2.1 Animals and Animal Model
All animal experiments were approved by the intramural Committee on Ethics Conduct of Animal Studies of Academy of Military Medical Sciences, China. 8-week-old male Sprague-Dawley rats weighing 200–220 g were housed at microisolator cages and allowed free access to water under standard laboratory conditions. Before experimentation, the rats were acclimated to the laboratory environment for three days. For exposure to hypobaric hypoxia rats were maintained for 1–5 day at an equivalent altitude of 7000 m in a hypobaric chamber (0.87%O2).
2.2 AAV9 Construction and Viral Delivery
Rats were injected with Scramble-eGFP and AAV9-ALOX15 knockdown virus, and 1.6E12 vg/rat were delivered via the tail vein, which was synthesized by Genomeditech Co., Ltd (Shanghai, China). Four weeks after virus delivery, an ALOX15 knockdown effect was detected and subsequent experiments was performed.
2.3 RNA-seq
Total RNA of right ventricular muscle was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA,USA) according to the manufacturer’s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel electrophoresis. All samples achieved a RIN valueN8; 500 ng of RNA was used for cDNA library establish. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP and Buffer. The ligation products were sequenced using Illumina HiSeq2500 by Gene Denovo Biotechnology Co.(Guangzhou, China).Principal component analysis (PCA), GO enrichment analysis and KEGG pathway enrichment was performed with corresponding R package.
2.4 Enzyme-Linked Immune Sorbent Assay
The concentrations of cTn-I, CKMB, HMGB-1, TNF-α, IL-6, LPO,CCL2, CCL5, 12/15s-HETE, MIP-1αand GM-SCF in serum or myocardium were measured by ELISA. Rat serum or myocardium supernatant were centrifuged at 3000 rpm for 10 min at 4 ℃, and detected by commercially available kit following the manufacturer's instructions.
2.5 Measurement of myocardial enzyme
The serum levels of creatine kinase MB isoenzymes (CKMB), was measured by automatic biochemical analysis apparatus (Roche Diagnostics; Germany)
2.6 Histopathologic Evaluation
After acute hypobaric and hypoxia stimuli exposure, rats were sacrificed and right ventricular muscle of rats in each group were collected and fixed in buffered 4% form aldehyde for Hematoxylin and eosin (H&E) staining.
2.7 Electron Microscopy
The myocardial tissue of rats in each group were immediately placed in microtubes, fixed with 2.5% glutaraldehyde, postfixed in 2% osmium tetroxide, and then polymerized using epoxy resin. The ultrastructure of the tissue was observed via high-resolution H-7650 TEM.
2.8 Leukocyte Assay
Blood samples were obtained from adult rat by abdominal artery under isoflurane anesthesia. Blood was collected in EDTA microtainer tubes (Becton Dickinson) via heparin-coated capillary tubes. Undiluted blood samples were run on the XN-V series multispecies hematology analyzer (XN-1000V; Sysmex) immediately following collection.
2.9 Immunofluorescence
Immunofluorescence was performed using 5µm-thick sections of 4% paraformaldehyde-fixed paraffin-embedded tissue samples, and the slides were incubated with primary antibodies for 12 h and with secondary antibodies for 2 h. The antibodies are shown in Additional file 1: Table S1. Immunofluorescence images were obtained with a laser scanning confocal microscope.
2.10 Detection of Reactive Oxygen Species (ROS)
Cells were incubated with 2, 7-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe for 20 min (Beyotime, Nanjing, China). After incubation, the cell was washed twice with PBS buffer, and then, ROS levels were detected using immunofluorescence microscopy
2.11 Flow cytometric analysis
Spleen was ground to prepare single-cell suspensions in PBS buffer (Servicebio, Wuhan, China). Bone marrow samples from femurs were prepared as described previously[25]. Whole blood was collected from the orbit to an EDTA vacuum tubes. Red blood cells were lysed in ACK lysis buffer (BD Biosciences) according to the manufactures protocol. 1×106 cells resuspended in 100 µl PBS buffer were prepared for subsequent flow cytometry. Cells were then washed twice with staining buffer, incubated with a mixture of antibodies at 4°C for 1 h (STable1). The gating strategy for mouse monocytes was based on the CD11b and Ly6c staining intensities[26]. Flow cytometric data was analyzed using FlowJo software.
2.12 Liquid chromatography and mass spectrometry
12/15s-HETE were extracted from 20 mg of heart tissue using methanol and acetonitrile 5:3 v/v and spiked with the internal standard LTB4-d5 (5ng). All separations were performed using a ACQUITY Premier (Waters). For optimal chromatographic separation, a Waters ACQUITY Premier HSS T3(100×2.1 mm, 1.7 m)was used. Column using a flow rate of 0.4mL/min at 40°C .The gradient elution was as follows: 0-5min from 50% B to 95% B, 5-6min 95% B,6-6.1 min from 95% B to 50%,6.1-9min 50%B, while using the solvents A, 0.1% formic acid, and B, acetonitrile. Electrospray ionization was performed in the negative ion mode, and the capillary of ESI source was set to 100℃, with the spray voltage at 2.5 kV. The nebulizer gas temperature was 450℃ with a flow rate of 850L/H.
2.13 Cell culture and treatments
HUVEC cells was cultured in DMEM medium and supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin and 10% fetal bovine serum (Lonza, Levallois, France), at 37 ℃under 5% CO2/95% air environment. For 12/15s-HETE (Cayman Europe) treatment, cells attached to the plate bottom were exposed to 0.5ng/ml for 24h.
2.14 Western Blotting Assay
Whole cell lysates were prepared with RIPA lysis buffer and the BCA protein detection kit was used to measure protein concentrations. Thirty micrograms of protein lysate was subject to 10% SDS-PAGE transferred onto polyvinylidene fluoride membranes. After blocking in 5% bovine serum albumin for 3 h at room temperature, the membranes were incubated with primary antibodies (STable1) at 4°C overnight, followed by incubation with horseradish peroxidase (HRP)-linked secondary antibody at room temperature for 1 h. The Omin-Enhanced Chemiluminescence Detection Kit was used to detect and visualize protein bands.
2.15 Statistical analysis
Data are expressed as means ± SD. One-way ANOVO test was used to evaluate difference between groups and p < 0.05 was chosen as the threshold for statistical significance. Statistical analysis was performed using GraphPad Prism version 8 (GraphPad Software, La Jolla, CA, USA).