Reagents
Roswell Park Memorial Institute 1640 Medium, 0.25% trypsin-EDTA, and FBS were purchased from HyClone (Logan, USA.). Nrf2 siRNA vector and Nrf2 high expression vector were designed and synthesized by RiboBio Company (Guangzhou, China) and Gene Chemical Technology (Shanghai, China). Recombinant vector “pGL3-NOX1-1500” and “pGL3-NOX1-1489” were synthesized by GenePharma (Suzhou, China). LipofectamineTM2000 was bought from Invitrogen (California, USA.). PrimeScriptTM RT (RR037A) and SYBRR premix Ex TaqTM (RR420A) reagent kits were purchased from TaKaRa (Osaka, Japan). Primers of human GAPDH, Nrf2, NOX1, and NF-κB were ordered from Sangon Biotech (Shanghai, China). Primary monoclonal antibodies of human GAPDH (ab181602), Nrf2 (ab62352), and NOX1 (ab78016) were purchased from Abcam (Cambridge, UK) and NF-κB p65 (8242S) was purchased from Cell Signaling Technology (Massachusetts, USA). Goat anti-rabbit horseradish peroxidase-conjugated secondary antibody was bought from Beyotime (Shanghai, China). TNF-α was purchased from PeproTech (New Jersey, USA). IL-6 and IL-8 ELISA kits were bought from R&D (Minnesota, USA). Reactive Oxygen Species Kit, Lipid Peroxidation MDA Kit, and Total Antioxidant Capacity Assay Kit were ordered from Beyotime (Shanghai, China).
Cell culture
A549 cells were cultured in RPMI-1640 medium with 10% FBS and kept in an environment containing 5% CO2 at 37◦C. They were passaged every three days and cells used for experiments were incubated in six-well plates. According to different purposes, cells were randomly divided into three parts. Specifically, cells in first part were randomly assigned into the control group and TNF-α treated group; those in second part were randomly divided into naive group, TNF-α-treated group, negative control group, Nrf2 siRNA group, Nrf2 overexpression group, and empty vector group; and those in third part were randomly divided into pGL3 group, pGL3-NOX1-1500 group and pGL3-NOX1-1489 group.
Cell treatments
Cells in control group were cultured in normal medium while cells in TNF-α-treated group were cultured with 2.5, 5, 10, 20 and 40 ng/ml TNF-α for 24 hours or cultured with 10ng/ml TNF-α for 0, 6, 12, 24, 36 and 48 hours, respectively.
Cells in naive group were cultured in normal medium, while cells in TNF-α-treated group were stimulated by 10ng/ml TNF-α for 24 hours. Vector transfection was performed using lipofectamineTM 2000. Cells in the remaining groups were transfected with negative siRNA vector (50nmol/L), Nrf2 siRNA vector (50nmol/L), Nrf2 high expression vector (4ug), and empty vector (4ug) for six hours, followed by stimulation of 10ng/ml TNF-α for 24 hours.
Cells in third part were transfected with pGL3 basic plasmid, and those in pGL3-NOX1-1500 plasmid and pGL3-NOX1-1489 plasmid, respectively.
ELISA assay
Cell supernatants in each group were collected and levels of IL-6 and IL-8 were measured by ELISA assay in accordance with the manufacturer’s protocol, each supernatant sample was measured in duplicates.
ROS detection
Intracellular ROS were determined by non-fluorescent probe 20, 70-dichlorofluorescin diacetate (DCFH-DA) in accordance with the manufacturer’s protocol. Briefly, cells were digested by 0.25% trypsin-EDTA, followed by being treated with DCFH-DA (10mM) in the dark at 37◦C for 30min, gently washed by PBS and observed under a fluorescence microscope. Cellular fluorescence intensity was determined in a microplate reader (RT-7300, Rayto, Guangdong, China) at an excitation of 485nm and an emission of 538nm.
MDA and total antioxidation capability (T-AOC) measurement
Total cellular protein was extracted by cell lysis buffer supplemented with protease inhibitors and quantified via BCA assay. Afterwards, MDA concentration was measured by an MDA kit according to its instruction. Briefly, cellular protein samples were mixed with reagents provided in the kit and kept in a boiling water bath at 100◦C for 15min. Then the mixtures were centrifuged and supernatants were measured at 532nm with a microplate reader. MDA level was expressed as nmol/ml. T-AOC was determined via Rapid ABTS method using a T-AOC assay kit according to the enclosed guidelines. Briefly, cellular protein samples were mixed with required reagents and incubated at room temperature for 5min; then the mixtures were measured at 405nm by a Varioskan Flash spectral scanning multimode reader. T-AOC level was expressed as umol/mg.
Quantitative real-time polymerase chain reaction (qRT-PCR) analysis
Total RNA was extracted from cell pellets using trizol reagent based on the manufacturer’s instructions. RNA concentration and purity were measured with a spectrophotometer prior to cDNA synthesis. Afterwards, cDNA was synthesized by reverse transcription of 600ng total RNA according to the guideline of a PrimeScriptTM RT reagent kit. Finally, genes of interest were amplified in accordance with SYBRR premix Ex TaqTM reagent kit’s instructions under an ABI PRISM 7500 sequence detection system (Applied Biosystems, USA). Relative expression of target genes was normalized to that of housekeeping gene GAPDH and calculated by the 2−ΔΔCt method. Primers used for qRT-PCR analysis were shown in Table 1.
Table 1: Primers used for qRT-PCR analysis
Western blot analysis
Whole cell protein extractions and quantifications were performed as described above. Subsequently, cell lysates with an equal amount of total protein (40μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skimmed milk for four hours, and then were incubated with rabbit anti-human primary antibodies of GAPDH (1:5000), Nrf2 (1:2000), NF-κB p65 (1:1000), and NOX1 (1:1000) overnight at 4◦C. Afterwards, the membranes were washed and incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:6000) for one hour. Lastly, proteins of interest were visualized via an ECL detection reagent by an ImageQuant LAS4000 chemiluminescence detection system. All images were analyzed using the Quantity One software. GAPDH served as a loading control and expressions of target proteins were normalized.
Luciferase activity assay
AliBaba2.1 database (http://gene-regulation.com/pub/programs/alibaba2/) was applied to predict potential transcription factor binding site in the promoter of NOX1. Subsequently, the fragment in 5′-flanking region of NOX1 promoter (1500bp) and that of deletion mutant (1489bp), of which the predicted binding site was deleted were cloned, amplified and inserted into pGL3 basic plasmid to construct recombinant plasmids, namely “pGL3-NOX1-1500” plasmid and “pGL3-NOX1-1489” plasmid. After being transfected with these plasmids, cells were stimulated by TNF-α and then luciferase activity was monitored on a MD SpectraMax M5 enzyme-labeled instrument and calculated as a ratio of firefly luciferase relative to Renilla luciferase luminescence.
Statistical analysis
Statistical analyses were performed using SPSS21.0 (IBM Analytics, Armonk, NY, USA). Data were represented as means ± standard deviation, and analyzed by independent-samples t-test and one-way ANOVA. P values<0.05 was considered to be statistically significant.