Cell lines
The lung fibroblast cell line LL 29 was obtained from ATCC. The cells were cultured in F12K minimum essential medium containing 15% fetal bovine serum (Gibco, USA) in an environment containing 5% CO2 at 37 °C.
Pulmonary fibrosis model and experimental design
BALB/c mice weighing 18–22 g was purchased from Charles River Laboratories. The mice were randomly divided into groups with eight mice each. Mice anesthetised with sodium pentobarbital (40 mg/kg) were administered with 5 mg/kg of bleomycin through the trachea. Control mice were treated with an equal volume of saline. A fibrosis model was successfully established after bleomycin treatment for 30 days and pathological examination of the lungs. We continuously administered ginsenoside Rg3 (5mg/kg body weight) to the lung fibrosis mice. After 28 days, the mice were euthanized through the intravenous injection of pentobarbital sodium at a final concentration of 100 mg/kg. The mice were checked, and death was confirmed by observing lack of respiration and cardiac output. Tissues were fixed or frozen for subsequent pathology testing. Euthanasia methods was in accordance to the proper practice of AVMA 2020. All procedures involving animals were performed in accordance with the ethical standards of the Institutional Animal Care and Use Committee (IACUC) at Tianjin International Joint Academy of Biomedicine.
Pathological analysis
After the tissue was cut into 4μm sections and dewaxed, the sections were treated with 3% H2O2 for 10 min to block endogenous peroxidase activity. After antigen repair, the sections were incubated with normal goat serum at 37 °C for 10 min. Afterwards, the sections were incubated with the following primary antibodies: SMA (Affinity, Changzhou, China), HIF-1α (Affinity, Changzhou, China), E-cadherin (Affinity, Changzhou, China) and Vimentin (Affinity, Changzhou, China). After 2 h, the sections were washed and incubated with a secondary antibody (Zhongshan Biotechnology Co., Ltd., Beijing, China) at 37 °C for 30 min. After staining with 3,3′-diaminobenzidine, the sections were fixed for observation.
Cell migration assay
The treated cells were seeded onto a 24-well plate and grown to 70% density at 37 °C with 5% CO2. A straight scratch was then made in the middle of the plate. After 24 h, cell migration and the migration rates were recorded with a microscope.
Cell invasion assay
Matrigel was evenly spread on the bottom of a Transwell chamber. The treated cells were seeded into the Transwell chamber and cultured in a serum-free medium. Then, the 24-well plate was added with 10% FBS medium. After 16 h of incubation in an environment containing 5% CO2 at 37 °C, the cells inside the small chamber were removed; the cells outside were stained with crystal violet, photographed and analysed for cell invasion ability.
Colony formation assay
The treated cells were uniformly seeded onto a 6-well plate at a final concentration of 1,000 cells per well and then incubated for 2 weeks at 37 °C in an environment saturated with 5% CO2. After clones were formed, the cells were washed with 1× PBS and subsequently fixed and stained. The number of clones in each group was determined.
Western blot
The Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology Co., Ltd. P0027) was used to extract nuclear and plasma proteins from the tissues and cells. All lysates contained protease inhibitors. The quantified proteins were separated by 10% SDS-PAGE. After transferring the protein to the PVDF membrane and blocking with 5% BSA, the PVDF membrane was blocked at room temperature with the following primary antibodies: HIF-1α (Affinity, Changzhou, China), TGFΒ1 (Affinity, Changzhou, China), SAMD3 (Affinity, Changzhou, China), p-SMAD3 (Affinity, Changzhou, China) E-cadherin (Affinity, Changzhou, China), Vimentin (Affinity, Changzhou, China), Lamin B (Affinity, Changzhou, China) and GAPDH (Affinity, Changzhou, China). GAPDH and Lamin B were used as loading controls. After 4 h, the excess primary antibody was removed, and the PVDF membrane was incubated with HRP-labelled secondary antibody at room temperature for 2 h. Protein intensity was detected with an Image Lab instrument (Bio-Rad, USA).
Biacore assay
Human HIF-1α cDNA was synthesized by Genwiz (Beijing, China) and cloned into PET-His prokaryotic protein expression plasmid using BamHI and NheI endonucleases. Biacore assay was performed with a Biacore 3000 instrument (GE Healthcare, Piscataway, NJ, USA). 50mM NHS and 200mM EDC were mixed in equal volumes and injected into a closed CMD500M chip (XanTec Bioanalytics) at a rate of 10 μl/min. The purified HIF-1α protein was diluted with sodium acetate buffer pH 5.0 and injected into the chip, and the remaining active groups were blocked with 1M ethanolamine. Subsequently, RG3 was injected into the CM5 sensor chips at a rate of 30μl/min. Data analysis was conducted using the BIA evaluation software.
Molecular docking
The crystal structure of HIF-1α was downloaded from the PDB database and used to perform molecular docking with Rg3 by using the Sybyl X1.1 software.
Scanning electron microscope inspection
After treatment, the cells were grown on climbing films. After 24 h, the cells were fixed and dehydrated in acetone/isoamyl acetate (1:1) and dried with a gradient concentration of acetonitrile. The cells were then coated with gold and photographed using a scanning electron microscope (LEO 1530 VP, Germany).
Statistical analysis
Data were analysed with the SPSS 18.0 statistics software (SPSS Inc., Chicago, IL, USA) and presented as the mean ± standard deviations of the mean. Significant differences between two groups were compared using a Student’s t-test. Comparisons among three or more groups were conducted using ANOVA with post hoc contrasts by Student–Newman–Keuls test. p<0.05 was considered to indicate a statistically significant difference.