixed in 4% formaldehyde and permeabilized with 0.1%Triton X. The cells were probed with Nrf2 antibodies followed by Alexa Fluor 488-conjugated secondary antibodies (Thermo Fisher Scientific, USA). To visualize the nuclei, cells were then treated with 1 µg/ml DAPI for 10 min and then washed by PBS. Finally, added the anti-fade mounting medium and collected images using Zeiss LSM 880 laser confocal fluorescence microscope (Carl Zeiss, Oberkochen, Germany).
To observe the expression of Nrf2 in mouse lung tissue. Paraffin slides were rehydrated in alcohol with decreasing concentrations and then placed in 10 mM sodium citrate buffer heated to 95 ℃ for 30 min for antigen retrieval. Then, the slides were permeabilized with 0.5% Triton-X and blocked with 5% BSA for 2 hours. The slides were probed with Nrf2 antibodies followed by Alexa Fluor 555-conjugated secondary antibodies (Thermo Fisher Scientific, USA). The slides were incubated in DAPI for 10 minutes and then washed. Finally, the slides were mounted with an anti-fade mounting medium and collected images using Zeiss Axio Imager 2 fluorescence microscope (Carl Zeiss, Oberkochen, Germany).
2.14 Western blot
The samples were homogenized in ice-cold RIPA buffer with protease inhibitor mixture, and the supernatant was collected after centrifugation (12,000 rpm, 10 minutes and 4 ℃). The protein concentrations were measured using a BCA kit, and proteins were then denatured at 100 ℃ for 5 min. Proteins were loaded onto a 10% SDS-PAGE gel and transferred to a nitrocellulose filter membrane. Blots were blocked at room temperature for 1 h and then incubated with primary antibodies at 4 °C overnight. After blocking and washing, the blot was incubated with the secondary antibody for 2 hours. Protein bands were visualized and analyzed by the ChemiDocTM XRS Plus luminescence image analyzer (Bio-Rad, USA) using the ECL system (Millipore, USA).
2.15 Reverse transcription-quantitative polymerase chain reaction
Total RNA isolation and quantitative real-time PCR were performed using the procedure described previously (26). The primers used in this study were synthesized by GENEWIZ Biotechnology Co., Ltd. (Suzhou, China) (Table 1).
| Gene | Forward | Reverse |
|---|---|---|
| Nrf2 | 5′-AAAATCATTAACCTCCCTGTTGAT-3′ | 5′-CGGCGACTTTATTCTTACCTCTC-3′ |
| Ptgs2 | 5’-AAGTGCGATTGTACCCGGAC-3′ | 5′-GTGCACTGTGTTTGGAGTGG-3′ |
| GPX4 | 5’-CTTATCCAGGCAGACCATGTGC-3’ | 5’-CCTCTGCTGCAAGAGCCTCCC-3’ |
| FTH1 | 5’-GCACACTCCATTGCATTCAGCC-3’ | 5’-GCCGAGAAACTGATGAAGCTGC-3’ |
| GAPDH | 5′-TGTGATGGGTGTGAACCACGAGAA‐3′ | 5′-GAGCCCTTCCACAATGCCAAAGTT-3′ |
2.16 Protein–Protein interaction Analysis
Search Tool for the Retrieval of Reciprocity Genes (STRING) online database (http://string-db.org/) was used to evaluate protein–protein interaction network of Nrf2, Ptgs2, GPX4 and FTH1. Organism was selected “Mus musculus”. The online database provided assessment and integration of protein interactions, including direct (physical) and indirect (functional) correlations (27).
2.17 Statistical analysis
All experiments were performed at least three times. Measurement data were expressed as means ± standard deviation (SD). Comparisons between groups were carried out by analysis of variance (ANOVA) with GraphPad Prism. P < 0.05 was considered statistically significant.
The cell morphology of MLE-12 cells exposed to seawater (0–12 h) was observed by bright field microscopy. The results showed that seawater exposure caused a decrease in the number and changes in cell morphology of MLE-12 cells (Fig. 1A). CCK8 assay showed a time-dependent decrease in MLE-12 cell viability after seawater exposure (Fig. 1B). The results of flow cytometry detection of death also showed an increase in the proportion of cell death as the seawater exposure time prolonged (Fig. 1C and D) (20). These results indicate that seawater exposure causes MLE-12 cell damage and death. Based on the above experiments, this study selected seawater stimulation for 6 hours as the follow-up test conditions. As shown in Fig. 1E, seawater treatment induced a significant increase in Ptgs2 mRNA in MLE-12 cells, a putative ferroptosis molecular marker (28, 29). Nrf2 mRNA and protein expression in MLE-12 cells also increased after 6 hours of seawater stimulation (Fig. 1F and G).
To investigate whether ferroptosis participated in MLE-12 cell death induced by seawater drowning, we pretreated cells with the ferroptosis specific inhibitor Fer-1. MLE-12 cells were treated with Fer-1 at a final concentration of 0 µM to 40 µM for 24 hours (Fig. 2A). It was found that the concentration of 10 µM did not significantly inhibit cell viability, and 10 µM was used as the concentration for subsequent experiments of Fer-1(19). As shown in Figures. 2B-D, the percentage of cell death after Fer-1 treatment was reduced and cell viability was increased relative to the SW group. The fluorescent indicator (DCFH-DA) and dihydroethidium (DHE) fluorescent probes were used to monitor the generation of intracellular ROS in seawater-treated MLE-12 cells. The two detection methods consistently showed that seawater exposure caused a significant increase of intracellular ROS in MLE-12 cells, which was decreased by Fer-1 treatment (Fig. 2E and F). Lipid ROS accumulation is a typical feature of ferroptosis (30). The results of flow cytometry after BODIPY® 581/591 C11 staining showed that lipid ROS levels were significantly reduced after Fer-1 treatment compared with SW group (Fig. 2G and H). Glutathione depletion and lipid peroxide accumulation are important features of ferroptosis (31). MDA is the most common by-product of lipid peroxidation (32). Fer-1 treatment reversed the decrease of GSH level and SOD activity caused by seawater drowning (Fig. 2I and K). Fer-1 also reduced the content of MDA (Fig. 2J). These results suggest that ferroptosis is present in MLE-12 cell damage induced by seawater stimulation.
To investigate the effect of Nrf2 on MLE-12 cell damage induced by seawater drowning, we treated cells with DMF (20 µM) and ML385 (20 µM). As shown in Figure.3A and B, DMF did significantly increase the expression of Nrf2 whereas ML385 treatment reduced the expression level of Nrf2. As a transcription factor, translocation into the nucleus is a key step for Nrf2 to play a regulatory role. Immunofluorescence images showed that DMF significantly promoted the translocation of Nrf2 into the nucleus, while ML385 suppressed this phenomenon (Fig. 3C). Flow cytometry results showed that DMF reduced the percentage of cell death caused by seawater stimulation, whereas ML385 reversed this effect (Fig. 3D and E). The results of CCK8 also showed that compared with the SW group, the cell viability of the SW + DMF group increased, while the cell viability of the SW + ML385 group and the SW + DMF + ML385 group decreased (Fig. 3F). These results indicated that Nrf2 activation could attenuate cell death induced by seawater stimulation in MLE-12 cells.
We further explored the relationship between Nrf2 activities and seawater-induced ferroptosis in MLE-12 cells. As shown in Figures.4A-C, DMF increased the GSH content and SOD activity, and decreased the content of MDA, whereas ML385 reversed these effects. Furthermore, the detection of intracellular ROS by the fluorescent dye DCFH-DA showed that seawater-induced ROS levels were reduced after DMF treatment, while ML385 increased ROS levels (Fig. 4D). The same results were obtained for the fluorescent images of DHE staining (Fig. 4E). The results of flow cytometry for detecting lipid ROS also indicated that ML385 abolished the effect of DMF on reducing lipid ROS accumulation induced by seawater stimulation (Fig. 4F and G).
Mitochondria are important organelles of oxidative metabolism and play a crucial role in ferroptosis (33). Mitochondrial membrane potential (MMP) was detected by JC-1 staining fluorescence image and rhodamine 123. As shown in Figures. 5A and C, DMF has a protective effect on seawater-induced mitochondrial membrane potential reduction (mitochondrial depolarization), while ML385 reverses this effect. MitoSox Red is a mitochondria-targeted ROS dye. Fluorescence images and intensity results indicate that ML385 reverses the effect of DMF on decreasing mitochondrial ROS levels. (Fig. 5B and D). GPXs can use reduced GSH to eliminate peroxides, of which glutathione peroxidase 4 (GPX4) is the main neutralizer of lipid peroxides (34). We found that DMF treatment had an effect of improving seawater-induced decrease in GPX4 mRNA levels in MLE-12 cells, whereas ML385 caused a decrease in GPX4 mRNA (Fig. 5E). FTH1 is an important gene for storing iron and maintaining iron homeostasis (35, 36). As shown in Fig. 5F, DMF promoted the expression of FTH1 mRNA, whereas ML385 reversed this effect. As a potential marker of ferroptosis, Ptgs2 also showed a significant decrease in mRNA level relative to SW group after DMF treatment, while the levels of SW + ML385 group and SW + DMF + ML385 group were further increased (Fig. 5G). A network of Ptgs2, GPX4 and FTH1 that significantly interacted with Nrf2 was constructed using the String database (protein–protein interaction). The network graphic showed that ferroptosis-related genes FTH1, GPX4 and Ptgs2 were clearly associated with Nrf2 (Fig. 5H). The results of the above various tests indicated that DMF promoted Nrf2 expression inhibited ferroptosis in the MLE-12 cell drowning model, and this beneficial effect was abolished by the Nrf2 inhibitor ML385.
To further validate the above findings, we performed in vivo experiments by using a mouse drowning lung injury model. As shown in Fig. 6A, on the third day after drowning, obvious edema and hemorrhage occurred in the gross anatomy of the lung, and the ferroptosis inhibitor Fer-1 treatment improved this condition. Compared with the SW group, the SW + Fer-1 group showed a significant decrease in lung wet-to-dry ratio, which indicated a reduction in pulmonary edema (Fig. 6C). H&E-stained sections showed that Fer-1 improved lung injury caused by seawater drowning (Fig. 6B and D). We further tested mouse lung injury by Micro CT. As shown in Fig. 6F, seawater drowning caused obvious damage and deformation of the mouse lung, but Fer-1 treatment ameliorates this pathological change. The lung volume of the mice was calculated by the Analyze 12.0 software based on the Micro CT data. Compared with the SW group, the lung volume of the SW + Fer-1 group also recovered significantly (Fig. 6E). We further tested the content of GSH and MDA and SOD activity in lung tissue, and the results showed that Fer-1 treatment improved the reduction of GSH content and SOD activity and decreased MDA levels (Fig. 6G-I). These results indicated that inhibition of ferroptosis could improve lung injury in mice caused by seawater drowning.
As shown in Fig. 7A and B, DMF promotes the expression of Nrf2 in mice lung tissue. Immunofluorescence images also showed the same conditions as Western blots (Fig. 7C). Compared with the SW group, the SW + DMF group had a lower lung wet-to-dry ratio, indicating an improvement in pulmonary edema (Fig. 7D). Gross anatomy images show that DMF treatment improves lung tissue hemorrhage and edema caused by seawater drowning (Fig. 7F). H&E slice staining images also showed that DMF treatment improved lung pathological damage in mice (Fig. 7E and G). As shown in Figures.7H and I, Micro CT results showed that lung injury and deformation were improved in the SW + DMF group compared with the SW group, and the lung volume was also restored in the SW + DMF group. The results of GSH, MDA in lung tissue showed that compared with SW group, the content of GSH in SW + DMF group increased, and the content of MDA decreased (Fig. 7J and K). qPCR results showed that seawater caused an increase of Ptgs2 mRNA expression in mouse lung, and DMF treatment reduced Ptgs2 mRNA levels (Fig. 7L). These data indicate that Nrf2 agonist DMF treatment inhibited ferroptosis and improved lung injury in mice caused by seawater drowning.
To confirm the protective effect of Nrf2 on drowning lung injury, we used Nrf2−/− mice for further validation. As shown in Fig. 8A and 8B, we verified the knockout effect of Nrf2 in mouse lung tissue by Western blotting. The same result was obtained with the immunofluorescence image (Fig. 8C). The results of lung wet-to-dry ratio showed that knocking out Nrf2 promoted pulmonary edema (Fig. 8D). Gross anatomy and H&E staining images showed that seawater drowning caused more severe bleeding and pathological damage in Nrf2 knockout mice compared to wild-type mice (Fig. 8E-G). Similar results were obtained by Micro CT. The Nrf2 KO + SW group had more severe lung injury than the SW group, and the lung volume decreased more severely (Fig. 8H and I). Biochemical indicators showed that the lower levels of GSH and the higher MDA in Nrf2−/− mice compared with those in wild-type mice (Fig. 8J and K). In addition, the expression of Ptgs2 mRNA in the Nrf2 KO + SW group was also higher than that in the SW group. (Fig. 8L). These results indicated that Nrf2 deficiency aggravated lung injury in mice induced by seawater drowning.
Drowning is one of the three major causes of unintentional injury death in the world (37). Approximately 50,000 people annually die mainly due to ALI or ARDS while no specific and effective treatments are currently available (5, 37). Thus, it is very important to understand the key mechanisms and look for strategies to treatment seawater-induced ALI. It is generally recognized that many factors such as oxidation, inflammation, apoptosis and delayed cell proliferation be involved in the pathogenesis of seawater drowning-ALI (4), but which is the key factor is still unknown. In this study, we demonstrated that seawater stimuli directly induced the levels of intracellular ROS and lipid ROS, and reduced GSH content and led to severe mitochondrial damage in MLE-12 cells and mice. These results demonstrated that severe oxidative stress injury is the key factor in seawater drowning-induced ALI.
Ferroptosis is an iron-dependent, lipid peroxide-driven form of cell death. The new cellular death phenotype is mechanistically and phenotypically distinct from other cell death processes, e.g. apoptosis, autophagy, pyroptosis (14, 38). However, whether and how ferroptosis plays a role in lung injury caused by seawater drowning has never been explored. In this study, we firstly found that seawater exposure induced MLE-12 cell damage and death and the ferroptosis inhibitor Fer-1 treatment improved cell viability and cell death. Furthermore, Fer-1 treatment reduced the levels of intracellular ROS and lipid ROS and MDA, and increased the levels of GSH and SOD. This indicated that ferroptosis participated in MLE-12 cell damage caused by seawater exposure. The mouse drowning model also verified that ferroptosis is present in lung injury caused by seawater drowning. Gross anatomical morphology, HE slices staining and micro CT results consistently showed that ferroptosis inhibition attenuated ALI in mice caused by seawater drowning.
Mitochondria are important organelles of oxidative metabolism and play a crucial role in ferroptosis (19). Abnormal mitochondrial metabolism significantly contributes to rapid glutathione depletion and subsequent lipid ROS generation and ferroptosis (33). In this study, we monitored mitochondria function using C11 BODIPY 581/591 and mitochondrial superoxide images by MitoSox Red fluorescent probe and DHE fluorescent probe and Mitochondrial membrane potential (MMP) assay. We found that seawater stimulation caused an increase in intracellular ROS, lipid ROS accumulation in MLE-12 cells. Furthermore, Fer-1 treatment reduced the levels of intracellular ROS and lipid ROS and MDA, and increased the levels of GSH and SOD. Here, we presented evidences to demonstrate that the mitochondrion is indeed a crucial player in ferroptosis induced by seawater simulation.
The mechanisms about the negative regulation of ferroptosis are being hot topic in recent years as therapeutic means for multiple pathologies (20, 21). Elucidating how ferroptosis provokes ALI will expose new therapeutic opportunities to treat these diseases. Nrf2 is a core player in the regulation of antioxidant molecules in cells, regulating a variety of genes (10, 39). Nrf2 activation has a beneficial effect on ischemia-reperfusion (I/R)-induced lung injury(40), lipopolysaccharide-induced ALI (41) and ventilator-induced lung injury (42). Nrf2 inhibition promoted erastin or artesunate-induced ferroptosis through regulating redox homeostasis (39, 43). In this study, we found that seawater exposure induced an increase in Nrf2 expression in MLE-12 cells (Fig. 1F and G). In addition, we used DMF to induce Nrf2 expression and translocation into the nucleus, which improved MLE-12 cell damage caused by seawater exposure and ML385 reversed this effect. Furthermore, induction of Nrf2 expression by DMF significantly attenuated GSH depletion and accumulation of MDA, ROS and lipid ROS induced by seawater exposure in MLE-12 cells. In addition, detection of mitochondrial membrane potential and mitochondrial ROS content also indicated that induction of Nrf2 expression improved mitochondrial function in MLE-12 cells. Ptgs2 mRNA results also showed that Nrf2 inhibited ferroptosis caused by seawater exposure in MLE-12 cells (Fig. 5G). DMF treatment of wild-type mice also significantly ameliorated lung injury induced by seawater drowning. The results of GSH, MDA and Ptgs2 mRNA also indicate that DMF-induced Nrf2 inhibited ferroptosis in the lungs of mice caused by seawater drowning (Fig. 7J-L). We further used Nrf2 knockout mice to verify the role of Nrf2 on ferroptosis in seawater drowned mice. Nrf2 knockout mice had more severe lung injury than wild-type mice. Seawater drowning caused more serious ferroptosis in Nrf2 knockout mice. These results demonstrated in vivo levels that Nrf2 inhibited ferroptosis and alleviated lung damage caused by seawater drowning.
There many important genes regulating ferroptosis process, such as GPX4, Ptgs2 and FTH1. Ferroptosis process often led GSH depletion which involves in abnormal GSH synthesis cysteine supply controled by system xc-, glutathione reductase and GPX4 (31). GPXs synthesis GSH to eliminate lipid peroxides and prevent GSH depletion (34). In this study, we found Nrf2 agonist DMF up-regulated GPX4 mRNA expression. Meanwhile, we used the String database (protein-protein interaction) and found that Nrf2 and GPX4 did has positive relations. The p62–Keap1–Nrf2 antioxidant system may be responsible for the promoted function of Nrf2 on GPX4 expression (21). Nrf2-mediated upregulation of the iron storage protein ferritin promoted cellular proliferation (36). In this study, we found that Nrf2 activation increased FTH1 mRNA expression. Ptgs2, also known as cyclooxygenase 2 (Cox-2), is considered to be a typical indicator of ferroptosis (38). The network graphic showed that ferroptosis-related genes FTH1, GPX4 and Ptgs2 were associated with Nrf2, which molecularly explain the beneficent role in seawater drowning-induced oxidative stress damage.
Although the pathogenetic role of ferroptosis in lung injury induced by seawater drowning is still elusive, its involvement in multiple human diseases has been established. Ferroptosis is a key mechanism for cell death associated with ischemic organ injury, neurodegeneration and cancer (14, 20, 34). GPX4 inhibition induced ferroptosis could prevent cancer resistant to chemotherapy (28, 44). Noteworthily, the latest research ferroptosis inhibitor attenuated cigarette smoke-induced chronic obstructive pulmonary disease (45) and radiation-induced lung fibrosis through the activation of Nrf2 pathway (46). These findings suggest that modulating ferroptosis might be potential therapeutic approache in treating lung diseases. Our study supported the notion that Nrf2 plays an important role in attenuating seawater drowning-induced lung injury (Fig. 9).
In this study, we firstly demonstrated in vivo and in vitro that Nrf2 can inhibit ferroptosis and alleviate ALI induced by drowning. These results elucidate a new mechanism underlying drowning-induced oxidative stress damage and identify Nrf2 as a potential therapeutic target for the treatment of lung injury.
Nrf2, nuclear factor (erythroid-derived 2)-like 2
SW, seawater
DMF, dimethyl fumarate
ALI, acute lung injury
ARDS, acute respiratory distress syndrome
Fer-1, ferrostatin-1
DHE, dihydroethidium
ROS, reactive oxygen species
SOD, superoxide dismutase
GSH, glutathione
MDA, malondialdehyde
Ptgs2, prostaglandin-endoperoxide synthase 2
FTH1, ferritin heavy chain 1
GPX4, glutathione peroxidase 4
MMP, mitochondrial membrane potential
Acknowledgments
Not applicable.
Authors' contributions
QFP, YBQandDZC made substantial contributions to the conception and design of the experiment. YBQ, BBW, GL, YXW and DC performed experiments. RQY, MDLand JLC did the statistical analysis and data interpretation. YBQ, DZC and QFP wrote the main manuscript text. All authors read and approved the final manuscript.
Funding
This work was supported byNational Natural Science Foundation of China (81871518, 81901522, 81702799); National first-class discipline program of Food Science and Technology (JUFSTR20180101); Wuxi health and family planning commission(Z201810); Public Health Research center at Jiangnan University (JUPH201805); Fundamental Research Funds for the Central Universities (JUSRP11955).
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Ethics approval
The study all experiments were conducted in accordance with established guidelines and approved by the Animal Care and Use Committee of Jiangnan University (JN.No20180615b0841230).
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Footnotes
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Contributor Information
Dao-zhen Chen,E-mail: [email protected];
Qing-feng Pang,E-mail: [email protected].