Cooking oil fumes
COFs samples were collected by referring to Ding et al (Ding et al. 2020). Briefly, The COFs was produced from heating peanut oil (200 mL) by an electric heater in iron pan and the temperature was kept at the smoke point (280 ± 10 °C). The fumes were collected into a brown bottle for preservation and stored at -80 °C. Different concentrations of COFs were dissolved in dimethyl sulfoxide (DMSO) when used.
Cells and culture conditions
Beas-2B cells were purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China) and cultured in high glucose DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 100 U/mL penicillin (C0222, Beyotime, Shanghai, China), 10% fetal bovine serum (FBS, Gibco, New York, NY, USA) and 100 μg/mL streptomycin (C0222, Beyotime, Shanghai, China) at 37 °C in 5 % CO2.
Cell viability assay
Beas-2B cells were seeded in 12-well plates at density of 3×105 cells/well, treated with COFs at concentration of 200 μg/mL for 12 h, 24 h and 48 h and observed with electron microscope (Olympus CX22LED microscope 10×magnification, Olympus Corporation, Tokyo, Japan) (Liu et al. 2018). The cells viability was detected with MTT assay (Cao et al. 2021). Briefly, Beas-2B cells were seeded at the density of 1×104 cells per well in 96-well plate, then the cells were cultured in a humidified atmosphere of 5 % CO2 at 37 °C (Lab-Line CO2 Incubator, Merlose Park, USA) and treated with COFs (100, 150, 200, 250, 300, 350, 400, 600, 800 μg/mL) for 24 h. Thiazolyl blue tetrazolium bromide (MTT) was added to each well. The absorption at 490 nm was measured with microplate reader (Varioskan® Flash, Thermo Fisher Scientific Inc.), the survival rate was calculated with OD experimental group/OD control group×100% (Park and Park 2009).
Apoptosis assay
After 24 h exposure to COFs (200, 400, 600 μg/mL), Beas-2B cells were digested with trypsin without EDTA, washed with 1 mL PBS twice and centrifuged 5 min at 1000 rpm. According to the manufacturer's protocols, apoptosis was measured with double stained with Annexin-V-FITC apoptosis detection kit and propidium (PI) (eBioscience, CA, USA). Briefly, 100 µL binding buffer, 1 µL Annexin V-FITC, and 1 µL PI staining solution were added to Beas-2B cells in 6-well plates, the cells incubated at room temperature for 5 min and the apoptosis was detected with a flow cytometry (BD, FACS CantoII) (Wang et al. 2019).
mRNA expression measurement
Total cellular RNA was extracted from Beas-2B cells with RNA isolation kit (Sigma-Aldrich) according to the manufacturer’s instructions. The mRNA was transcribed into cDNA with cDNA synthesis kit. Then, qRT-PCR was conducted by using the SYBR Premix Ex Taq (Tli RNaseH Plus) with Applied Biosystems 7500 Real-Time PCR system (Appbice Applied Biosystems Trading Co., Ltd., Shanghai, China). The PCR thermocycling conditions were as follows: denaturation at 95 °C for 40 seconds followed by 40 cycles of 95 °C for 10 seconds and 60 °C for 35 seconds (Zhong et al. 2019). The primer pairs for human IL-6, iNOS, ICAM-1, IL-1β, IL-8, COX-2, NLRP3, CRP, TNF-α, IL-17, IL-18 and β-actin were shown in table 1.
Table 1 Sequences of primers and probes used in the present study
Gene
|
Primer Forward(5→3)
|
Primer Reverse(5→3)
|
Product
|
TNF-α
|
AGGACGAACATCCAACCTTCCCAA
|
TTTGAGCCAGAAGAGGTTGAGGGT
|
92
|
iNOS
|
TGCAGACACGTGCGTTACTCC
|
GGTAGCCAGCATAGCGGATG
|
130
|
COX-2
|
TTCAAATGAGATTGTGGGAAAATTGCT
|
AGATCATCTCTGCCTGAGTATCTT
|
146
|
IL-1β
|
TCCAGGGACAGGATATGGAG
|
TCTTTCAACACGCAGGACAG
|
133
|
IL-6
|
ATGAACTCCTTCTCCACAAGCGC
|
GAAGAGCCCTCAGGCTGGACTG
|
166
|
IL-8
|
AGCTCTGTGTGAAGGTGCAG
|
AATTTCTGTGTTGGCGCAGT
|
148
|
ICAM-1
|
AGC TTC TCC TGC TCT GCA AC
|
GTC TGC TGG GAA TTT TCT GG
|
146
|
NLRP3
|
CTTCCTTTCCAGTTTGCTGC
|
TCTCGCAGTCCACTTCCTTT
|
212
|
CRP
|
GGGCCCTTCAGTCCTAATGTC
|
TTCGCCTTGCACTTCATACTT
|
156
|
β-Actin
|
ACCGCCGAGACCGCGTCCGCCCCGC
|
TGTTGCCGAGGCCGTACA
|
133
|
IL-17
|
TGGGAAGACCTCATTGGTGT
|
GGATTTCGTGGGATTGTGAT
|
84
|
IL-18
|
AGTCAGCAAGGAATTGTCTCC
|
GAAGCGATCTGGAAGGTCTG
|
135
|
Measurement of intracellular ROS
The levels of ROS were measured with the fluorescent probe 2’, 7’-dichlorofluorescein diacetate (DCFH-DA) (KeyGen Biotech, Jiangsu, China). In short, Beas-2B cells were treated with COFs (200, 400, 600, 800 μg/mL) for 24 h, then washed with PBS once and probed with 10 mM DCFH-DA for 30 min at 37 °C. The positive fluorescent signals were imaged under inverted fluorescence microscope (Park et al. 2008).
Measurement of intracellular NO
Intracellular NO was measured with the NO-specific cell-penetrable fluorescent probe 3-Amino, 4-aminomethyl-2, 7-difluorescein, diacetate (DAF-FM) (KeyGen Biotech, Jiangsu, China). Briefly, Beas-2B cells were seeded in 12-well plates at a density of 1 × 106 cells/well and treated with different concentrations COFs (200, 400, 600, 800 μg/mL) for 24 h. After removal supernatant, the cells was washed once and probed with 10 mM DAF-FM for 35 min at 37 °C. The positive fluorescent signals were observed under inverted fluorescence microscope ( × 10) (Li et al. 2020).
Cell signaling pathway analysis
Western blot analysis was carried out according to our previous study (Cao et al. 2020b). After treatment with COFs (0, 200, 400, 600 μg/mL) for 24 h, Beas-2B cells were harvested and lysed, total protein extracts and nuclear extracts were prepared with total protein and nuclear protein extraction kits. Then the protein concentration was measured by BCA method. Protein samples were separated by 10% SDS-PAGE gels and transferred to PVDF membranes according to standard electroblotting procedures. After blocked with 5% non-fat milk for 1 h, the membrane was incubated with anti-Bax, anti-Bcl-2, anti-cleaved caspase-1, anti-cleaved caspase-3, anti-IκB, anti-p-IκB, anti-p65, anti-PCNA, anti-β-actin, anti-JNK, anti-ERK, anti-p38, anti-p-JNK, anti-p-ERK, anti-p-p38 monoclonal antibodies (at 1:2000 dilutions), then incubated with secondary antibodies. Finally, bands were observed by using an enhanced chemiluminescence kit. The relative level of total protein was normalized to β‑actin and the relative level of nuclear protein was normalized to PCNA (Cao et al. 2020a; van der Stel et al. 2020).
Statistical analysis
The data were presented as means ± standard deviation (SD) at least three independently performed experiments. The date analyses were performed with one-way analysis of variance (ANOVA) with SPSS 21.0 (IBM SPSS, Armonk, NY, USA). P < 0.05 and P < 0.01 revealed statistical significance.