2.1 Reagents
Rutin was obtained from the China Biological Products Certification Institute. The antibodies against ATG5 (Boster, BA3525-2, Wuhan, China), ATG7 (Boster, BA3527-2, Wuhan, China), BECN-1 (Santa Cruz Biotechnology, 11-11427, Texas, U.S.A), and SQSTM1 (Boster, BA2849, Wuhan, China) were used. Additionally, the RFP-eGFP-LC3 plasmid (Beyotime, C3011, Shanghai, China) was used.
2.2 Animal model
Five- to six-week-old adult male SD rats (250 to 300 g) were obtained from the Experimental Animal Center in our university. Twenty-one SD rats were randomly and equally divided into three groups of normoxia, hypoxia, and hypoxia + rutin. All rats were provided with sufficient food and water. The 7 rats in the normoxic group were kept in a normoxic environment with a FiO2 of 0.21 for 21 days. The other fourteen rats in the hypoxic (7 rats) and rutin (7 rats) groups were housed in a hypoxic environment with a FiO2 of 0.12 for 21 days [33]. At the beginning of the exposure period, the rats in the rutin group were randomly induced with an intraperitoneal injection of rutin, which was dissolved in 5% cellulose sodium carboxymethyl (CMC) at a dose of 200 mgkg-1⋅d-1 for 21 days. The other rats in the normoxic and hypoxic groups were only induced by 5% CMC on the same days.
2.3 RVSP and ventricular weight measurements in rats
At the end of the exposure period, the RVSP of each rat was measured by right heart catheterization. Then, the animals were euthanized by decapitation. The rat hearts were quickly excised, the free wall of the RV was dissected, and each chamber was weighed. The (RV/LV+S) ratio was used as an index of RV hypertrophy. Finally, lung tissue was extracted and retained for subsequent experiments.
2.4 Morphological analysis of the pulmonary artery in rats
The rat lungs were washed with 1 × phosphate-buffered saline (PBS) and stored in the freezer at -80 °C. The remaining lung tissue was immersed in 4% paraformaldehyde, and then embedded in paraffin wax and cut into sections. The sections were stained with hematoxylin-eosin (H&E) staining (AR1180, Boster). The lung tissue sections were examined by digital photomicrography and analyzed with Image-Pro Plus 6.0.
2.5 Cell isolation and culture
PASMCs were prepared from rat pulmonary arterial (PA) tissue. The PA was slit to remove the pulmonary artery endothelial cells (PAECs) and washed with 1 × HEPES buffer. The PASMCs were digested by using 1 mg/mL collagenase II at 37 °C for 30 min and were washed once with 1 × phosphate buffered saline (PBS) to remove the collagenase II. Then, PASMCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (SH30022.01B, Hyclone) with 20% fetal bovine serum (FBS) (SV30087.03, Hyclone) and incubated at 37 °C. Finally, the cell culture medium was replaced, and the cells were ready to use in subsequent experiments. In vitro PASMCs were treated with 0.1 μmol/L rutin and exposed to hypoxia for 24 h by placing the cells in a hypoxia chamber (THERMO) with a water-saturated atmosphere comprising 3% O2, 5% CO2, and 91% N2.
2.6 Immunofluorescence analysis
The PASMCs were plated in 6 well plates and grown on coverslips to 80% confluence. The cells were randomly divided into three groups, including normoxia, hypoxia and hypoxia + rutin. The cells were permeabilized with 0.01% Triton X-100 after fixation. The cells were then incubated with primary antibodies overnight at 4°C. After being washed, cells were incubated with the corresponding Cy3 (A0516, Beyotime) or FITC (A0562, Beyotime) secondary antibodies followed by 4’, 6-diamidino-2-phenylindole (DAPI) (C1005, Beyotime) staining. The slices were then examined under a fluorescence microscope (Leica XF 6000).
2.7 Transmission electron microscopy
The PASMCs centrifuged at 3000 × g for 15 min and fixed with 2% glutaraldehyde (electron microscopy grade), and the photographing of autophagic vacuoles was consigned to the Electron Microscopy Center of our university.
2.8 Western blot analysis
The protein samples were extracted from PASMCs and lung tissues of rats as described previously [34]. Protein concentrations were determined by the Bradford assay using bovine serum albumin (BSA) (A8020, Solarbio) as a standard. The protein samples were separated on a 10-12% SDS-PAGE gel, transferred into a nitrocellulose membrane, and blocked in 5% nonfat milk. The membrane was incubated with specific antibodies against ATG 5 (1: 400), ATG7 (1: 400), BECN-1 (1: 400), SQSTM1 (1: 400), Mfn1 (1: 500), and β-actin (1: 1000) followed by reaction with horseradish peroxidase-conjugated secondary antibodies and visualization with enhanced chemiluminescence reagents.
2.9 siRNA design and transfection
Mfn1 and negative-control siRNAs were purchased from GenePharma (China). The siRNA sequences are as follows: siMfn1 (NM_138976.1): sense, 5’-CCAGCUGGUAUACAAUATT-3’, antisense,
5’-UAUUCUGUAUACCAGCUGGTT-3’. NC control: sense,
5’-UUCUCCGAACGUGUCACGUTT-3’, antisense,
5’-ACGUGACACGUUCGGAGAATT-3’.
The PASMCs were cultured in 6 well plates until 50-70% confluence. After growth arrest, 2 μg of small interfering RNA (siRNA) and 10 μL X-tremeGene of siRNA transfection reagents were diluted in serum-free Opti-MEM-1 medium. The siRNA-containing medium was added to a 6 well plate for 24 hours. The knockdown efficiency was determined by western blot (WB) analysis.
2.10 Statistics
The data are presented as the mean ± standard deviation (SD). The analysis was performed with Student's t-test or one-way analysis (ANOVA) by Dunnett's test, where appropriate. The statistical significance level was set at P < 0.05. Statistical analysis was performed with Statistical Product and Service Solutions version 13.0.