Two siRNAs targeting mouse Bach1 mRNA were designed using a software available at www.ambion.com via a cDNA sequence (SEQ ID, XM_006522879), and an empty adenoviral vector was used as a negative control. The specific siRNA coding sequences were designed to contain the target sequences and their reverse complement 19-nucleotide sequences which are separated by 9-nucleotide sequences of stem-loop structure (Table I).

The forward and reverse oligonucleotides were chemically synthesized and annealed to form a duplex. Two siRNA sequences were cloned into the plasmid vector, pGenesil-1. The siRNA expression cassette was subsequently excised from pGenesil-1 using EcoRI and HindIII, and also ligated into the linearized adenoviral shuttle vector, pShuttle-CMV. The ligation products were transformed into E. coli DH5α, and positive clones were selected. The recombinant adenovirus vectors were obtained by homologous recombination with pShuttle-CMV-Bach1-siRNA and the skeleton plasmid of pAdeasy-1 in bacteria BJ5183. The recombinant cosmids titled Ad-siBach1 carrying either Bach1 siRNA or adenoviral vector with green fluorescent protein (GFP) were linearized by PacI digestion and then transfected 293A cells (Wuhan Cell Marker Biotechnology, Wuhan, China) using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). The success of Bach1 siRNA insertion into adenoviral plasmid was confirmed by DNA sequencing and the titer of each virus stock was determined by plaque assay on 293A cells. Finally, the siRNA adenoviral vector was calculated as 1.1×10⁹ plaque-forming units (PFU)/ml and the empty adenoviral vector was 1.4×10⁹ PFU/ml. The virus was purified by double CsCl gradient ultracentrifugation used for in vivo experiments. The transfection efficiency was monitored by fluorescence microscopy and flow cytometry. The silencing efficiency of target gene and protein were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. The selected siRNA sequence with the highest inhibitory effect was used in the animal experiments.

Seven-week-old female C57BL/6 mice (body weight, 18–20 g) were purchased from Vital River Laboratories (Beijing, China). The animals were maintained under specific pathogen-free conditions and kept for 1 week prior to use. The room temperature was maintained at 22±3°C, with a 12-h/12-h day/night cycle and relative humidity of 50±10%. All animals had free access to rodent chow and water. The mice were randomly divided into 4 groups as follows: a control with saline (n=5), BLM (n=5), BLM + control siRNA (n=5) and BLM + Bach1 siRNA groups (n=5). For the model of PF, 5 mg/kg BLM (Nippon Kayaku Co., Ltd., Tokyo, Japan) in 50 μl phosphate-buffered saline (PBS) were administered intratracheally to the mice; the control animals were injected intratracheally with the same volume of sterile saline. BLM and saline were administered only once. The mice in the control siRNA and Bach1 siRNA group were injected with control siRNA or Bach1 siRNA, respectively, via the tail vein (one injection every other day), with a total dose of 1×10⁹ PFU viruses per mouse after 2 weeks of BLM administration. At the designated time points (28 days post-BLM administration), the mice were humanely sacrificed by an overdose of the anesthesia (inhalation of ether) for serum, BALF and histological measurements. We lavaged the lungs using 0.9% saline using a tracheal cannula for 3 times and collected 1.5 ml BALF in each mouse. The blood of the experimental mice was collected using a capillary through the conjunctiva and into the orbital sinus. Following centrifugation (2000 × g for 5 min at 4°C), the serum and supernatants were stored at −80°C until use. In addition, the lungs were removed and immediately frozen in liquid nitrogen, and then stored at −80°C until further processing. All procedures involving animals were approved by the Ethics Committee of Capital Medical University, Beijing, China and complied with guidelines on the Use of Experimental Animals.

The left lung tissues from the mice were dissected and fixed in 4% paraformaldehyde. After 24 h, the tissues were dehydrated and embedded in paraffin. Paraffin blocks of 3–4 μm thickness were cut and stained with hematoxylin and eosin (H&E) and Masson's reagent (Solarbio, Beijing, China) for the assessment of PF histopathology. The histological severity of pulmonary alveolitis was scored as previously described (28), and PF was scored as described in the study by Ashcroft et al (29).

The MLF cell line (MIC-CELL-0040) was purchased from PriCells Biomedical Technology Co., Ltd. (Hubei, China) and maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA), 100 U/ml penicillin and streptomycin. The cells were plated at 1×10⁵ cells/100 mm and cultured at 37°C in a 5% CO₂ humidified atmosphere. We used 0.25% trypsin for digestion and the cells were filled in 2 culture flasks for passage (1:2). All experiments proceeded using cells between 3 and 4 cell passages. For the induction of fibrosis using the recombinant protein, TGF-β1, the cells were seeded in 12-well plates at a density of 1×10⁵ cells/well and starved for 24 h and then incubated with TGF-β1 (5 ng/ml) in complete medium for 24 h. To knockdown Bach1 expression, the MLFs were infected with the Bach1 siRNA at a multiplicity of infection (MOI) of 50 for 2 h and were then washed to remove the virus. The cells in in the control group (control siRNA) were transfected with empty vector. The cells were then cultured for a further 48 h and then analyzed for their GFP intensity using a FACScan flow cytometer (Becton-Dickinson, Mountain View, CA, USA) and directly observed under a fluorescence microscope (Olympus BX51; Olympus, Tokyo, Japan).

Total RNA was isolated from the mouse pulmonary tissue samples and the MLFs using the simple Total RNA kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer's instructions. RNA was reverse transcribed using the Revert Aid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc., Shanghai, China) and quantitative PCR (qPCR) was performed using the SYBR PrimeScript RT-PCR kit (Takara Biotechnology Co., Ltd., Dalian, China). The initial denaturation step was at 95°C for 10 min, and then PCR amplification was achieved by 40 cycles at 95°C for 15 sec and 60°C for 1 min. The process of RT-qPCR was implemented by ABI Prism 7500 (Applied Biosystems, Foster City, CA, USA). The sequences of the primers used are listed in Table II.

The level of gene expression was quantified using a standard curve and the comparative CT method normalized to GADPH mRNA expression. We used the formula 2^−ΔΔCT to calculate the relative expression levels of genes. Both samples were examined in triplicate, and the experiment was repeated at least 3 times.

Cell pellets or lung tissues were lysed in RIPA lysis buffer containing 1 mM phenylmethanesulfonyl fluoride (Applygen Technologies, Inc., Beijing, China) on ice, and the concentration of the sample proteins was tested using the BCA method before being mixed in 5X SDS loading buffer. Following heat denaturation at 100°C for 5 min, equal amounts of lysate (60 mg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Applygen Technologies, Inc.). The membranes were blocked with 5% fat-free milk for 1 h at room temperature, followed by incubation with primary antibodies against β-actin (1:500; sc-130301; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Bach1 (1:300; ab49657), HO-1 (1:500; ab13248), GPx1 (1:1,000; ab140883) and NQO1 (1:500; ab28947) (all from Abcam, Cambridge, UK) at 4°C overnight. The following day, the membranes were incubated with fluorescent-labeled secondary antibodies (1:10,000; 600-101-096; Rockland, Inc., Gilbertsville, PE, USA) for 1 h at room temperature, and the bands were visualized using a double-infrared laser scanning imaging system (LI-COR Biosciences, Lincoln, NE, USA). Protein expression was analyzed and normalized to that of β-actin.

BALF and blood from the mice were sampled at the end of 4 weeks after the initiation of the animal experiment. The supernatants of MLFs were also collected after 48 h of the transfection of siRNA for measurements. We then used the BALF, serum and cell supernatants to analyze the levels of cytokines in lung fibrosis. ELISA was used to detect TGF-β1 and IL-6 using respective ELISA kits (R&D Systems, Minneapolis, MN, USA). The supernatants of MLFs, serum and BALF of mice were diluted according to different proportions along with reagents and standard dilutions prepared before the experiment. Standard, control and samples (100 μl/well) were incubated for 2 h at room temperature. They were then washed with wash buffer 5 times and 100 μl of conjugate was then added for 2 h followed by substrate solution for 30 min away from light. After the addition of 100 μl of stop solution, the sample concentrations were determined to measure the absorbance at a 450 nm using a microplate reader (Bio Rad, Hercules, CA, USA). Both assays were performed in duplicate.

Data were analyzed using a statistical software package (SPSS 13.0; SPSS, Inc., Chicago, IL, USA), and expressed as mean ± SD. One-way analysis of variance (ANOVA) was performed for multiple group comparisons. A value of P<0.05 was considered to indicate a statistically significant difference and a value of P<0.01 was considered to indicate a marked statistically significant difference.

The successful construction of Bach1 siRNA adenoviral vectors was determined by gene sequencing. To demonstrate the transfection efficiency of Bach1 siRNA on MLFs and its targeting in vivo, we used a fluorescent microscope and flow cytometric analysis. After the Bach1 siRNA was transfected into the MLFs, >90% GFP was expressed in the fluorescence states (Fig. 1A), and the results of flow cytometry also suggested that the transfection efficiency of Bach1 siRNA was 93.6% (Fig. 1B). After 2 weeks of siRNA administration, GFPs were observed in the tissue sections of the lungs of mice, suggesting that Bach1 siRNA targeted the lungs (Fig. 1C).

The MLFs were incubated with recombinant protein TGF-β1 (5 ng/ml) for the induction of fibrosis prior to transfection with control siRNA, or Bach1 siRNA#1 and #2 in vitro. Similarly, superior Bach1 siRNA (Bach#1) was injected into the mice with BLM-induced PF in vivo. The inhibitory effects of Bach1 siRNA on Bach1 mRNA and protein expression were analyzed by RT-qPCR and western blot analysis. As shown in Fig. 2A, the mRNA level of Bach1 in the TGF-β1 group was significantly increased compared with that of the blank group (P<0.05), suggesting that TGF-β1 promoted Bach1 generation. The mRNA expression of Bach1 in the MLFs following transfection with Bach1 siRNA#1 or #2 was significantly decreased compared with that of the TGF-β1 group or the control siRNA group (P<0.01), and was also significantly decreased by 15.3 and 22%, respectively (P<0.01) compared with the blank group (P<0.01) (Fig. 2A). Similarly, the results of western blot analysis also revealed that the protein expression of Bach1 in the MLFs following transfection with Bach1 siRNA#1 or #2 was markedly decreased (Fig. 2C). To further characterize Bach1 expression in vivo, we detected Bach1 expression in the lungs of mice and found that Bach1 mRNA and protein expression in the mice was decreased significantly following the administration of BLM and Bach1 siRNA, compared with the control group and the control siRNA group (Fig. 2B and D).

To examine the mechanisms through which Bach1 siRNA affects the expression of antioxidant factors, such as HO-1, GPx1 and NQO1, we examined their mRNA and protein expression levels in the MLFs and lungs of mice. The results of RT-qPCR revealed that the mRNA levels of HO-1 and GPx1 were decreased following exposure of the MLFs to TGF-β1 compared with the blank group (both P<0.05; Fig. 3A and B), and the knockdown of Bach1 significantly increased the HO-1 and GPx1 mRNA levels compared with the TGF-β1 group (P<0.01; Fig. 3A and B). The effect of Bach1 siRNA#1 was more prominent than that of Bach1 siRNA#2 in the MLFs. Furthermore, as shown in Fig. 3D and E, we observed a significant increase in the mRNA levels of HO-1 and GPx1 in the lung tissues of mice following treatment with Bach1 siRNA compared with the BLM group (P<0.05 and P<0.01). Concomitantly, the knockdown of Bach1 significantly increased the protein levels of HO-1 and GPx1 in the MLFs in the blank group or TGF-β1 group, and in the lung tissues from mice in the control group or BLM group (Fig. 3G and H). However, Bach1 siRNA did not alter the mRNA and protein levels of NQO1 neither in the MLFs nor in the lung tissues (Fig. 3C and F). The results of protein expression were similar to those of mRNA expression. All these results suggest that Bach1 siRNA promotes the generation of oxidation resistance factors, and it may thus inhibit the oxidative stress induced by TGF-β1 and BLM.

Due to the important role of TGF-β1 and IL-6 in the pathogenesis of PF, we analyzed their concentrations in the supernatant of MLFs, and in serum and BALF from mice. The levels of TGF-β1 and IL-6 in serum and BALF from mice with BLM-induced lung fibrosis were significantly increased compared with the control group (both P<0.01; Fig. 4A–D). Their concentrations in the supernatant of the MLFs following stimulation with TGF-β1 were significantly increased compared with the blank group (both P<0.01; Fig. 4E and F). Additionally, the levels of IL-6 and TGF-β1 in serum and BALF from the mice in the Bach1 siRNA group were significantly decreased compared with those of the mice BLM treated with control siRNA or in the control group (both P<0.01; Fig. 4A–D). The results of the analysis of the expression of IL-6 and TGF-β1 in the supernatant of MLFs were similar to those obtained from the serum and BALF of mice. These results suggest that the use of Bach1 siRNA suppresses the expression of TGF-β1 and that of related cytokines in BLM-induced fibrosis.

For histological analysis, the lung tissues of mice were stained with H&E and Masson's staining. The degree of lung fibrosis was accessed by alveolitis, fibering and the integrity of the structure. Intact alveoli, a normal interstitium and a few inflammatory cells in the lung tissues were observed in the control group (Fig. 5A). Nevertheless, H&E staining of the BLM-adminstered mice (at 28 days) revealed the extensive destruction of alveoli (collapsed and disappeared), the extensive thickening of the lung interstitium, peribronchial and interstitial infiltrations of inflammatory cells, predominant lymphocytes, and multiple focal fibrotic lesions. There were milder inflammatory infiltrations and the destruction of alveoli following treatment with Bach1 siRNA compared with the BLM group and control siRNA group (Fig. 5A). As shown by Masson's trichrome staining, we observed massive fibrosis (blue dye), the accumulation of inflammatory cells and the extensive destruction of alveoli in the BLM group (Fig. 5B). The same change was detected in the control siRNA group. However, treatment with Bach1 siRNA significantly attenuated these fibrosis-related changes in the mice with BLM-induced fibrosis (Fig. 5B).

The alveolitis and fibrosis scores indicated that the mice in the Bach1 siRNA group had significantly lower alveolitis and fibrosis scores than those in the BLM group and control siRNA group (P<0.05 and P<0.01; Fig. 5C). Therefore, treatment with Bach1 siRNA potentially inhibits the histopathological progress of BLM-induced lung fibrosis in mice.