Cell line, drugs, and reagents
The rat PMVECs used in this experiment were purchased from Beijing Beina Biotechnology Company (Beijing, China). DMEM medium (d5796, Sigma, St. Louis, MO, USA), trypsin digestion solution (c0201, Biyuntian, Shanghai, China), fetal bovine serum (FBS) (10099141, GIBCO, Invitrogen Inc., Carlsbad, CA, USA), phosphate-buffered saline (PBS) (sh30256.01, Hyclone, Thermo Fisher Scientific, Waltham, MA, USA), penicillin/streptomycin (sv30010, Biyuntian), T25 cell culture bottle (n707003, Nest, Shanghai, China), CCK8 Kit (nu679, Japan Tongren, Tokyo, Japan), FAM-YVAD-FMK pyrolysis kit (ab219935, Abcam, Cambridge, United Kingdom), IL-18 ELISA kit (ab0213909, Abcam), and IL-1 β ELISA kit (ab100767, Abcam) were purchased. The main equipment included an ultra-clean worktable (YT-CJ-2NB, Beijing Yatailong, Beijing, China), direct heating CO2 incubator (DH-160I, Santo Instrument Product, Beijing, China), inverted biological microscope (DSZ2000X, Beijing Zhongxian Hengye Instruments), low-speed centrifuge (SL02, Zhixin Instruments, Shanghai, China), flow cytometry (FACSCantoⅡ, BD Biosciences, Franklin Lake, NJ, USA), ultraviolet-visible photometer (Eppendorf, Hamburg, Germany), and Imark Microplate Reader (Bio-Rad, Hercules, CA, USA).
PMVECs were cultured in a DMEM medium containing 10% Gibico fetal bovine serum (FBS), 1% double antibiotics, and high glucose in a 5% CO2 incubator at 37℃. When confluence reached 70%-80%, the old medium was discarded for trypsinization and passage. The second- to third-generation cells with good growth were used for the experiments.
Preparation of CSE and LPS
CSE was prepared according to a published method . A filter-removed cigarette (Huangguoshu cigarette, tar 10 mg, nicotine 0.9 mg) was connected to a 50 ml syringe through a rubber tube. After it was ignited, negative pressure suction was performed. Each cigarette was smoked at a 50 ml/min rate six times. The smoke (300 ml) was introduced into 25 ml of serum-free DMEM medium. The pH was adjusted to 7.4 with 1 mM NaOH. The container was gently shaken to dissolve the smoke in the medium. The bacteria and particles were removed by filtering through a filter membrane of 0.22 µm. This solution was defined as the 100% CSE stock solution, stored at -20℃ for later use. The concentration of CSE was detected by ultraviolet spectrophotometry at a wavelength of 320 mm. The absorbance difference of different batches of CSE solutions was about ±0.2. The stock solution was diluted to different concentrations according to the experimental needs.
The LPS stock solution (5 mg/ml) was diluted 1000 times to prepare a solution with an intermediate concentration of 5 µg/ml. The solution with a 5 µg/ml concentration was diluted 250 times to get the final concentration of 20 ng/ml.
Screening of drug intervention concentration with the cell counting kit-8 (CCK8) method
The cells in the logarithmic growth phase were digested with 0.25% trypsin, and the cell suspension at a density of 5×104/ml was inoculated on 96-well plates. After the cells adhered to the wall, the culture medium was changed. Then, CSE was added at 0%, 1%, 2.5%, 5%, 10%, 20% and 25% for 24 h (six groups) in 100 µl of complete medium. Then, 10 µl of CCK8 solution was added to each well, and the plates were incubated at 37℃ for 2 h. The optical density (OD) value was detected at 450 nm using a microplate reader. The formula for cell viability was cell viability (%) = [A (dosing) - A (blank)] / [a (0 dosing) - A (blank)] ×100. The experiments were repeated three times.
Cell grouping and treatment
The cells were divided into four groups, with six wells in each group. In the blank control group, PMVEC cells were cultured without treatment. In the CSE group, the concentration of CSE was set as 20% for 12 h, according to the results of the cell viability experiment. In the LPS group, 20 ng/ml LPS was added for 12 h. In the CSE+LPS group, the cells were treated with 20% CSE and 20 ng/ml LPS for 12 h. The cell supernatant was collected for measurements.
Cell morphology observation
Cell growth and morphological changes were observed under a microscope.
Double staining with propidium iodide (PI) and carboxyl fluorescein (FAM-YVAD-FMK)
The cells were digested with trypsin (without EDTA) and collected, and then inoculated at 1×105/well in 96-well plates. The culture medium was discarded after centrifugation. The cells were rinsed twice with PBS, centrifuged at 2000 rpm for 5 min, and about 3×105 cells were collected. The PI/ FAM-YVAD-FMK staining solution was prepared with the cell staining buffer solution. Then, 5 µl of PI staining solution and 5 µl of FAM-YVAD-FMK staining solution were added to each well. The cells were resuspended with the prepared staining solution and incubated at room temperature for 1 h in the dark. Flow cytometry was used for observation within 1 h.
IL-1β and IL-18 by ELISA
PMVECs in the logarithmic phase were selected, cultured for 24 h, and centrifuged at 3000 rpm for 5 min. The supernatant was taken. ELISA was performed according to the manufacturer’s instructions. The OD value of each well was measured at a wavelength of 450 nm within 5 min after stopping the reaction.
SPSS 21.0 (IBM, Armonk, NY, USA) was used for statistical analysis. The continuous data were expressed as mean ± standard deviation. Since the data were consistent with a normal distribution and homogeneity of variance, ANOVA with the Dunnett post hoc test for pairwise comparison. The independent sample t-test was used for comparison between two groups. P-values <0.05 were considered statistically significant.