2.1 Cell culture
The human bronchial epithelial cell line 16HBE cells (Cell Bank of Chinese Academy of Sciences, Shanghai, China) were cultured in RPMI-640 medium (Hyclone, Logan, UT, USA). The medium was supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) and 1% antibiotics (Gibco). The 16HBE cells were cultivated at 37°C in a humidified atmosphere of 5% CO2.
2.2 Cell transfection
To obtained Nrf2-knockdown 16HBE cells, the 16HBE cells were transfected with si-Nrf2 (Invitrogen, Carlsbad, CA, USA). The 16HBE cells transfected with si-NC were used as negative controls. To generate the TLR4-overexpressing 16HBE cells, the 16HBE cells were transfected with pcDNA3.0-TLR4. The 16HBE cells transfected with pcDNA3.0 were used as negative controls. After post transfection, the transfection efficiency was confirmed by western blot analysis.
2.3 Preparation of CSE
CSE was prepared from Daqingshan cigarettes (Tar: 12.5, Hohhot Cigarette Factory, Hohhot, China; model group) as previously described [15]. A 1 cm cigarette without filter was combusted with a variable speed pump (Thermo Fisher Scientific, Waltham, MA, USA). The smoke was bubbled through 15 ml double-distilled water and the solution was filtered through a 0.22-μm pore filter to remove large particles. This resulting suspension was used as 100% CSE mother solution and then diluted to the concentration of 5% CSE with culture medium.
2.4 Cell viability assay
The cell viability of 16HBE cells was evaluated by MTT assay, which depends on a colorimetric method. The cells were treated with 10 μl of MTT reagent (5 mg/ml). After incubation for 4 h at 37°C, the culture medium was replaced with 100 μl of DMSO. Finally, sample absorbance was analyzed by the ELISA-reader (Bio-Tek Instruments, Winooski, VT, USA) at a wavelength of 490 nm.
2.5 Measurement of ROS production
ROS production was assessed using the 2’,7’-dichlorodihydrofluorescein diacetate (H2DCF-DA), which can diffuse into the cells and go through deacetylation and oxidization by cellular enzymes into 2’,7’-dichlorofluorescein (DCF) in the presence of ROS. In brief, the treated 16HBE cells were collected and incubated with H2DCF-DA for 30 min at room temperature in the dark. Then, the cells were washed for three times and the fluorescence intensity in each group was analyzed using flow cytometry with the excitation wavelength 488 nm and the emission wavelength 525 nm.
2.6 Detection of glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities
After treatment, the activities of GPx and SOD in the supernatants were measured using corresponding commercial kits (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s protocol.
2.7 Quantitative real-time PCR (qRT-PCR)
Total RNA was isolated from 16HBE cells by the single-step method using Trizol reagent (Applied Biosystems, Waltham, MA, USA) following the manufacturer’s instructions. Then the total RNA was transcribed with cDNA transcription reagent (Applied Biosystems) to obtain cDNA. The expressions of IL-6, IFN-γ, and TNF-α at mRNA levels were determined by qRT-PCR using SYBR Green Master Mix (Applied Biosystems) with the use of an ABI PRISM 7500 Sequence Detection System (Applied Biosystems). The levels of target mRNA were normalized to β-actin mRNA, which is an internal control. β-actin primers were: forward: 5’-ATCA CCAT TGGC AATG AGCG-3’ and reverse: 5’-TTGA AGGT AGTT TCGT GGAT-3’; TNF-α primers were: forward: 5’-TTC TGT CTA CTG AAC TTC GGG GTG ATC GGT CC-3’ and reverse: 5’-GTA TGA GAT AGC AAA TCG GCT GAC GGT GTG GG; IFN-γ primers were: forward: 5’-ACT GGC AAA AGG ATG GTG AC-3’ and reverse: 5’-TGA GCT CAT TGA ATG CTT GG; IL-6 primers were: forward: 5’-AAC GAT GAT GCA CTT GCA GA-3’ and reverse: 5’-GAG CAT TGG AAA TTG GGG TA-3’.
2.8 Western blot analysis
After addition with appropriate amount of lysis buffer (Beyotime Biotechnology, Shanghai, China) and incubation for 30 min, the resultant lysis solution was collected and centrifugated at 12,000 rpm for 15 min at 4°C. The supernatant was collected for the determination of protein concentration by the BCA method. The protein samples were subjected to denaturation in the 98°C metal bath for 5 min and then separated by 12% SDS-PAGE. Afterwards, the proteins were transferred onto the PVDF membrane, followed by blocking with 5% skim milk for 1 h and incubation with the corresponding primary antibodies overnight at 4°C. The primary antibodies against bax, bcl-2, cleaved caspase-3, nuclear Nrf2, HO-1, TLR4, p65, p-p65 and IκBα were used in this study (Abcam, Cambridge, MA, USA). Next, the membrane was incubated with secondary antibody at room temperature for 1 h. The secondary antibody was used at a dilution of 1: 3000 (Abcam). The bands on the membrane were then visualized by the chemiluminescence method using chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ, USA). The gray value was analyzed via the Image-J software (National Institutes of Health, NIH, Bethesda, MD, USA).
2.9 Cell apoptosis assay
Caspase 3 activity in cellular lysates was assessed using the Caspase 3 Activity Assay Kit purchased from the Beyotime Institute of Biotechnology. Briefly, each sample was incubated with caspase 3 substrate (Ac-DEVD-pNA) at 37°C for a further 2 h. Finally, the reaction products were measured with Microplate Spectrophotometer (Thermo Fisher Scientific) at an absorbance of 405 nm.
2.10 ELISA
Supernatants from experimental cultures of 16HBE cells were collected and stored at -80°C until use. The levels of IL-6, IFN-γ, and TNF-α in the supernatants were determined by using ELISA kits (R&D Systems, Minneapolis, MN, USA) in accordance with the manufacturer’s instructions. Finally, the detection of absorbance at 450 nm was conducted by using a microplate reader (Bio-Tek).
2.11 Statistical analysis
Data are expressed as means ± SEM. Experiments with multiple comparisons were analyzed by one-way ANOVA followed by Tukey post hoc test using IBM SPSS statistics version 22.0. A p value less than 0.05 (p<0.05) was regarded as statistically significant.