2.1 Reagents and antibodies
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).
2.2 Cell culture and treatment
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).
2.3 Cell viability assay
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).
2.4 Determination of the levels of SOD, GSH, and MDA
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.
2.5 Detection of intracellular ROS by fluorometric intracellular ROS kit
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).
2.6 Collection of intracellular ROS images by DHE fluorescent probe
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).
2.7 Mitochondrial membrane potential assay
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.
2.8 Collection of mitochondrial superoxide images by MitoSox Red fluorescent probe
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).
2.9 Flow cytometric analysis of cell death and lipid ROS
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).
2.10 Seawater drowning model and treatments
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).
Table 1
Primers used in this study for PCR
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.