Contributed equally
Nicotine is one of the primary components in cigarettes, which is responsible for addiction. Numerous studies have investigated the effects of nicotine on pulmonary disease. The health of epithelial cells is important in the development of chronic obstructive pulmonary disease (COPD). Accumulating evidence has suggested that epithelial cell death may initiate or contribute to the progression of a number of lung diseases via airway remodeling. Pyroptosis is a unique form of inflammatory cell death mediated by the activation of caspase-1 and the NOD-like receptor protein-3 (NLRP3) inflammasome. The present study aimed to evaluate whether pyroptosis of epithelial cells was involved in the progression of COPD. The normal human bronchial epithelial cell line 16HBE was treated with 0.1 or 1 µM nicotine. Then the proliferation ability of 16HBE cells was detected by CCK-8. Cell death was detected by flow cytometry analysis and TUNEL assay. Subsequently, the levels of pro-caspase 1, caspase 1, IL-1β, IL-18, NLRP3, ASC and cleaved GSDMD were examined by western blotting. It was revealed that nicotine treatment significantly induced cell death and suppressed proliferation of 16HBE cells. Furthermore, nicotine exposure increased the expression levels of caspase-1, IL-1β, IL-18, NLRP3, apoptosis-associated speck-like protein and gasdermin D in 16HBE cells. Therefore, the present study concluded that nicotine treatment induced pyroptosis in 16HBE cells, which may be associated with the progression of COPD.
Chronic obstructive pulmonary disease (COPD) is responsible for >1,400,000 annual mortalities worldwide, and is characterized by persistent airflow limitation and limited therapeutic options (
Overwhelming evidence has suggested that cigarette smoking is the leading cause of COPD worldwide (
Inflammation and cell death are the two critical pathological mechanisms of COPD (
Bronchial epithelial cells are the first anatomical barrier exposed to noxious gases and particles of cigarette smoke, which can initiate airway remodeling in COPD (
The present study aimed to investigate whether epithelial cells undergo pyroptosis in COPD progression. These findings may improve understanding of the underlying mechanisms of COPD and provide valuable information for the diagnosis and treatment of COPD.
The normal human bronchial epithelial cell line 16HBE is a common cell line used to study COPD pathogenesis
The proliferation of 16HBE cells was evaluated using the Cell Counting Kit-8 (CCK-8) assay (Dojindo Laboratories, Inc.). 16HBE cells were seeded in 96-well plates (1.0×104 cells/well), and were exposed to 0, 0.1 or 1 µM nicotine for 0, 48, 72 and 96 h at 37°C (
The death of 16HBE cells (1×106/100 µl) was analyzed using an Annexin V-FITC and PI staining kit (cat. no. A211-01/02; Vazyme Biotech Co., Ltd.) according to the manufacturer's instructions. Flow cytometry was performed on a FACSCalibur flow cytometer (BD Biosciences) and the rate of cell death (% of PI+ cells) was analyzed with FlowJo software (FlowJo, LLC, version 10.6.0).
Total RNA was isolated from each group of 16HBE cells using TRIzol® (Thermo Fisher Scientific, Inc.; cat. no. 15596026). The concentration of RNA was determined using an ND-2000 Spectrophotometer (Thermo Fisher Scientific, Inc.). RNA was reverse transcribed into cDNA by M-MLV Reverse Transcriptase kit (Elk Wuhan Biotechnology Co., Ltd. cat. no. EQ002) with the following temperature protocol: 42°C for 50 min for the reverse transcription reaction; 99°C for 5 min to inactivate the reverse transcriptase; and 4°C to store the reverse transcription product. qPCR was performed with the KAPA SYBR FAST qPCR Kit (Kapa Biosystems; Roche Diagnostics) using a 7300 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). Data were normalized to the housekeeping gene GAPDH. The mRNA levels were measured using the 2−ΔΔCq method of quantification (
Proteins were extracted from treated 16HBE cells by RIPA lysis buffer (Beyotime Institute of Biotechnology; cat. no. P0013B) and were quantified using the BCA method. Proteins (30 µg per lane) were separated by SDS-polyacrylamide gel electrophoresis on 10% gels and were electrophoretically transferred to polyvinylidene fluoride membranes, which were blocked with 5% skimmed milk for 1 h at room temperature. The following primary antibodies were used overnight at 4°C: Rabbit anti-pro-caspase-1 (1:1,000 dilution, cat. no. ab179515; Abcam), rabbit anti-caspase-1 (1:500 dilution, cat. no. 22915-1-AP; Proteintech Group Inc.), rabbit anti-IL-1β (1:500 dilution, cat. no. A1112; ABclonal Biotech Co., Ltd.), rabbit anti-IL-18 (1:500 dilution, cat. no. A1115; ABclonal Biotech Co., Ltd.), rabbit anti-NLRP3 (1:1,000 dilution, cat. no. ab263899; Abcam), rabbit anti-ASC (1:1,000 dilution, cat. no. ab283684; Abcam), rabbit anti-cleaved N-terminal GSDMD (1:1,000 dilution, cat. no. ab215203; Abcam) and mouse anti-GAPDH (1:500 dilution, cat. no. 60004-1-lg; Proteintech Group, Inc.). Horseradish peroxidase-conjugated goat anti-rabbit/mouse IgG (1:5,000 dilution, cat. no. BA1054/BA1051; Boster Biological Technology) was used as a secondary antibody for 1 h at room temperature. Immunoreactive protein bands were detected by ECL hypersensitive chemiluminescence kit (cat. no. P0018M; Beyotime Institute of Biotechnology) with an Odyssey Scanning System (version 3.0, LI-COR Biosciences).
Immunofluorescence staining of 16HBE cells was performed as previously described (
16HBE cells (4×104 cells/cm2) were seeded onto cover glasses. After 24 h, the cells were treated with 0.1 or 1 µM nicotine for another 48 h followed by fixing with 4% (w/v) paraformaldehyde for 15 min at room temperature. Then the cells were subjected to a TUNEL assay (DeadEnd Fluorometric TUNEL System; Promega Corporation) according to the manufacturer's instructions. Briefly, cells were incubated with 250 µl of TUNEL labeling solution in one well of a 24-well plate for 1 h at 37°C covered with aluminum foil. Then cells were incubated with PBS containing DAPI nucleic acid stain (dilution, 1:1,000; 1 mg/ml solution, cat. no. 62248; Thermo Fisher Scientific, Inc.) for 10 min at room temperature to stain nuclei. TUNEL positive (TUNEL+) cells were calculated by counting the number of stained cells in five separate field per slides using a Leica confocal microscope TCS SP2 (Leica Microsystems GmbH).
All experiments were repeated at least three times
The present study used nicotine-treated 16HBE cells as an
To elucidate the relationship between nicotine and pyroptosis, 16HBE cells were used for
COPD is a multifactorial disease associated with numerous mechanisms (
Cigarette smoke can cause inflammation, which is an important injury factor in lung diseases such as COPD (
Bronchial epithelial cells serve an integral role in the airway defense mechanism via the mucociliary system and mechanical barriers, which serve significant roles in the progression of various lung diseases in addition to COPD, such as asthma, lung cancer, pneumonia and pulmonary fibrosis (
Finally, the present study had some limitations. The
In conclusion, the results of the present study suggested that nicotine exposure suppressed the proliferation and promoted the death of 16HBE cells, which may be associated with the increased expression levels of caspase-1, IL-1β, IL-18, NLRP3, ASC and GSDMD. These findings indicated that nicotine treatment induced pyroptosis in 16HBE cells, which may be associated with the progression of COPD.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
YC and YD were responsible for the conception of the present study. RM, YC and YD confirm the authenticity of all the raw data. RM and JZ conducted the experiments, analyzed and interpreted the data. JZ was responsible for statistical analysis. All authors read and approved the final manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
Nicotine suppresses the proliferation of 16HBE cells. 16HBE cells were seeded in 96-well flat-bottom plates at 2×103 cells/well and treated with 0.1 or 1 µM nicotine for 0, 48, 72 and 96 h. Cell proliferation of nicotine-treated 16HBE cells was examined by Cell Counting Kit-8 assay. Data are presented as the mean ± SD (n=3). **P<0.01 vs. 0 h. OD, optical density.
Nicotine induces 16HBE cell death. 16HBE cells were treated with 0.1 or 1 µM nicotine for 48 h. Nicotine-treated 16HBE cell death was evaluated by flow cytometry measuring Annexin V and PI. The number in each quadrant represents the percentage of cells in that compartment. Data are presented as the mean ± SD (n=3). *P<0.05, ***P<0.001 vs. Control.
Nicotine induces 16HBE cell death. 16HBE cells were treated with 0.1 or 1 µM nicotine for 48 h. Nicotine-treated 16HBE cell death was examined by TUNEL staining (n=3).
Epithelial cells display characteristic features of pyroptosis after nicotine exposure. 16HBE cells were treated with 0.1 or 1 µM nicotine for 48 h. (A) mRNA expression levels of caspase-1, IL-1β and IL-18 in nicotine-treated 16HBE cells were measured by reverse transcription-quantitative PCR. (B) Protein expression levels of pro-caspase-1, caspase-1, IL-1β and IL-18 in nicotine-treated 16HBE cells were measured by western blotting. GAPDH was used as a loading control. Data are presented as the mean ± SD (n=3). *P<0.05, **P<0.01, ***P<0.001 vs. Control.
Nicotine increases the expression levels of NLRP3, ASC, GSDMD and caspase-1 in 16HBE cells. 16HBE cells were treated with 0.1 or 1 µM nicotine for 48 h. (A) Protein expression levels of NLRP3, ASC and GSDMD were examined by western blotting. Data are presented as the mean ± SD (n=3). ***P<0.001 vs. Control. (B) Caspase-1 expression was detected by immunofluorescence analysis (n=3). ASC, apoptosis-associated speck-like protein; NLRP3, NOD-like receptor protein 3; GSDMD, gasdermin D.