Hyperoxia exposure upregulates Dvl-1 and activates Wnt/β-catenin signaling pathway in newborn rat lung

Reagents and antibodies

The following were obtained: sheep anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody and RIPA lysis buffer (Cell Signaling Technology, MA, USA); BCA protein and, StarSignal chemiluminescent assay kits (Vazyme, Nanjing, China); StarScript II First-strand cDNA Synthesis Mix with gDNA Remover, 2 × RealStar Green Fast Mixture (with ROXII), and TRIGene (TaKaRa, Dalian, China); polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, USA); GSK3β, p-GSK3β, cyclin D1, Dvl-1, and β-catenin primary antibodies (ZENBIO Biotech, Chengdu, China); GAPDH primary antibody (Goodhere Biotech, Hangzhou, China); PCR primers (Genscript Biotech, Nanjing, China); MDA and SOD assay kits (Solarbio Life Sciences, Beijing, China); immunohistochemistry kit (MXB, Fujian, China), rat AECII cells (Bnbio, Beijing, China); Roswell Park Memorial Institute (RPMI) 1640, trypsin, penicillin, and streptomycin (Gibco, Thermo Fisher Scientific Inc., USA); fetal bovine serum (Biological Industries, Beth Haemek, Israel); lipofectamine 3000 (Invitrogen, Thermo Fisher, USA); AnnexinV-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit and Cell Counting Kit-8 (Vazyme, Nanjing, China); and MSAB (Wnt/β-catenin pathway inhibitor (Selleck, Shanghai, China).

Animal procedures and treatment

All animal experimental procedures were approved by the Ethics Committee of Animalsof the Affiliated Wuxi Children’s Hospital of Nanjing Medical University(No:WXCH2020-04-003 at April 15, 2020). Sprague Dawley (SD) rats were obtained from the Nanjing Agriculture University. A total of 36 newborn SD rats were randomly selected into two groups, hyperoxia group (85% O2 from the beginning of birth) and the control group (normoxia, 21% O2). The number of rats used in each group is shown in Table 3. Rats had free access to food and water. On the 3rd, the 7th and the 14th days after birth, nine newborn rats from the two groups were anesthetized with sodium pentobarbital, and their lungs were collected. The right lung was fixed with paraformaldehyde (PFA) for immunohistochemistry, the upper lobes of the left lung were used for real-time qPCR and oxidative stress index test, and the lower lobes of the left lung were used for Western blotting.

Table 3 Experimental groupsOxidative stress index test

The upper lobes of the left lung were homogenized with cold normal saline and centrifuged (4 °C, 12,500 g, 10 min), and the supernatant was collected for assays. MDA and SOD assays were performed using assay kits, according to their manufacturers’ instructions.

Western blot analysis

Total protein was separated from lung tissues with radio immunoprecipitation assay (RIPA), and quantified with a bicinchoninic acid assay (BCA) protein assay kit. After dilution with loading buffer, the separated proteins were boiled for 5 min. Twenty micrograms of protein samples were separated using 12.5% SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes. The membranes were probed overnight at 4 °C with primary antibodies against Dvl-1 (1:1000 dilution), β-catenin (1:1000 dilution), GSK3β (1:1000 dilution), p-GSK3β (1:1000 dilution), and CTNNBL1 (1:1000 dilution) and cyclin D1 (1:1000 dilution) and GAPDH (1:1000 dilution) proteins and subsequently hybridized with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. The protein bands were detected using an ECL advanced system (Millipore) and quantified using Photoshop software.

Immunohistochemistry (IHC)

The lungs were fixed with 4% formalin for 24 h at room temperature. The fixed lungs were sequentially dehydrated, vitrified, embedded in paraffin, fixed, cut into 5 μM thick sections. The sections were dewaxed with dimethylbenzene, hydrated with gradient alcohol according to the manufacturer’s instructions and treated with 3% H2O2 to block endogenous peroxidase activity. The treated sections were placed into an EDTA-Tris buffer solution and microwaved for 20 min, blocked with serum, and incubated overnight at 4°Cwith Dvl-1 primary antibody (1:200 dilution), β-catenin primary antibody (1:200 dilution) and cyclin D1 primary antibody (1:200 dilution). After sequential incubation with a biotin-labeled secondary antibody and streptavidin-peroxidase, sections were developed using 3,3′-diaminobenzidine (DAB) and dehydrated. IHC score was determined semi-quantitatively by multiplication of the positive fraction with the grayscale value according to the following system: a) positive fraction was categorized as 0 (no staining), 1+ (≤10%), 2+ (>10% and <50%), and 3+ (≥50%); b) intensity was graded as 0 (no staining), 1 (low staining), 2 (medium staining), and 3 (strong staining).

Real-time polymerase chain reaction (RT-PCR)

Total RNA was extracted using TRIGene reagent according to the manufacturer’s instructions. The purified mRNA was reverse transcribed into cDNA using PrimeScript™ RT reagent kit with gDNA Eraser and real-time PCR was performed using TB Green® Premix Ex Taq™ II (Tli RNaseH Plus). Data were standardized to the endogenous expression of GAPDH. The sequences of the primers are listed in Table 4. Real-time PCR was performed according to the method provided by QuantStudio 3 (Applied Biosystems, USA) in a 20 μl volume using 1 μl cDNA, l μl forward primer, 1 μl reverse primer and 10 μl 2 × RealStar Green Fast Mixture (with ROX II). The thermal cycling conditions were Stage 1 (1 cycle, 30 s at 95 °C), Stage 2 (45 cycles, 10 s at 95 °C, 30 s at 60 °C), and Stage 3 (1 cycle, 15 s at 95 °C, 1 min at 60 °C).

Table 4 Sequences of the real-time PCR primersCell culture, transient transfection, and hyperoxia treatment

Rat AECII cells were cultured in RPMI-1640 medium at 37 °C in a 5% CO2 atmosphere. The cell culture medium included 10%(v/v) FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. AECII cells were divided into the following five group: AECII cells group (control group), AECII cells hyperoxia exposure group (hyperoxia group), AECII cells Dvl-l downregulation treatment group (si-Dvl-l group), AECII cells Dvl-l downregulation + hyperoxia exposure group (si-Dvl-l + hyperoxia group), and AECII cells Dvl-l downregulation + hyperoxia exposure + 5 μM MSAB treatment group (si-Dvl-l + hyperoxia + MSAB group). When reaching 70% confluency, the transfected AECII cells were treated with lipo3000 and Dvl-l siRNA oligonucleotides and then cultured in serum-free RPMI-1640 medium for 6 h. The cell culture plate was then placed into a sterile modular incubator chamber (MIC-101, Billups-Rothenberg, Del Mar, CA) and exposed to hyperoxia (85% O2 and 5% CO2) for 24 h.

Cell counting Kit-8 assay

The proliferation rates of AECII cells in the five groups were detected using CCK-8 assay. AECII cells (1 × 105 cells/mL), after respectively different treatments, were cultured for 24 h, and then prepared as cell suspensions using culture medium. The cell suspension (100 μL) was inoculated into 96-well plates and cultured for 24 h. The solution (10 μL) was added to each well and incubated for 2 h at 37 °C. The absorbance of each well was measured by microplate reader (Shanghai Flash Spectrum Biotechnology, China) at a wavelength of 450 nm. The data analysis was reported in a previous study [29].

Scratch wound assay

After five different treatments, the AECII cells were cultured for 24 h and then inoculated into 6-well plates and cultured until 100% confluency. A uniform straight scratch was formed in the cells’ monolayer by a 200 μL pipette tip and then washed gently. After culturing for 24 h or 48 h, the scratch wounds were photographed.

Cell apoptosis analysis

Apoptosis was determined with FITC/PI double staining using a flow cytometer. After different treatments for 24 h, the cells were trypsinized, centrifuged, and washed with cold 1 × PBS. The cells were then resuspended in 200 μL binding buffer, mixed with 5 μL Annexin V + FITC and 5 μL PI, and incubated in the dark at room temperature for 15 min. Finally, the cells were mixed with 300 μL binding buffer. The experimental methods and analyses were reported in a previous study [30].

Statistical analysis

The experimental data were expressed as mean ± standard deviation values. Statistical analysis was conducted by SPSS 21.0 software (SPSS Inc., Chicago, IL, USA). The unpaired Student’s t-test was used to compare differences between the two groups. A P value < 0.05 was considered to indicate a statistically significant difference. One-way analysis of variance for multiple-group comparisons was performed using GraphPad Prism 8 (GraphPad Software, San Diego, USA). A P value < 0.05 was considered statistically significant.

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