A total of 32 specific pathogen free 6–8-week-old male Sprague Dawley (SD) rats (body weight, 180–220 g) were purchased from the Animal Care and Use Committee of the Army Medical University (Chongqing, China). The experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (10). All experimental procedures were approved by the Ethics Committee of Guizhou Provincial People's Hospital (Guiyang, China). All efforts were made to minimize animal suffering and to reduce the number of animals used.

All animals were maintained in a controlled environment (22–25°C, 40–60% relative humidity, with a 12-h light-dark cycle with the lights on at 08:00 am) with food and water available. Animals were allowed to acclimatize for 1 week to the experimental chamber. The rats were allocated randomly to four groups: Control group (n=8), IH group (n=8), IH+25 mg/kg Mdivi-1 group (n=8) and IH+50 mg/kg Mdivi-1 group (n=8). The rats in the IH group, IH+25 mg/kg Mdivi-1 group and IH+50 mg/kg Mdivi-1 group were exposed to IH conditions (21% O₂, 5% CO₂ and balanced N₂) for 90 sec and hypoxia (8% O₂, 5% CO₂ and balanced N₂ for 30 sec) from 08:00 am to 04:00 pm daily for 4 consecutive weeks (11–13). The chambers had a rear N₂ inlet and a front air extractor, which enabled recovery to normoxia. A computerized system controlled the valve inlets and the alternating cycles of the extractors. The CO₂ in the chamber was maintained at a low level by continuous air extraction (14). Rats in the control group were maintained under normoxic conditions during the experiment. The treatment groups were injected intraperitoneally with Mdivi-1 (MedChemExpress, Monmouth Junction, NJ, USA) prior to oxygen treatment every day (15). A general clinical pain score for scoring cyanosis condition for decisions about endpoint was use as presented in Table I (16).

Trained personnel euthanized the experimental animals via CO₂ inhalation following IH exposure. Rats were left in the chamber until clinical death could be ensured. The flow rate of CO₂ displaced 20% of the volume chamber per minute. If no breath was seen for 60 sec the rodent was removed from the chamber. To ensure death, the rat's chest was felt to determine that there is no longer a heartbeat. Death was verified following euthanasia and prior to disposal. The lungs were dissected out and the right lower lobes were fixed in 4% paraformaldehyde at room temperature for 48 h. The remaining lung tissues were frozen in liquid nitrogen and then stored at −80°C until further analysis.

The right lower lobes were fixed in 4% paraformaldehyde at room temperature for 24 h, embedded in paraffin blocks and slices into 6-µm-thick sections. The sections were then stained with haematoxylin for 5 min and eosin for 90 sec at room temperature, and observed by light microscopy. Damage to the lung tissue was scored by the pathologist on a scale of 1 (no injury) to 4 (worst), as described previously (17).

WTRL1 cells (a rat pulmonary alveolar epithelial cell line; http://www.cellresource.cn/fdetail.aspx?id=1349) were purchased from the National Infrastructure of Cell Line Resource, (Beijing, China). The cells were grown in RPMI-1640 medium (HyClone, Logan, UT, USA) with 10% FBS (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), penicillin (100 units/ml) and streptomycin (100 µg/ml) and were cultured at 37°C in 5% CO₂ in a humidified incubator. WTRL1 cells in the logarithmic growth phase were divided randomly into five groups (3×10⁶ cells/well) in 6-well plates: Control, IH, IH+10 µM Mdivi-1, IH+50 µM Mdivi-1 and IH+100 µM Mdivi-1. The control group was exposed to air, while the IH group, IH+10 µM Mdivi-1 group, IH+50 µM Mdivi-1 group and IH+100 µM Mdivi-1 group were placed in a low oxygen culture box containing 5% O₂ for 1 h. The box was then reoxygenated with 21% O₂ for 30 min and this process was repeated for 12 h. The treatment groups were incubated with Mdivi-1 for 30 min prior to IH culture.

WTRL1 cells were grown logarithmically and placed on 60-mm Petri dishes in a low oxygen culture box containing 5% O₂ for 1 h. The box was reoxygenated with 21% O₂ for 30 min and this process was repeated for 6, 12, 24 and 48 h. Next, the cells were resuspended in Annexin V-PI from a dual-staining assay kit (Nanjing KeyGen Biotech. Co. Ltd., Nanjing, China) for 10 min at room temperature. The fluorescence signals from Annexin V and PI were measured by flow cytometry with a FACSCalibur^™ flow cytometer (BD Bioscience), and the data were analysed using Cell Quest Pro software (version 5.0; BD Bioscience). The flow cytometry results were measured in 1 h.

Total RNA was extracted from lung tissues or cells with TRIzol (Thermo Fisher Scientific, Inc.). The D260/D280 ratio was measured and samples with a ratio between 1.8 and 2.0 were used for reverse transcription with a High Capacity cDNA Archive kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The reverse transcription protocol were as follows: 25°C for 10 min, 37°C for 120 min and 85°C for 5 min. qPCR was applied to analyze the cDNAs by using the 2X Maxima SYBR-Green/ROX qPCR Master Mix kit (Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. The amplification conditions were as follows: 95°C for 10 min for denaturation, followed by 40 cycles of 95°C for 15 sec, 60°C for 30 sec and 72°C for 60 sec. qPCR for Bcl-2, caspase-3, caspase-9, Cyt-C and Drp-1 was performed using TaqMan gene expression assays and the 2^−ΔΔCq method with GAPDH as the endogenous control (18). The primers were synthesized by Sheng Gong (Shanghai) Co., Ltd. (Shanghai, China) and the sequences are presented in Table II.

Total protein cell lysates were isolated from rat lungs or from cells as described previously (19). Protein quantification was assessed using a bicinchoninic acid protein quantification assay and absorbance read at 562 nm was used to determine sample volumes for further analysis. A total of 50 µg total protein from each tissue homogenate was separated by 30% SDS-PAGE and then transferred to polyvinylidene difluoride membranes, which were blocked with 5% skim milk at room temperature for 1 h. Membranes were washed twice with TBST and incubated with primary antibodies at 4°C overnight against Bcl-2 polyclonal antibody (cat. no. YT0470), caspase-3 polyclonal antibody (cat. no. YT0656), caspase-9 polyclonal antibody (cat. no. YT0662), Drp1 Polyclonal antibody (cat. no. YT1414; all 1:1,000; ImmunoWay Biotechnology Company, Plano, TX, USA), Cyt-C antibody (cat. no. 10993-1-AP; 1:1,000; ProteinTech, Group, Inc., Chicago, IL, USA), mouse monoclonal β-actin antibody (1:4,000; cat. no. YM3138; ImmunoWay Biotechnology Company). Membranes were washed twice and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (1:6,000; cat. no. PMK-014-091; Bioprimacy, Wuhan, China) and goat anti-rabbit IgG (1:8,000; cat. no. PMK-014-090; Bioprimacy, Wuhan, China) for 2 h at room temperature. The blots were visualized with eECL western blot kit (Beyotime Institute of Biotechnology, Jiangsu, China). The intensities of the bands were measured using ImageLab 5.1 Software (Bio-Rad Laboratories, Inc.). Rat β-actin was used as the loading control in western blotting.

All experiments were repeated three times. All data are presented as mean ± standard deviation. Comparisons of two or three experimental conditions were evaluated using an unpaired Student's t-test or one-way analysis of variance and the Bonferroni t-test followed by the Bonferroni post-test using GraphPad Prism 5.0 software (GraphPad Software Corp, San Diego, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

Histopathological examinations were performed for the lungs of rats subjected to normoxia, IH, IH+25 mg/kg Mdivi-1 and IH+50 mg/kg Mdivi-1. IH caused severe interstitial pneumonia and a large number of inflammatory cells, while following Mdivi-1 intervention, interstitial pneumonia and inflammatory cells were reduced. The effect in the IH+50 mg/kg Mdivi-1 was more obvious compared with in the IH+25 mg/kg Mdivi-1 group (Fig. 1). Consistent with these histopathological observations, the lung injury score in the IH group was significantly increased compared with the control group (P<0.005) and the score in the IH+50 mg/kg Mdivi-1 group was significantly decreased compared with in the IH group (P<0.005).

After SD rats were exposed to IH for 4 weeks and after WTRL1 cells were exposed to IH for 12 h in culture, the expression of apoptosis markers was increased at the gene level in the rat lung tissue cells and WRTL1 cells. In addition, Mdivi-1 treatment effectively reduced the apoptosis rate of the hypoxia-exposed cells. After SD rats were exposed to IH for 4 weeks, Bcl-2 mRNA levels were significantly decreased (P<0.05; Fig. 2A), but caspase-3 and caspase-9 mRNA levels were significantly increased compared with the control group rats (P<0.05; Fig. 2B and C). After Mdivi-1 intervention, Bcl-2 mRNA levels were significantly increased and caspase-3, and Drp-1 mRNA levels were significantly decreased in the IH+50 mg/kg Mdivi-1 group compared with the IH group (P<0.05; Fig. 2A, B and D). However, no significant difference in Cyt C levels was identified between the groups (Fig. 2E). After WTRL1 cells were exposed to IH for 12 h, Bcl-2 mRNA levels were significantly decreased, but caspase-3, caspase-9, Cyt-C and Drp-1 mRNA levels were significantly increased compared with the control group (P<0.05; Fig. 3A-E). After Mdivi-1 intervention, Bcl-2 mRNA levels were significantly increased and caspase-3, caspase-9, Cyt-C and Drp-1 mRNA levels were significantly decreased compared with in the IH group (P<0.05).

After SD rats were exposed to IH for 4 weeks and after WTRL1 cells were exposed to IH for 12 h in culture, the expression of apoptosis markers was increased at the protein level in rat lung tissue cells and WRTL1 cells. In addition, Mdivi-1 treatment reduced the apoptosis rate of the hypoxia-exposed cells. Western blotting results for Bcl-2, caspase-3, caspase-9, Cyt-C and Drp-1 protein expression in rat lungs are presented in Fig. 4. After SD rats were exposed to IH for 4 weeks, the protein expression of Bcl-2 in the IH group was significantly decreased, but the protein expression of caspase-3, caspase-9 and Drp-1 was significantly increased compared with the control group (P<0.05; Fig. 4A-E). After Mdivi-1 intervention, the protein expression of Bcl-2 was significantly increased and the protein expression of caspase-3, caspase-9, and Drp-1 was significantly decreased in the IH+50 mg/kg Mdivi-1 group compared with the IH group (P<0.05). After WTRL1 cells were exposed to IH for 12 h, Bcl-2 protein expression was significantly decreased in the IH group and caspase-3, caspase-9 and Drp-1 protein expression was significantly increased (P<0.05; Fig. 5A-E). After Mdivi-1 intervention, Bcl-2 protein expression was significantly increased and caspase-9, Cyt-C, and Drp-1 protein expression was significantly decreased in the IH+100 µM Mdivi-1 group (P<0.05; Fig. 5 A, B and D-F).

The apoptosis rate of WTRL1 cells was positively correlated with the time of exposure to IH. After WTRL1 cell exposure to IH for 6, 12, 24 and 48 h, the rate of apoptosis in WTRL1 cells increased gradually with the IH exposure time, and the apoptosis rate was the highest at 48 h (P<0.05; Fig. 6).