Benign lung disease samples were collected for the analysis CS-induced DDR and airway inflammation. 10 patients with smoking and 6 non-smokers were included in our studt. Patients were excluded if they had a previous history of cancer. The study protocol was approved by the Research Ethics Committees of the local ethics committee.
Human bronchial epithelial cells (16HBE cells) were purchased from the American Type Culture Collection (ATCC, USA) and were cultured in RPMI 1640 (Sigma-Aldrich, St. Louis, USA) supplemented with 10% fetal calf serum. Cells were seeded at 1×104 cells/well in 24-well plates and cultured at 37 °C in a humidified atmosphere containing 5% CO2/95% air for 24 h before exposure.
C57BL/6 mice (male, aged 8-10 weeks) were purchased from Slac Laboratory Animal Center (Shanghai, China). IL-17 KO mice (C57BL/6 background) were purchased from the Center for Experimental Medicine and Systems Biology (Institute of Medical Science, University of Tokyo, Japan). Mice were randomly assigned to different treatments (air or cigarette smoke) at the time of purchase to minimize any potential bias. All protocols in this study were approved by the Ethics Committee for Animal Studies of Zhejiang University, China.
CS exposure and CS extract (CSE) preparation
Mice were exposed to cigarette smoke in a chamber using a smoking machine (Model TE-10, Teague Enterprises). The total particulate matter concentrations of the chamber atmosphere were 160–180 mg/m3. Mice were exposed 2 hours a day for 5 d/week in consecutive 12 or 24 weeks. The control group was exposed to filtered air under identical conditions. To prepare CSE, smoke of 1 cigarette was bubbled slowly through 10 ml of RPMI 1640, which was considered as 100% CSE solution, and then sterilized.
Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissue sections. Briefly, 4 µm thick sections were deparaffinized in xylene, rehydrated in graded ethanol, and washed twice with PBS. Endogenous peroxidase activity was blocked by incubating sections with 3% hydrogen peroxide in the dark for 10 min. Slides were incubated overnight at 4°C with the primary antibody (gH2AX, 1:200, Abcam; IL-17, 1:400, Cell Signaling). The sections were washed and incubated at room temperature for 1 h with the secondary antibodies. Finally, the slides were exposed to a substrate chromogen mixture and counterstained with Hematoxylin & Eosin (H&E). Stained slides were analysed on an Olympus optical microscope and scored according to the number of positively stained cells.
2×104 HBE cells were treated with or without CSE or IL-17 as indicated concentrations. The cells were washed 3 times with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 30 min at room temperature and permeabilized with 0.2% Triton X-100 for 5 min at 4°C. After blocking with 5% bovine serum albumin PBS for 30 min, the cells were incubated with primary antibody against gH2AX diluted in PBS containing 5% BSA (1:1,000) overnight at 4°C, washed three times in PBS, and then incubated with secondary antibody diluted in PBS containing 5% bovine serum albumin for 30 min at room temperature. DNA was counterstained with 1 mg/ml 4’,6-diamidino-2-phenylindole (DAPI) for 10 min at 37°C. Cells mounted on cover slips were observed with a fluorescence microscope or confocal laser scanning microscope.
Western blot analysis
Cells were lysed in RIPA buffer. Equal amounts of protein were loaded onto SDS-polyacrylamide gels (PAGE), fractionated by electrophoresis, and transferred to nitrocellulose membranes (Bio-Rad). The membranes were blocked for 1h with 5% fat-free milk prepared in PBS containing 0.05% Tween-20. Membranes were incubated overnight at 4°C with IL-17 antibody (Cell Signaling) or anti-β Actin antibody (Cell Signaling). Then membranes were then blotted with corresponding secondary antibodies (1:2000 dilution) for 2 hours and protein bands were visualized using enhanced chemiluminescence.
Prism version 5 (GraphPad Software, Inc. La Jolla, CA) and SPSS for Windows, version 13.0 (SPSS Inc., Chicago, IL, USA) was used for data collection and presentation. The data shown are presented as mean ± standard error. Statistical significance was considered at P<0.05 using Student’s t test. Different levels of significance are indicated as *P<0.05, **P<0.01, ***P<0.001.