A total of ten pregnant Wistar rats (weight, 200–250 g; 10 weeks old) were purchased from the Animal Center of Nanjing Medical University. Animals were housed at 25±5°C, with 50% humidity and 12 h light/dark cycles with ad libitum supply of food and water. A rat model of BPD was established according to a previous study (26). Briefly, the 15 pups of the pregnant Wistar rats were randomly divided into the following three groups (n=5): i) BPD model group; ii) BPD + MK group and; iii) control group. The model group rats were maintained in a sealed Plexiglass chamber with 85% oxygen for 14 days from the date of birth. The oxygen concentration in the chamber was continuously recorded using an analyzer with a strip-chart recorder (Servomex). The control group rats were maintained in room air. All animal procedures were performed according to the Institutional Animal Care and Use of Laboratory Animals guidelines by the National Institutes of Health. The present study was approved by the Animal Ethics Committee of The First Affiliated Hospital of Bengbu Medical College.

The BPD + MK group were administered with an intraperitoneal injection of 10 mg/kg MK (Sigma-Aldrich; Merck KGaA) every other day from day 2 to day 14 post-hyperoxia induction (27). The control and BPD model groups were administered with an intraperitoneal injection of the same amount of saline (0.9% NaCl). All efforts were made to alleviate the pain of the rats throughout the experiment. In addition, all experiments were stopped when the rats lost >15% of their body weight. The health and behavior of all rats were monitored every 2 days, and no rats died prior to the end of the experiment. Rats were anaesthetized by pentobarbital intraperitoneal injection (40 mg/kg) and subsequently sacrificed via cervical dislocation (rats without a heartbeat and not breathing were confirmed as dead) at 14 days after hyperoxia induction, for further experiments.

Tissue samples from the middle lobe of the right lungs in the rats were aerated with PBS under a pressure of 18 cm H₂O. Subsequently, tissue samples were fixed in 4% paraformaldehyde (Beyotime Institute of Biotechnology) at 4°C overnight. The lung tissues were embedded in paraffin and cut into 4-µm thick sections, following which they were rehydrated using an alcohol gradient (absolute ethanol for 5 min; 90% ethanol for 2 min; 80% ethanol for 2 min; 70% ethanol for 2 min; distilled water for 2 min), and stained with hematoxylin and eosin at room temperature for 10 min. The mean linear intercept (MLI), which represents the average alveolar diameter, was measured under a fluorescent microscope at ×200 magnification. The number of alveoli transected by the line from the center of the most peripheral bronchiole to the pleura, which is defined as the radial alveolar count (RAC), was calculated to evaluate the stage of lung development. In addition, the right upper lung lobes of the rats were collected and weighed to determine the lung weight/body weight (LW/BW) ratio.

AEC II cells were isolated from three rat pups from the control group within 24 h of birth (28). Briefly, the lungs of newborn rat pups were collected and digested with 0.25% Trypsin-EDTA (Gibco; Thermo Fisher Scientific, Inc.) at 37°C. Subsequently, the lungs were digested with 0.1% collagenase I (Gibco; Thermo Fisher Scientific, Inc.) and filtered through a cell strainer (70 µm). The isolated cells were resuspended and purified by centrifugation (1,000 × g, 4°C, 5 min). Cells were incubated in minimum essential medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂. At passage 3, the purity and viability of AEC II cells was determined according to a previous study (28,29), and cells with 90% purity and >95% viability were used for subsequent experiments.

Cells were randomly divided into the following two groups: Hyperoxia-induced group (HOX group; cells cultured at 37°C for 48 h in an 85% O₂−5% CO₂ incubator) and the normoxic control group (NOX group; cells cultured at 37°C for 48 h in a 21% O₂−5% CO₂ incubator). The NOX group was further divided into three subgroups: i) Control; ii) transforming growth factor (TGF)-β1 (Sigma-Aldrich; Merck KGaA); and iii) TGF-β1 + MK (10 µM). Cells in the TGF-β1 group were cultured with TGF-β1 (3 ng/ml) at 37°C for 48 h to induce EMT. Cells in the TGF-β1 + MK group were cultured with TGF-β1 (3 ng/ml) at 37°C for 48 h to induce EMT and then treated with 10 µM MK at 37°C for 24 h. The HOX group was further divided into five subgroups: i) Control; ii) hyperoxia; iii) hyperoxia + TGF-β1 antibody; iv) hyperoxia + MK; and v) hyperoxia + TGF-β1 antibody + MK. Cells in the control group did not undergo any treatment.

The protein expression levels of surfactant protein C (SP-C), E-cadherin, N-cadherin, Vimentin, collagen I (Col I), matrix metallopeptidase (MMP)1/3, TGF-β1 and Smad3 in the tissue samples and cells were determined by western blotting. Briefly, total protein was extracted using a total protein extraction kit (Beyotime Institute of Biotechnology), and quantified using the Bicinchoninic Acid Protein kit (Pierce; Thermo Fisher Scientific, Inc.). Proteins (40 µg per lane) were loaded on a 10% gel, resolved via SDS-PAGE under reduced conditions, and subsequently transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk and PBS with 0.5% Tween-20 (PBST) solution at room temperature for 1 h. Subsequently, the membranes were incubated at 4°C overnight with primary antibodies targeted against: SP-C (1:1,000; cat. no. ab211326; Abcam), E-cadherin (1:1,000; cat. no. ab233766; Abcam), N-cadherin (1:1,000; cat. no. ab76011; Abcam), Vimentin (1:1,000; cat. no. ab137321; Abcam), Col I (1:1,000; cat. no. ab34710; Abcam), MMP-1 (1:1,000; cat. no. ab137332; Abcam), MMP-3 (1:1,000; cat. no. ab52915; Abcam), TGF-β1 (1:1,000; cat. no. ab92486; Abcam), Smad3 (1:1,000; cat. no. ab40854; Abcam) and GAPDH (1:1,000; cat. no. ab181602; Abcam). Following primary incubation, the membrane was washed three times with PBST and incubated with a horseradish peroxidase-conjugated secondary antibody (1:1,000; cat no. ab7090; Abcam) for 1 h. Protein bands were visualized by enhanced chemical luminescence (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Protein expression levels were semi-quantified using ImageJ software version 1.46 (National Institutes of Health) with GAPDH as the loading control.

Total RNA was isolated from the tissues and cells using an RNA Isolation kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol, and reverse transcribed into first strand cDNA using the cDNA Synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The temperature protocol for the reverse transcription reaction were as following: annealing at 25°C for 5 min, cDNA synthesis at 42°C for 60 min and termination at 80°C for 2 min. Subsequently, mRNA expression levels were assessed by qPCR using a Prism 7000 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) with SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd.), according to the manufacturer's protocol. The primer pairs used for qPCR were purchased from Sangon Biotech Co., Ltd. The following primer sequences were used: SP-C forward, 5′-TTGGTCCTTCACCTCTGTCC-3′ and reverse, 5′-CTCCCACAATCACCACGAC-3′; Vimentin forward, 5′-GACGCCATCAACACCGAGTT-3′ and reverse, 5′-CTTTGTCGTTGGTTAGCTGGT-3′; E-cadherin forward, 5′-CGAGAGCTACACGTTCACGG-3′ and reverse, 5′-GGGTGTCGAGGGAAAAATAGG-3′; N-cadherin forward, 5′-TTTGATGGAGGTCTCCTAACACC-3′ and reverse, 5′-ACGTTTAACACGTTGGAAATGTG-3′; GAPDH forward, 5′-CTTTGGTATCGTGGAAGGACTC-3′ and reverse, 5′-GTAGAGGCAGGGATGATGTTCT-3′. The following thermocycling conditions were used for qPCR: 5 min at 95°C; followed by 40 cycles of 95°C for 10 sec and 60°C for 30 sec. mRNA expression levels (SP-C, E-cadherin, N-cadherin, Vimentin) were quantified using the 2^−ΔΔCq method (30) and normalized to the internal reference gene GAPDH.

Data are presented as the mean ± standard deviation of at least three independent experiments. One-way ANOVA followed by Tukey's post hoc test was used for comparisons between multiple groups. Statistical analyses were performed using SPSS software (version 18; SPSS, Inc.). P<0.05 was considered to indicate a statistically significant difference.

To assess the effects of MK on lung function, the MLI, RAC and LW/BW ratio of the lungs were assessed in the different groups. The MLI of the BPD model group was longer compared with the control group, and MK treatment significantly decreased the MLI in the BPD model group (Fig. 1A). Similarly, MK markedly significantly the RAC and decreased the LW/BW ratio in the BPD model group (Fig. 1B and C).

The lung tissues of the rats were isolated at day 14 post-hyperoxia induction, and the protein and mRNA expression levels of epithelial cell markers E-cadherin and SP-C, as well as mesenchymal cell markers N-cadherin and Vimentin, were measured by western blotting and RT-qPCR, respectively. The protein expression levels of E-cadherin and SP-C in the BPD model group were markedly decreased, whereas the protein expression levels of N-cadherin and Vimentin were increased compared with the control group (Fig. 2A). The mRNA expression levels of E-cadherin and SP-C in the BPD model group were significantly decreased, whereas the mRNA expression levels of N-cadherin and Vimentin were significantly increased compared with the control group (Fig. 2B-E). MK treatment notably inhibited BPD-induced effects on protein and mRNA expression levels (Fig. 2A-E).

In addition, western blotting was performed to measure the relative expression of TGF-β signaling pathway-related proteins Col I, MMP1/3, TGF-β1 and Smad3. Compared with the control group, the expression levels of Col I, MMP1 and MMP3 in the BPD model group were significantly increased, and MK treatment significantly inhibited these effects (Fig. 3A-D). Similarly, the expression levels of TGF-β1 and Smad3 in the BPD model group were increased compared with the control group, which were significantly decreased by treatment with MK (Fig. 3A, E and F).

AEC II cells were isolated from the normal rats and incubated with TGF-β1 to induce EMT. The protein and mRNA expression levels of E-cadherin, SP-C, N-cadherin and Vimentin were measured by western blotting and RT-qPCR. Compared with the control group, the protein expression levels of E-cadherin and SP-C in the TGF-β1 group were markedly decreased, whereas the protein expression levels of N-cadherin and Vimentin were notably increased, and these alterations were inhibited by treatment with MK (Fig. 4A). Compared with the control group, the mRNA expression levels of E-cadherin and SP-C in the TGF-β1 group were significantly decreased, whereas the mRNA expression levels of N-cadherin and Vimentin were significantly increased, and these alterations were inhibited by treatment with MK (Fig. 4B-E).

Furthermore, western blotting was performed to detect the protein expression levels of TGF-β signal pathway-related proteins Col I, MMP1/3, TGF-β1 and Smad3. The results indicated that the expression levels of Col I, MMP1 and MMP3 in the TGF-β1 group were significantly increased compared with the control group, which were significantly decreased by MK treatment (Fig. 5A-D). The expression levels of TGF-β1 and Smad3 in the TGF-β1 group were also increased compared with the control group, which were significantly decreased by treatment with MK (Fig. 5A, E and F).

To further explore the effect of MK on EMT in AEC II cells derived from a hyperoxia-induced rat model of BPD, the effects of MK on hyperoxia-induced AEC II cells were investigated. AEC II cells were divided into five groups (hyperoxia group, hyperoxia + TGF-β1 antibody group, hyperoxia + MK group, hyperoxia + TGF-β1 antibody + MK group, and control group), and the protein and mRNA expression levels of E-cadherin, SP-C, N-cadherin and Vimentin were detected by western blotting and RT-qPCR, respectively. The results suggested that, compared with the control group, the protein expression levels of E-cadherin and SP-C in the hyperoxia group were reduced, and the protein expression levels of N-cadherin and Vimentin were notably increased (Fig. 6A). Compared with the control group, the mRNA expression levels of E-cadherin and SP-C in the hyperoxia group were significantly reduced, and the mRNA expression levels of N-cadherin and Vimentin were significantly increased (Fig. 6B-E). Furthermore, hyperoxia-induced alterations to protein and mRNA expression were inhibited by treatment with MK or TGF-β1 antibody, with the TGF-β1 antibody + MK group displaying the most effective inhibition (Fig. 6A-E).

The hyperoxia group displayed a significant increase in the expression levels of Col I, MMP1 and MMP3 compared with the control group, which was notably inhibited by treatment with MK or TGF-β1 antibody. The combined treatment of MK and TGF-β1 was more effective compared with either treatment alone (Fig. 7A, B and F). Furthermore, compared with the control group, the expression levels of TGF-β1 and Smad3 in the hyperoxia group were markedly increased, and hyperoxia-induced effects were suppressed by treatment with MK or TGF-β1 (Fig. 7A, C and D).