Our animal study is reported in accordance with the ARRIVE guidelines for reporting experiments involving animals. Experiments were carried out in a randomized and blinded manner and statistical analyses were performed prior to revealing the treatment groups. Rats were assigned an identity number and assigned to groups randomly so that the experimenter was blinded to treatment.
Materials
Animal
We purchased 120 healthy SD rats (8-weeks old) from the Experimental Animal Center of Xinjiang Medical University (male: 60; female: 60; weight: 160–200 g), animal license number: SCXK (Xinjiang) 2016-0003.
Instruments
Cryostat; Optical microscope(Leica); refrigerated centrifuge (Eppendorf); Vertical electrophoresis tank (Shanghai Tianneng Technology Co., Ltd. Shanghai, China); Constant temperature mixer (ABSON); Ultrasonic cell disruptor (Jiangsu Wavefield Intelligent Technology Co., Ltd.Jiangsu, China); Microplate reader softmax Pro (Molecular Devices); Automatic digital gel image analysis system (Shanghai Tianneng Technology Co., Ltd. Shanghai, China); Analytical balance (METTLER TOLED0); High performance liquid phase spectrometer (Waters); Series Mass spectrometer (Thermo Fishier Scientific), Northwest Special Environmental Artificial Test Chamber (Xinjiang Special Environmental Medicine Key Laboratory, located in Urumqi, Xinjiang). BC-5300Vet Automatic Hemacytometer and its ancillary reagents from WanRui Co., Shenzhen, China.
Pharmaceutical reagents
DML (produced in Xinjiang Madison Pharmaceutical Company Decoction Piece Factory, batch number: M30062307, Xinjiang, China); The extraction of TFDM: DML powder is added with ethanol (60%) at a solid-to-liquid ratio of 1:40, and refluxed and infiltrated at 50 °C. The total time is 50 min; Nifedipine tablets (produced by Shanxi Yunpeng Company, batch number: F160601, Shanxi, China); Ammonia, IAM iodoacetamide, Triethylammonium bicarbonate buffer, Trypsin protease, Sodium lauryl sulfate SDS, Acetone, BCA protein concentration Assay kit (enhanced), SDS-PAGE gel preparation kit, Coomassie brilliant blue staining solution (conventional method), iTRAQ 8 PLEX, Protein Ladder (non-prestained), Protease Inhibitor Cocktail, TCEP, Water LC/MS, Acetonitrile LC/MS, Methanol LC/MS, Formic acid LC/MS, ketamine, diazepam, atropine.
Methods
Model building
The experimental conditions for rats in the control group (CG) were as follows: a simulated altitude of 720 m, a temperature range of 18–26 °C, a humidity range of 40–60%, a pressure of 93.2 kPa, and an oxygen partial pressure of 19.54 kPa. The control rats were fed under these conditions for 45 days with free access to food and no drug intervention. During the experiment, we made a series of observations relating to behavior, autonomous activities, food intake, drinking, hair, feces, urine, eyes, ears, nose, and mouth. We also checked for secretions and weighed each rat each day.
The experimental conditions for rats in the plateau model group (MG) were as follows: a simulated altitude of 5000 m, a temperature range of 18–26 °C, a humidity range of 40–60%, a pressure of 54.1 kPa, and an oxygen partial pressure of 10.84 kPa. The rats were fed for 30 days under this condition. During the experiment, we made a series of observations relating to behavior, voluntary activities, food intake, drinking, hair, feces, urine, eyes, ears, nose, and mouth; we also weighed each rat on a daily basis. The experiments were carried out in Northwest Special Environment Artificial Test Cabin, Xinjiang Special Environmental Medicine Key Laboratory, Urumqi, Xinjiang).
Experiment grouping and intervention measures
We randomly divided 120 rats into six groups. Using the stratified randomization method, we stratified the rats according to gender. We randomized models for 60 female rats and 60 male rats and a random ranking table was used to generate random numbers so that we could divide the rats into six groups. Finally, each group contained the same number of rats in each group; 20 rats in each group; half were male). The specific groupings were as follows:
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Plain control group (CG): No drug intervention was given and rats were kept in a plain environment for 45 days.
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Plateau model group (MG): No drug intervention was given and the rats were kept in a plateau environment for 45 days.
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Nifedipine group (NE): housed in a plateau environment for 45 days, treated with 2.7 mg/kg of nifedipine once a day on last 15 days.
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TFDM low-dose group (DML·L): housed in a plateau environment for 45 days, treated with 15 mg/kg of TFDM once a day on last 15 days.
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TFDM medium-dose group (DML·M): housed in a plateau environment for 45 days, treated with 30 mg/kg of TFDM once a day on last 15 days.
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TFDM high-dose group (DML·H): housed in a plateau environment for 45 days, treated with 60 mg/kg of TFDM once a day on last 15 days.
The determination of pulmonary artery pressure
The rats were anesthetized by intraperitoneal injection of 0.75 mL/100 g solution of atropine (0.5 mg/mL), ketamine (50 mg/mL), and diazepam (5 mg/mL). The rats were then fixed in the supine position, the neck and chest skin was cut and the thoracic cavity was opened along the midline of the sternum to expose the lungs and heart completely. An A7-gauge needle, filled with normal heparinized saline, was then inserted into the pulmonary artery to measure pulmonary artery pressure. The position of the needle was observed directly with the naked eye. The other end of the needle was connected to a pressure transducer with a self-made catheter, and a biosignal recorder was used to monitor pressure changes.
The determination of serum levels of Hb, Hct
Previous reports showed that the serum levels of Hb and the Hct were significantly increased in CMS rats6. Therefore, to verify that we had successfully established a rat model of CMS model, we measured the serum levels of Hb and the Hct by using the BC-5300 Vet Automatic Hemacytometer and ancillary reagents (WanRui Co., Shenzhen, China). The tests were completed within 0.5—1 h of blood sampling.
Pathological examinations of the lungs and pulmonary artery
Rats were euthanized 24 h after the final drug administration. (Rats were fixed on the lid of the rearing box, grasp the tail of the rat with one hand, pull it back with a slight force, and press the head with the thumb and index finger of the other hand quickly, use surgical scissors or the tweezers quickly pressed the rat’s neck, and both hands used force to dislocate the cervical spine, which caused the spinal cord and the brain to break.) The lungs were then removed and washed in normal saline. Lung tissues were then fixed with 10% formalin solution, dehydrated with a gradient series of ethanol, cleared with xylene, and embedded in paraffin. Tissues were then cut into 5 µm sections and stained with hematoxylin and eosin. We then used a light microscope to identify morphological changes in the cells and tissues. We also evaluated pulmonary arteries with diameters < 100 µm because previous reports showed that rats with pulmonary hypertension exhibited a significantly medial thickness in arteries that were < 100 µm in external diameter33. Vessel dimension was taken as the mean of two measurements made at right angles to each other. Wall thickness (WT) was estimated as the intima plus media; we also calculated the proportion of external diameter that was occupied by the WT (%WT) as follows: (2 × wall thickness)/external diameter × 10034.
Protein identification and quantification
Total protein was extracted from blood plasma with urea lysis buffer (7 M urea, 2 M thiourea, and 1% SDS) containing a protease inhibitor. Protein concentrations were then determined with a BCA Protein Assay Kit (Pierce, Thermo, USA). Following reduction, cysteine alkylation, and digestion, samples were then labeled with iTRAQ reagents (Applied Biosystems, 4390812) in accordance with the manufacturer’s instructions. After being desalted with a C18 solid-phase extraction, peptides were then tested by Nano Liquid Chromatography-Mass Spectrometry/Mass Spectrometry analysis35.
RAW data files were analyzed using ProteomeDiscoverer (Thermo Scientific, Version 2.2) against the Rattus database (http://asia.ensembl.org). The MS/MS search criteria were as follows: a mass tolerance of 10 ppm for MS and 0.02 Da for MS/MS tolerance; trypsin as the enzyme with 2 missed cleavage events allowed; carbamido methylation of cysteine and the trimethyltin (TMT) of the N- terminus and lysine side chains of peptides as a fixed modification; and methionine oxidation as a dynamic modification. The false discovery rate (FDR) for peptide identification was set as ≤ 0.01. A minimum of one unique peptide identification was used to support protein identification.
GO function classification and KEGG metabolic pathway analysis
Annotation of all identified proteins was performed using GO (http://www.blast2go.com/b2ghome; http://geneontology.org/) and KEGG pathway (http://www.genome.jp/kegg/) analysis. DEPs were then used for GO and KEGG enrichment analysis. The functional network prediction of protein–protein interaction (PPI) provided by STRING database (version 11, http://string-db.org/)36.
Immunohistochemical detection
The separated lung tissue was embedded with O.C.T. and then quickly frozen in liquid nitrogen. Next, we prepared frozen sections (10 μm). After antigen retrieval, the sections were incubated at 4 °C overnight with primary antibodies against α-1-acid glycoprotein, collagen, fibulin, haptoglobin, PLTP, and TAGLN2. The next day, the sections were washed, and secondary antibodies were added dropwise. Sections were then incubated with diaminobenzidine (DAB), counterstained with hematoxylin, mounted with neutral gum, and images were acquired by microscopy.
Statistical analysis
The data were expressed as the mean ± standard deviation, with one-way ANOVA and Tukey’s post hoc test used for multiple comparisons by SPSS (IBM, version 22.0), in which P < 0.05 was considered statistically significant.
