Cell culture and treatment
We used 10μg/mL lipopolysaccharide (LPS) to simulate Human alveolar type Ⅱ epithelial cells A549 cells for 16 h to simulate sepsis-induced lung injury . A549 cell line was purchased from the Shanghai Cell Collection (Shanghai, China), and cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) at 37˚C in a humidified atmosphere of 5% CO2.
A549 were plated in 96 well plates (1 × 106 cells/mL) for12h. After Puerarin treatment, MTT (5 mg/mL in PBS, 10 μL, Thermo fisher scientific, Rockford, IL, USA) was added to each well and incubated at 37 °C for 3 h in the dark. After incubation, the culture medium was replaced with 100 μL DMSO, and the absorbance was quantitated at 570 nm using a multi-well spectrophotometer (Molecular Devices, Sunnyvale, CA, USA).
TNF-α ELISA kits (ab181421, Abcam, UK), IL‐8 ELISA kit (ab46032, Abcam, UK) and IL‐1β ELISA kit (ab214025, Abcam, UK) were used to measure the cell TNF‐α, IL‐8 and IL‐1βfollowing the instructions.
Lipid peroxidation assay
GSH enzyme activity was measured with a GSH-Px Assay Kit (Cayman Chemical, Ann Arbor, MI, USA), which is commercially available. It was used in accordance with the manufacturer’s protocol. Malondialdehyde (MDA) activity was measured with TBARS quantitative kit (C10445, Thermo fisher scientific, USA) according to the manufacturer’s protocol.
Iron concentration detection
To detect iron concentration in the cells during sepsis, an iron assay kit (MAK025, Sigma-Aldrich) was used according to the manufacturer's protocol.
To detect Fe2+ content in the cells, an iron assay kit (ab83366, Abcam, UK) and presented as nanogram Fe2+ per milligram of protein according to the manufacturer's protocol.
ROS levels of cells were detected using a fluorescent probe, 2′,7′-dichlorodihydrofluorescein (DCHF) (Sigma), which could be rapidly oxidized into the highly fluorescent 2′,7′-dichlorofluorescein (DCF) in the presence of intracellular reactive oxygen species (ROS). Fluorescence was monitored with a laser scanning confocal microscope (Leica, Germany) at 488 nm. The amount of ROS was quantified as the relative fluorescence intensity of DCF per cell in the scan area.
A549 cells were collected and lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology) at 4°C for 30 min. Then proteins were detected using a BCA protein assay kit (Bio-Rad Laboratories, Inc.). Loading buffer was added to cytosolic extracts, and after boiling for about 5 min, 30 µg of protein of each sample were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then the total gel was transferred into polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 10% skimmed milk for 2 h at room temperature, followed by incubation with anti- SLC7A11 (ab175186, Abcam, UK), anti- GPX4 (ab125066, Abcam, UK), anti- FTH1 (ab75972, Abcam, UK), anti- NOX1 (ab78016, Abcam, UK) and anti-GAPDH (ab8245, Abcam, UK) primary antibodies overnight at 4°C with 1 : 1000 dilution followed by incubation with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG,1:5000, ab172130, Abcam). The signals were detected using enhanced chemiluminescence reagent (GE Healthcare) and Image J software (version 146; National Institutes of Health, Bethesda, MD, USA) was used to analyze the fold-changes of protein levels.
All data were analyzed with the GraphPad Prism 7.0 software (GraphPad Software, Inc.). Student's t-test was applied to compare the differences between diferent groups. Comparisons among multiple groups were analyzed using one-way ANOVA followed by Tukey’s post hoc test. P<0.05 was considered to indicate a statistically significant difference. The data in this study are expressed as the mean ± standard deviation (SD). All experiments were repeated three times independently.