Cell strains

Human lung carcinoma epithelial A549 cells and BEAS-2B were purchased from the American Type Culture Collection (ATCC) cell bank (China).

CSE preparation

CSE was prepared as previously described [12]. Briefly, the cigarette filter was removed, and one end of the cigarette (Jiangsu Zhongyan Industry Co., China) was connected to a Pasteur pipette containing 10 mL serum-free DMEM (Beijing Solarbio, China) and the other end was connected to a 20 mL syringe. Next, the piston was pulled back once every minute to draw 20 mL, which was held in place for 2 s. Thereafter, the glass bottle was agitated to dissolve the smoke until one cigarette was utilized completely. Each cigarette corresponded to 10 mL of culture media and was defined as 100% CSE. Subsequently, 0.22 μm filters were used to remove bacteria, followed by aliquoting and storage at − 80 °C. Prior to experimentation, samples were rapidly thawed and used within 30 min. Based on experimental grouping, culture media was used to dilute 100% CSE to the required concentrations.

Cell model

Briefly, A549 and BEAS-2B cells were thawed and cultured in DMEM containing 10% FBS (Gibco, USA) at 37 °C in a 5% CO2 incubator. Media was replaced once daily. Then, log-phase cells were collected and assigned to groups based on pre-experimental results and previous reports. The grouping was as follows: control group: A549 and BEAS-2B cells were cultured using the conventional method; experimental group: cultured A549 and BEAS-2B cells were treated with CSE for 24 h; low-dose group: cultured A549 and BEAS-2B cells were pretreated with 0.5 mM NaPyr(Jiangsu Changtai Pharmaceutical Co., China) for 2 h, followed by CSE for 24 h; medium-dose group: cultured A549 and BEAS-2B cells were pretreated with 1 mM NaPyr for 2 h, followed by CSE for 24 h; high-dose group: cultured A549 and BEAS-2B cells were pretreated with 2 mM NaPyr for 2 h, followed by CSE for 24 h; inhibitor group, cultured A549 and BEAS-2B cells were treated with CSE and Fer-1 (Aladdin Biochemical Technology Co., China).

CCK8 assay for cellular activity

Briefly, log-phase A549 and BEAS-2B cells were cultured in a 96-well plate (3 × 103 each well) containing 100 µL DMEM and treated based on the established grouping. Subsequently, the supernatant was collected, and 100 µL DMEM containing 10% CCK8 (Dojindo Laboratories Co., Japan) solution was added. A microplate reader (Bio-Rad Co., USA) was used to measure the absorbance (Ab) value at 450 nm wavelength. Cell activity (%) = (Ab value of treated well – Ab value of blank well)/(Ab value of control well – Ab value of blank well) × 100.

Measurement of intracellular free iron concentrations

Briefly, A549 and BEAS-2B cells were placed in culture plates and assigned to predetermined groups. Following treatment, cells were harvested, and the number of cells was calculated. A microplate reader was employed to measure the free iron concentrations according to the total iron assay kit (Elabscience, China).

Measurement of MDA and GSH content to assess lipid peroxidation

Briefly, 100 µL of each cell supernatant was mixed with 400 µL of MDA working reagent (Beijing Solarbio, China). The mixture was incubated at 100 °C for 30 min, cooled on ice, and centrifuged at 10,000 × g for 10 min at 4 °C temperature. Subsequently, 200 µL of supernatant was placed in a 96-well plate, and the absorbance was measured at 532 nm. The MDA concentration was calculated based on the measured absorbance. Similarly, GSH content in each group was detected according to the instructions of the GSH assay kit (Beijing Solarbio, China).

Measurement of MitoSOX to assess mitochondrial oxidation levels

Briefly, cells were seeded into 6-well plates and treated according to established experimental grouping. Cells were rapidly rinsed with Hanks’ balanced salt solution (Wabcan, China). Next, 5 µM mitochondrial probe MitoSOX (Thermo Fisher Scientific, USA) was added to plates, followed by incubation in the incubator for 10 min. After fixing with 40 g/L triformol, nuclei were stained with DAPI (Beijing Solarbio, China) for 10 min and rinsed three times with methanol. After sealing, blue and red fluorescence intensities were observed using the fluorescence microscope (Ghuin Co., Japan). Finally, MitoSOX fluorescence intensity was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD), reflecting mitochondrial oxidation levels.

Measurement of ROS to assess mitochondrial oxidation levels

Briefly, cells were seeded in a 6-well culture plate and treated according to established experimental grouping. After removal of the culture medium, 1 mL of 10 µmol/L DCFH-DA (KeyGEN BioTECH Co., China) was added to each well, and plates were incubated for 20 min. The cells were then washed with serum-free culture medium to remove the DCFH-DA not taken up by cells. After sealing, green intensity (excitation wavelength: 485 nm, emission wavelength: 530 nm) was observed using a fluorescence microscope and analyzed using ImageJ software, reflecting cellular oxidation levels.

JC-1 assay for mitochondrial membrane potential

Briefly, cells were seeded in a 6-well culture plate and treated according to established experimental grouping. The cells were washed with phosphate-buffered saline, followed by the addition of 1 mL culture medium to each well. Subsequently, 1 mL JC-1 working stock (Beijing Solarbio, China) was added to each well and mixed thoroughly. The plates were incubated in an incubator at 37 °C for 20 min. After incubation, the supernatant was removed, and the cells were rinsed with JC-1 dye buffer two times. Then, 2 mL of cell culture medium was added. The cells were observed under a fluorescence microscope using red (excitation wavelength: 488 nm, emission wavelength: 590 nm) and green (excitation wavelength: 485 nm; emission wavelength: 530 nm) filters.

ELISA

Briefly, cells were seeded in a 6-well culture plate at a concentration of.

600,000 per well and treated according to established experimental grouping. The supernatant of each group was collected after 24 h, followed by centrifugation at 2500 × g for 20 min. TNF and IL-8 expression levels were measured according to the ELISA assay kit instructions (Yi Fei Xue Biotechnology, China).

Quantitative PCR (qPCR)

Briefly, total RNA was extracted using the TRIzol assay kit (KeyGEN BioTECH Co., China). cDNA was synthesized using the cDNA synthesis assay kit (Tiangen Biotech, China). qPCR was performed using SYBR-Green Supermix (Invitrogen Life Technologies). Corresponding expression levels were calculated using the 2–∆∆Ct method and GAPDH as the internal control. The primers used are summarized as follows.

Sequences

Nrf2

F: 5′-AACACAAGAGCCCCTGTGTGGC-3′

R: 5′-TGCCCCTGAGATGGTGACAA-3′

GPX4

F: 5′-ACAAGAACGGCTGCGTGGTGAA-3′

R: 5′-GCCACACACTTGTGGAGCTAGA-3′

COX2

F: 5′-CTGGCGCTCAGCCATACAG-3′

R: 5′-CACCTCGGTTTTGACATGGGT-3′

GAPDH

F: 5′-GGTTGTCTCCTGCGACTTCA-3′,

R: 5′-TGGTCCAGGGTTTCTTACTCC-3′

Western blotting to measure protein expression

For each group of A549 and BEAS-2B cells, total protein was extracted using RIPA lysis buffer, and the total protein concentration was measured using the bicinchoninic acid (BCA) method. Then, 20 µg of protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein was transferred to a polyvinylidene difluoride (PVDF) membrane by wet transfer and blocked in 50 g/L non-fat milk at room temperature for 2 h. Subsequently, the primary antibody was added (GPx4, Nrf2, COX2, and GAPDH [1:1000], respectively)(Cell Signaling Technology, China), and the membrane was incubated overnight at 4 °C. After washing, the membrane was incubated in a secondary HRP-conjugated antibody for 2 h. After washing thoroughly, the blots were developed with substrate solution (Pierce Biotechnology, USA), and the chemiluminescent imaging system was used to examine fluorescence (Bio-Rad Co., USA). Fluorescence intensity was analyzed using Image Lab software.

Statistical analysis

Data analyses were performed in SPSS version 18.0 (IBM Corp., Chicago, IL, USA). All experiments were performed in duplicate and repeated at least 3 times. The results are presented as mean ± SD (standard deviation). The experimental data were assessed for a Gaussian distribution. Two-tailed unpaired Student’s t tests were applied for comparison of two normally distributed groups; comparisons between more than two normally distributed groups were made by one-way ANOVA followed by pairwise multiple comparison (Student-Newman-Keuls method, q-test). Differences were considered statistically significant at P < 0.05.