2.1 Histological sampling
We collected surgical resected paraffin-embedded human fibrotic lung tissues specimens (10 cases) and pathologically normal para-tumor lung tissue specimens (10 cases) from the Department of Pathology, the First Affiliated Hospital of Xi’an Jiaotong University, with the approval of the Institutional Review Board. Immunoreactions were performed on selected lung sections.
2.2 Preparation of recombinant AAV
Self-complementary recombinant adeno-associated virus were constructed by applying an AAV Helper-Free System (Cell Biolabs, SanDiego, CA, United States). The coding DNA of human Tβ4 (GenBank NM_021109.3) was inserted into pscAAV-MCS to yield the pscAAV- Tβ4 plasmid. Recombinant AAV containing Tβ4 (AAV-Tβ4) was generated via co-transfection of pscAAV-Tβ4, pHelper and pAAVRC5 into AAV-293 cells using polyethylenimine (PEI). Recombinant AAV carrying LacZ (AAV-LacZ) was constructed as a control virus. 72 hours after transfection, cells were collected for viral particle isolation, purification and quantitative analysis.
TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, United States) was employed to determine the recombinant AAV (rAAV) titters and the abundance of the rAAV in the lung. The primers against the cytomegalovirus promoter region were as follows: 5’-AGACTTGGAAATCCCCGTGAGT-3’ (forward) and 5’-CGTATTAGTCATCGCTATTACCATGGT-3’ (reverse). The sequence of the probe was 5’-6FAM-AACCGCTATCCACGCCCATTGATG-TAMRA-3’. The collected data were analyzed by the standard curve method.
Specific pathogen free, 6-week-old male ICR mice, weighing 25-30g were obtained from the Experimental Animal Center, School of Medicine, Xi'an Jiaotong University. The mice were housed under pathogen free conditions under a 12 hours light/dark cycle at constant temperature (22±2°C) and humidity, with free access to water and standard laboratory chow. All mice were acclimatized to the abovementioned conditions for one week before initiating experiments. All efforts were undertaken to minimize the suffering of the mice.
To test the transduction efficiency of repeated intraperitoneal (i.p.) rAAV injection, twenty-four mice were divided into 3 groups: PBS, AAV-LacZ and AAV-Tβ4. Mice in PBS group were injected with PBS, mice in AAV groups were injected were given AAV-LacZ [4x1010viral genome (vg)] or AAV-Tβ4 (4x1010vg) on day 0. Two mice from each group were randomly euthanized on day 14 and day 28. The remaining mice were injected again with AAV-LacZ and AAV-Tβ4 on day 28 and were sacrificed on day 42. The lungs of euthanized mice were harvested for further examination.
2.4 AAV-mediated Tβ4 expression upon LPS-induced lung injury and fibrosis
To verify the expression of Tβ4 in mouse lung after LPS treatment, thirty-five mice were divided into normal saline (NS, n=5) and LPS (n=30) groups. Septic lung injury model was established by i.p. injection of 5mg/kg LPS for five consecutive days. Five mice from the LPS group were euthanized on days 7, 14, 21, 28, 35 and 42, while all the mice in the NS group were euthanized on day 7. Mouse lungs were collected for HE and picrosirius red staining, western blotting, and other experiments.
To investigate the effects of Tβ4 on acute lung injury and fibrosis, forty mice were equally assigned into four groups: NS, NS+LPS, LPS+AAV-LacZ and LPS+AAV-Tβ4. Mice in AAV groups were i.p. injected with AAV (AAV-LacZ or AAV-Tβ4, 4x1010 vg) for the first time, while mice in the other two groups were injected with an equal volume of NS. Two days later (Day 0), the mice were i.p. instilled with NS or LPS. Five mice in each group were sacrificed on day 7. The remaining mice received the second i.p. administration of AAV or NS on day 26 (four weeks after the first adenovirus administration) and were sacrificed on day 42, when the lungs and serum were harvested for subsequent experiments. The mice were weighed during LPS modeling, and their lung coefficient was calculated (lung coefficient=lung wet weight/body weight×100).
2.5 Bronchoalveolar lavage (BAL)
BAL was carried out on day 7 following LPS injection. After the mice were sacrificed, their lungs and trachea were extracted immediately, and a 20G intravenous catheter was inserted into their trachea. 1mL PBS was instilled into the lungs and withdrawn three times via the catheter. More than 85% of the fluid was recovered as bronchoalveolar lavage fluid (BALF), which was then centrifuged at 1000rpm for 10minutes at 4°C. The supernatants were collected and stored at -80℃, and the precipitate was washed with red blood cell lysis buffer and resuspended in 500µL PBS for further tests.
2.6 Measurement of malondialdehyde (MDA) and myeloperoxidase (MPO)
MDA content and MPO activity in mouse lung tissue were detected with commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s protocols.
2.7 Measurement of hydroxyproline content
Pulmonary hydroxyproline content was detected with commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s protocols.
2.8 Measurement of IL-1β
IL-1β level was detected by enzyme-linked immunosorbent assay (ELISA) using commercially available kits (eBioscience, San Diego, CA, USA) according to the manufacturer’s protocols.
2.9 Cell culture, proliferation assay and reagents treatment
The HPAEpiC were cultured in DMEM, while HIF-1 cells were cultured in F-12K medium supplemented with 10% foetal bovine plasma and 2mM L-glutamine at 37°C in a 95% air, 5% CO2-humidified atmosphere.
Cells were trypsinized, and 500 cells were seeded onto 96-well plates and allowed to adhere for 24hours. Cells were then treated with Tβ4 at different concentrations (0, 75, and 150nM) and incubated for another 72hours. Cell viability was assessed using CCK-8 (Dojindo, Kyushu, Japan) assay at 24, 48, and 72hours according to the manufacturer’s protocols.
Cells were trypsinized, and 5×105 cells were seeded onto plastic dishes and then treated with H2O2 (0, 100, 200 and 400µM), LPS (1µg/mL), NAC (10mM), FCCP (10µM), Oligomycin (10µM) or TGF-β1 (5ng/mL).
2.10 Western Bloting
Protein extracts were prepared from cells and mouse lung tissues by RIPA Lysis Buffer supplemented with Complete EDTA-free protease inhibitor cocktail tablets (Roche Applied Science, Basel, Switzerland) and phosphatase inhibitor cocktails (Sigma-Aldrich). Protein samples (50µg) were loaded onto SDS-PAGE gels and transferred onto PVDF membranes. After blocking in 5% evaporated milk at room temperature for 2hours, the membranes were then incubated with the indicated primary antibodies in 5% evaporated milk in TBS plus 0.1% Tween 20 overnight at 4°C. The following primary antibodies were used: anti-Thymosin β4 (ab167650, Abcam, Cambridge, UK), anti-α-SMA (#56856, Cell Signaling Technology, Danvers, MA, USA), IL-1β (#12703, Cell Signaling), PINK1 (#6946, Cell Signaling), anti-Tom40 (H-300, Santa Cruz Biotechnology, Santa Cruz, CA), and β-actin as a loading control (no. 4970; Cell Signaling). and β-Actin as a loading control (#4970, Cell Signaling). Signals were developed using a chemiluminescent substrate and visualized through X-ray films.
Immunoreactions were performed on selected lung sections. Antigens were detected by the following primary antibody, followed by appropriate secondary antibodies: anti-Thymosin β4 (ab167650, Abcam, Cambridge, UK) and anti-α-SMA (#56856, Cell Signaling Technology, Danvers, MA, USA). The slides were then observed under a Nikon Eclipse microscope (Tokyo, Japan) coupled to a digital camera.
2.12 Statistical analysis
The results are expressed as the means ± standard deviation. Statistical analysis was performed using SPSS software 13.0 (SPSS, Inc., Chicago, IL, USA). The Shapiro-Wilk test and Levene statistic were used to evaluate the normality and homogeneity, respectively, of the variance. According to the situation, t-tests or Mann-Whitney U tests were used to evaluate differences between two groups; correlations between two quantitative groups were analysed with Pearson or Spearman correlation tests. The χ2 test was used for comparisons between two groups. The reported P-values are two-sided, and P-values <0.05 were considered statistically significant.