Subjects
Normal lung tissue specimens of 13 non-smokers, 11 smokers and 22 COPD patients were collected from Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China (Fundamental Characteristics of study subjects showed in Table S1 (see Additional file 1). In our study, COPD was diagnosed according to the global initiative for chronic obstructive pulmonary disease (GOLD). Subjects with respiratory infection or other chronic pulmonary diseases (asthmas, bronchiectasis and interstitial lung diseases (ILDs)), having systemic steroid use within the previous 4 weeks, or who had a history of other cancers were excluded. All samples collected were stored in a freezer at a temperature of -80°C until use.
We obtained approval from the ethics committee of Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hubei, China (TJ-IRB20210346). Each subject signed an informed consent form before the collection of samples.
A Mouse Model Of Cigarette Smoke Exposure
Dach1flox/flox mice were generated via CRISPR–Cas9 system by Nanjing Institute of Biomedicine of Nanjing University (Nanjing, China). Two loxP sites were inserted in upstream and downstream of exon 2 of Dach1, respectively. To generate club cells-specific knockdown Dach1 mice (Scgb1a1-cre+-Dach1flox/flox, hereinafter referred to as Dach1-CKO), the Scgb1a1-Cre transgenic mice were purchased from the Jackson laboratory (Bar Harbor, ME, USA) and then crossed with Dach1flox/flox mice. The littermates (Scgb1a1-Cre-Dach1flox/flox, hereinafter referred to as Dach1-C) of Dach1-CKO were used as controls. Dach1-CKO and Dach1-C mice (6 to 8 weeks) were administered tamoxifen (65 mg/kg, i.p.) for seven days to induce the expression of cre recombinase and the knockdown of Dach1. 14 days after the last day of tamoxifen injection, Dach1-CKO and Dach1-C mice were exposed to cigarette smoke (CS) or air (exposure twice a day, each exposure for 2 hours) for six months. All mice were housed in a specific pathogen-free (SPF) facility at the Tongji Medical College with a 12-hour light/12-hour dark cycle. All experimental procedures were approved by Huazhong University Animal Experiment Ethics Committee and were conducted in accordance with the animal experimentation guidelines of Huazhong University.
To test the effect of Dach1 on experimental COPD model, mice were intratracheally administered with Dach1-expressing AAV2/9 viral genome particle (total amount of titer 2 × 1011 vg per mouse) 21 days before CS or porcine pancreatic elastase (PPE) exposure.
A Mouse Model Of Emphysema Induced By Porcine Pancreatic Elastase
Porcine pancreatic elastase (PPE) was purchased from Sigma (E1250) and then dissolved in phosphate buffer solution (PBS). 2U PPE or PBS were intratracheally injected to AAV-Con or AAV-Dach1 mice. 42 days after injection, mice were tested for lung function and then sacrificed to acquire bronchoalveolar lavage fluid (BALF) and lung tissue.
Pulmonary Function Test
Briefly, COPD model mice were first anesthetized with 1% pentobarbital sodium 10 ml/kg body weight intraperitoneally and then were implemented with endotracheal intubation. Subsequently, mice pulmonary function was measured using the FlexiVent system (SCIREQ, Montreal, Quebec, Canada). Lung function parameters FEV0.1s/FVC was calculated.
Reagents And Antibodies
Cigarette smoke extraction (CSE) were prepared as previously described[20]. Antibodies against β-actin (Anti-β-Actin, 66009-1-Ig), Dach1 (Anti-Dach1, 60082-1-Ig), scgb1a1 (Anti-Scgb1a1, 10490-1-AP), Nrf2 (Anti-Nrf2, 16396-1-ap) were purchased from Proteintech. HO-1 (Anti-HO-1, GB11845) were purchased from Servicebio. Antibodies against Dach1 (Anti-Dach1, ab226176) for immunoprecipitation assay was purchased from Abcam. Antibodies against IgG (Anti-IgG, #2729) for immunoprecipitation assay was purchased from Cell Signaling Technology (CST).
Cells Culture And Stimulation
Human airway epithelial cell line (16HBE) was purchased and cultured as previously described[20]. The cells were incubated at 37°C with 5% CO2. For experimentation, 16HBE were grown in 12-well plates until 70–80% confluence and then exposed to Cigarette smoke extraction (CSE) with different concentrations and stimulation times.
The primary small airway epithelium (SAE, 10th-12th generation bronchi) was collected by fiberoptic bronchoscopy from one healthy nonsmoker (male, 50 years old). Freshly ten brushed cells were washed with cell culture medium (DMEM, Lonza, Walkersville, MD) with 10% fetal bovine serum (FBS, Gibco) and Penicillin-streptomycin (1:100). The collected cells were then centrifuged at 1000 RPM for 5 minutes and suspended with bronchial epithelial basal medium BEBM (Lonza, Walkersville, MD). Suspended cells (Passage zero) were then inoculated into cell culture dishes and fluid was changed every two days. Cell passage was performed when the degree of cell fusion reached 90 to 100%.
Histopathology, Immunohistochemical And Immunofluorescence Analysis
Human lung and mice left lung tissues were collected and placed in fresh 4% neutral-buffered paraformaldehyde for 24 hours at room temperature, then embedded in paraffin and subjected to the histological analysis as previously reported. Hematoxylin-eosin (H&E) staining were performed on mice lung sections. Immunofluorescence co-staining of scgb1a1 (Anti-Scgb1a1, 1:400) and Dach1(Anti-Dach1 ,1:200) were performed on human and mice lung sections.
Airway And Vascular Inflammation Score
Airway and vascular inflammation score were done following previous study[21].
Lentiviral Transfection
16HBE cells transfected with 60 MOI Lv-con or Lv-Dach1 for 48 hours were treated with 4ug/ml Puromycin to produce cells stably overexpressed Dach1. 4ug/ml polybrene was used to promote transfection efficiency.
Quantitative Rt-pcr Analysis
Quantitative RT-PCR analysis
Human lung tissues RNA was extracted by Trizol reagent method (Invitrogen). Total RNA was used for first-strand cDNA synthesis with M-MLV reverse transcriptase (Promega, Madison, WI). qRT-PCR was performed utilizing SYBR Green Master Mix (Takara, Otsu, Shiga, Japan) on the iCycler iQ system (Bio-Rad). PCR conditions included initial denaturation at 95°C for 5 minutes, 95°C for 45 seconds, and 60°C for 1 minute for 45 cycles. Gene expression levels were normalized to ß-Actin. The primers for Dach1 showed in Table S2 (see Additional file 1).
ELISA
Cells culture supernatant and bronchoalveolar lavage fluid (BALF) of mice were collected after centrifugation at 1000 RPM for 5 minutes. ELISA kits for human IL8 (DY208), human IL6 (DY206), mouse CXCL1 (DY453) and mouse IL6 (DY406) were purchased from R&D systems.
Western Blotting
Total protein from mice right lung tissue or 16HBE cells homogenate was extracted by RIPA lysis buffer containing a protease inhibitor cocktail and phosphatase inhibitors (Roche, Mannheim, Germany). The proteins were separated by 12% SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Germany). The membranes were blocked for 1–2 hours in 5% milk melted in Tris-buffered saline containing 0.05% Tween 20 (TBST) and then incubated with the primary antibody (Anti-β-Actin, 1:4000; Anti-Dach1, 1:2000; Anti-Nrf2,1:2000; Anti-HO-1, 1:2000)
Reactive Oxygen Species (Ros) Detection
Reactive oxygen species (ROS) detection was performed by ROS assay kit (Servicebio, G1706-100T). 16 HBE cells transfected with Lv-con or Lv-Dach1 were seeded in 12-well plates. After treatment of CSE for 12 hours, cells were washed twice with phosphate buffer and then incubated with DCFH-DA probe (diluted in 1:1000 with 1640 medium) at 37°C for 30 minutes. Thereafter, after twice washing with phosphate buffer, the fluorescence intensity of the cell was directly observed with a fluorescence microscope or a cell flow cytometer.
Immunoprecipitation Assay (Ip)
Protein G magnetic beads were purchased from Cell Signaling Technology (CST, #70024s). IP experiments were performed according to the recommended steps on the official website. First, 16HBE cells were seeded in 10-centimeter cell culture dishes until 70–80% confluence and then exposed to 10% CSE for 24 hours. Then, washed cells were harvested and lysed in NP-40 lysis buffer containing a protease inhibitor cocktail and phosphatase inhibitors (Roche, Mannheim, Germany). After centrifugation, the supernatants were collected and pre-cleared with 20ul protein G magnetic beads for 2 hours at 4°C. pre-cleared supernatants then were incubated overnight with rotation at 4°C with Anti-Dach1 (ab226176) or Anti-IgG (#2729) to generate immunocomplex. The next day, immunocomplex solutions and 20ul pre-cleared protein G magnetic beads were incubated with rotation at 4°C with for 3–3 hours. Finally, immunocomplex were eluted from protein G magnetic beads and further analyzed by western blotting analysis.
Chromatin Immuneprecipitation (Chip) Assay
ChIP assays were conducted using the ChIP Assay Kit (CST, #6003). Briefly, 1 × 107 16HBE cells transfected with Lv-con or Lv-dach1 were cross-linked with 1% formaldehyde (Sigma-F8775), and chromatin fragmentation was carried out according to the manufacturer’s protocol provided. Then, 10ug prepared chromatin fragmentation solution was incubated with an Anti-Flag (1:50), normal rabbit immunoglobulin G (IgG) or antibody against histone H3 (Anti-H3) overnight at 4°C with rotation. The DNA pulled down were subsequently used to performed CHIP-PCR. The PCR-amplified products were simultaneously identified by a 3% agarose gel. Primers to detect DNA enrichment are listed in Table S3 (see Additional file 1). Percent (%) input was analyzed by following standard formula.
Sirna Transfection
16HBE cells cultured in 1640 supplemented with 10% FBS were transfected with a Nrf2 siRNA or Scrambled siRNA (Ribobio, Guangzhou, China) using the Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA). 48 hours after tansfection, the transfected cells were exposed to 10%CSE for 24 hours.
Statistical analysis
Data from n independent experiment were presented as means ± SEM. Normality analysis was performed via the Shapiro-Wilk test. Differences were evaluated using unpaired Student's t test between two groups before any testing. One-way ANOVA was performed followed by Bonferroni post hoc test for comparisons between > 2 groups. The non-normal distributed data were analyzed using non-parametric testing (Mann-Whitney U test for two groups and Krushal-Wallis H test for > 2 groups). P values less than 0.05 were considered statistically significant. Statistical analysis was performed using GraphPad Prism 8.0.1 (GraphPad Software Inc., San Diego, CA).