Salidroside attenuates HALI via IL-17A-mediated ferroptosis of alveolar epithelial cells by regulating Act1-TRAF6-p38 MAPK pathway

Animals

Eight-week-old KM mice (purchased from the animal center of Jinzhou University, Liaoning, China) were used to conduct in vivo experiments. Mice were housed under controlled temperature (20 °C ± 2 °C) and humidity (60% ± 10%) on a 12 h light/dark cycle. Animals were fasted for 48 h before experiments and give free access to water. All experiments were conducted in accordance with the National Institutes Health guidelines and approved by the laboratory Animal Ethics Committee of the Jinzhou Medical University for Animal Research.

Hyperoxia-induced lung injury

In our study, the 32 mice were randomly divided for four groups (n = 8 per group): (1) room-air-expose (sham), (2) hyperoxia-expose with Sal (Sal + Hyperoxia), (3) hyperoxia-exposed (Hyperoxia), (4) hyperoxia-exposed with Y-320 (an inhibitor of IL-17) (Y-320 + Hyperoxia). The mice exposed to normoxia groups were placed in room air with 21% oxygen, and the mice exposed to hyperoxia were placed in over 90% oxygen for 24 h. The continue exposure to over 90% oxygen was achieved in a self-made airtight box which attached to a medical oxygen cylinder, and the O2 level inside was continuously monitored with O2 analyzer, mice had free access to food and water. In the first three days before exposure to the hyperoxia, mice in the Sal + Hyperoxia group or Y-320 + Hyperoxia group were treated with Sal (100 mg/Kg) or Y-320 (2 mg/Kg) once orally every day, while the rest of groups were given equal isotonic saline. Based on the above experiments, eight 8-week-old KM mice were randomly divided into two groups: Sal + Hyperoxia group and Sal + Hyperoxia + IL-17A group. Sal + Hyperoxia + IL-17A group, mice were i.v. injected with 50 μg/kg of recombinant mouse IL-17A (210–17, Pepro Tech, USA). Animal were sacrificed following reperfusion, and lungs were stored at − 80 °C until further experimental analysis.

Lung wet/dry weight ratio

Fresh lung tissue was drained of surface moisture and weighed immediately to obtain the wet weight. Then the lungs were placed in a dry oven for 60 °C for 48 h until the dry weight was obtained. The wet to dry ratio was calculated to reflect the degree of lung edema.

Histopathological scoring of lung injury

Lung tissues were lavaged with saline, dissected and fixed in 4% paraformaldehyde solution at 4 °C for 48 h. Embedded 5 µm tissue sections were deparaffinized with xylene followed by hydrated through graded alcohols. Then the sections were stained with hematoxylin and eosin. Histological sections were scored blindly by 5 readers to the level of induced injury. Briefly, (1) vascular congestion, (2) hemorrhage, (3) alveolar and interstitial inflammation, and (4) thickening of the alveolar wall were each scored on a scale of 0–4. 0, normal lung; 1, injury involving less than 25% of the lung; 2, injury involving 25–50% of the lung; 3, injury involving 50–75% of the lung; and 4, injury involving 75% or more of the lung. The sum of scores were calculated.

Masson trichrome staining

Masson staining was used for detection of collagen fibers in lung tissues. The lung tissue sections were deparaffinized with xylene and followed by rehydrated in a graded alcohol. Tissues sections were stained using the Masson Trichrome Staining kit (G1340, Solarbio) following the manufacture’s instruction. After stained, the tissue sections were observed under a light microscope.

Immunohistochemistry

Lung tissue sections were deparaffinized followed by hydrated. And then antigen was retrieved using 10 mM sodium citrate and endogenous peroxidase activity was quenched with 3% H2O2 for 10 min in the dark. Sections were washed with PBS and then blocked with sheep serum (ZSGB-BIO, ZLI-9056) for 30 min. Every slide was incubated respectively with mouse monoclonal primary antibody: IL-17RA (Bioss, bs-2606R) diluted with TBST in a ratio of 1:300 and secondary antibody: Biotin-Goat Anti Rabbit IgG (proteintech, SA00004-2) diluted with TBST in a ratio of 1:200. After washing, slides were incubated with DAB (ZSGB) followed by stained with hematoxylin and then observed under a light microscope.

Immunofluorescence staining

Lung tissue sections were deparaffinized and rehydrated, and then heated for antigen retrieval in the citrate buffer. After that, they were permeabilized with 0.1% Triton X-100, and blocked with 5% goat serum before incubation with anti-IL-17 (WL02981, 1:200, Wanleibio) and anti-GPX4 (ab125066, 1:200, Abcam) antibodies overnight at 4 °C. Then, the sections were leaved at room temperature for 2 h. After five times washing in phosphate buffered saline (PBS), these sections were incubated with green-fluorescent Alexa 488 goat anti-mouse IgG antibody (K1204, 1:400, Apexbio) and red-fluorescent Dylight 649 goat anti-rabbit IgG antibody (E032620, 1:400, Earthox) for 2 h at room temperature. Counterstaining of nuclei with DAPI (ab228549, Abcam) was also performed. Then slides were observed, and the images were captured with a fluorescence microscope.

Transmission electron microscopy

The lung tissues were fixed for 2 h in 2.5% glutaraldehyde in a 0.05 M sodium cacodylate buffer at a pH of 7.2 at 25 °C, followed by 2 h in 1% OsO4 in a 0.1 M sodium cacodylate buffer and 18 h in 1% aqueous uranyl acetate. After dehydration through an ethanol series, the specimens embedded in Epon 812 and ultrathin sections were collected on copper grids. After staining with uranyl acetate and lead citrate, the sections were examined using a Jeol1230 transmission electron microscope.

Western blotting

Lung tissue samples of 4 different groups were dissected and homogenized in RIPA lysis buffer (Solarbio, R0010) containing with phenylmethanesulfonyl fluoride (PMSF; Solarbio, P0100). Place the samples on ice to sufficiently lysis it and then the homogenate was centrifuged for 30 min at 4 °C. After centrifugation, supernatants were collected and the protein concentration was measured using a BCA kit (Solarbio, PC0020). The proteins were denatured at 100 °C for 5 min. Protein samples (20 ug/lane) were separated on a 10% SDS‐polyacrylamide gel by electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked with 5% nonfat milk at room temperature for 2 h and diluted in TBST, and further incubated with primary antibodies overnight at 4 °C. Herein, various primary antibodies were: rabbit monoclonal anti-GPX4 (ab125066, abcam, 1:2000), anti-TRAF6 (ab40675, abcam, 1:2000), anti-IL-17A (ab79056, abcam, 1:2000), anti-IL-17RA (bs-2606R, Bioss, 1:1000), anti- Act1 (A6776, ABclonal, 1:2000), anti-IL-6 (bs-6309R, Bioss, 1:1000), anti-TGF-β1 (orb11468, Biorbyt, 1:200), anti-p38 MAPK (AF6456, Affinity, 1:1000), anti-phospho-p38 MAPK (AF4001, Affinity, 1:1000), anti-β-actin(E021020, Earthox, 1:5000). After washing 4 times with TBST for 20 min, the membranes were incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibodies. Finally, the protein bands were detected using an ECL detector. Band density was quantified by Image J software.

Iron assay

The concentrations of endogenous ferrous iron levels of the lung tissue were measured with the iron assay kit (I291, Dojindo, Japan). Lung tissue was fully washed with cold PBS, and homogenized in iron assay buffer on the ice, and then centrifuged at 16,000 × g for 10 min. The supernatant was collected for subsequent experiments. The 100 μL standard (1 mmol/L) was added with 900 μL assay buffer to made into 1000 μmol/l standard solution and then diluted to prepare standard solution (50, 25, 12.5, 6.25, 3.125, 1.5625, 0 μmol/l). The 5% volume of reducer solution was added to each of standard solution, and 5% volume of assay buffer for ferrous iron along with blank tube. All tubes were incubated for 15 min at 37 °C. The 100 μL iron probe was added to each reaction, then mixed and incubated for a further 60 min at 37 °C in a 96-well plate. The absorbance at 593 nm was measured by microplate reader to calculate the iron level.

MDA assay

In order to evaluate the level of ferroptosis in each group, the concentration of malondialdehyde (MDA) in cell lysates was assessed with MDA assay kit (WLA048, Wanleibio, China) according to the manufacture’s instruments.

Statistical analysis

The results are presented as mean ± SEM. Differences were analyzed using an unpaired two-sided Student’s t-test for a two-group comparison or one way ANOVA test followed by a Bonferroni post hoc test for multiple comparisons. P values < 0.05 were considered to indicate a statistically significant difference. Statistical analyses were carried out with SPSS 20.0.

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