Chemicals and Reagents

Safflor yellow A (purity ≥ 98%, lot No. 157723) was provided by TargeMol (Wellesley Hills, MA). 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), LPS, and sulforaphane (SFN) were purchased from Sigma-Aldrich (St. Louis, MO). ML385 was obtained from Meilunbio (Dalian, China). ROS assay kit, 4′,6-diamidino-2-phenylindole (DAPI) staining solution, bicinchoninic acid (BCA) protein assay kit, nuclear and cytosolic protein extraction kit, SOD activity assay kit, CAT activity assay kit, GSH Px activity assay kit, malondialdehyde (MDA) assay kit, and horseradish peroxidase conjugated secondary antibody, enhanced chemiluminescence (ECL) assay kit together with enzyme-linked immunosorbent assay (ELISA) kits including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) were provided by Beyotime Biotechnology Institute (Shanghai, China). The ELISA kits for HO-1 and NQO1 were products of Colorfulgene Biotechnology (Wuhan, China). The primary antibodies for cleaved cysteinyl aspartate specific proteinase-3 (cleaved caspase-3) (AF7022), cysteinyl aspartate specific proteinase-3 (caspase-3) (AF6311), B-cell lymphoma-2 (Bcl-2) (AF6139), and Bcl-2-associated X protein (Bax) (AF0120) were supplied by Affinity Biosciences (Cincinnati, OH). And others including phosphorylated nuclear factor κB p65 (p-NF-κB p65) (ab76302), nuclear factor κB p65 (NF-κB p65) (ab32536), phosphorylated inhibitor of NF-κB α (p-IκBα) (ab133462), inhibitor of NF-κB α (IκBα) (ab32518), Nrf2 (ab92946), GAPDH (ab9485), and lamin B1 (ab16048) together with DyLight594 conjugated secondary antibody (ab96885) were obtained from Abcam (Cambridge, UK).

Cell Culture and Treatment

Human lung bronchial epithelial Beas-2B cells were obtained from American Type Culture Collection (ATCC) and maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C under a humid condition of 5% CO2. Cells were set as control group, LPS group, and drug groups. The LPS group was exposed to 0.1 mg/ml LPS in DMEM for 24 h. In addition to 100 μg/ml LPS, the drug groups were incubated with certain SYA or SFN for 24 h. The cells in the control group were cultivated in normal DMEM.

Cell Viability

Beas-2B cells were seeded in 96-well culture microplates at the density of 5 × 103 cells per well. After treated as above 20 μl MTT solution (5 mg/ml) were added and incubated for 4 h. Then, the medium was removed and 200 μl DMSO was added to dissolve the formazan crystals. The absorbance was determined on a microplate reader (BioTek, Winooski, VT) at 570 nm. To present the trends of cell viability, the concentrations of SYA were expressed as logarithm.

ROS Production

To detect the ROS level in Beas-2B cells, DCFH-DA in the assay kit was employed. In brief, the cells were treated as above and then exposed to DCFH-DA (0.5 mg/ml) for 20 min. The fluorescence intensity was read on a fluorescence microplate (Molecular Devices, San Jose, CA) with the excitation wavelength of 488 nm and emission wavelength of 525 nm.

MDA Content

The MDA content in Beas-2B cells was detected using a commercially available assay kit. According to the supplier’s instruction, the cells were treated as above and lysed on ice. After centrifugation at 1600 × g, the supernatant was collected and exposed to the working solution in the assay kit. Then the sample was boiled for 15 min and cooled to room temperature. The absorbance was recorded on a microplate reader at 532 nm.

SOD, CAT, and GSH Px Activity

To detect the activity of SOD, CAT, and GSH Px in Beas-2B cells, the colorimetric method was employed using the commercially available assay kits. In brief, the treated cells were homogenized at 4 °C and the supernatant was collected as samples for further analysis after centrifugation at 12,000 × g and 4 °C for 10 min. Then the protein concentration was quantified using a BCA assay kit, and enzyme activity was determined according to the supplier’s protocols on a microplate reader.

Immunofluorescence Staining

To reveal the location of intracellular Nrf2 in Beas-2B cells, immunofluorescence staining was performed. The cells were seeded in 12-well microplates with a coverslip in each well and treated as above indication. Then the cells were fixed with 4% paraformaldehyde and permeated with PBS containing 0.1% Triton X-100. Then the cells were exposed to the primary antibody of Nrf2 (1:200) overnight. DyLight594 conjugated secondary antibody was used to detect the protein. After staining with DAPI in the dark, the images were captured using a Nikon fluorescence microscope (Tokyo, Japan).

ELISA

To uncover the levels of TNF-α, IL-1, IL-6, HO-1, and NQO1 in Beas-2B cells, ELISA was implemented using the assay kits. For the secretion of pro-inflammatory cytokines, the treated cells were centrifuged at 500 × g for 5 min and the supernatant was collected for further analysis. For the intracellular enzymes, the treated cells were lysed at 4 °C and then the supernatant was also collected following centrifugation at 1000 × g for 20 min. Then the samples were handled in light of the suppliers’ protocols, and the absorbance was recorded on a microplate reader at 450 nm.

Western Blot Analysis

The total proteins were extracted using RIPA lysis buffer while the nuclear proteins were obtained using the nuclear and cytosolic protein extraction kit according to the supplier’s instructions. The protein concentrations were determined using BCA protein assay kit, and the proteins were separated on 10% SDS-PAGE and transferred to PVDF membranes. After blocking with non-fat milk, the membranes were incubated with primary antibodies including cleaved caspase-3 (1:1000), caspase-3 (1:1000), Bcl-2 (1:1000), Bax (1:1000), p-IκBα (1:10,000), IκBα (1:1000), p-NF-κB p65 (1:1000), NF-κB p65 (1:1000), Nrf2 (1:1000), GAPDH (1:2500), and lamin B1 (1:1000) at 4 °C overnight. After being rinsed with TBST buffer, the membranes were cultured with horseradish peroxidase conjugated secondary antibodies at room temperature for 1 h. The bands were visualized using an ECL substrate on a Bio-Rad imaging system (Hercules, CA). GAPDH and lamin B1 were used as internal controls. ImageJ software (NIH, Bethesda, MD) was used for densitometric analysis.

Molecular Docking

To elucidate the effect of safflor yellow A on Keap1-Nrf2 interaction, molecular docking was performed as our previous description (Zheng and Chen 2017). In brief, the 3D structure of safflor yellow A was established on SYBYL sketch and optimized by Tripos force field and Gasteiger-Huckel charges. The crystal structure of Keap1 Kelch domain was obtained from RSCB Protein Data Bank (PDB code: 4IQK). The protomol file was generated to produce the docking envelope and Surflex-Dock program with default parameters was operated for docking calculations.

Statistical Analysis

The data was provided as mean ± standard deviation and analyzed by GraphPad Prism 8.0 (San Diego, CA). The differences among groups were assessed using one-way analysis of variance (one-way ANOVA) followed by the Tukey test for multiple comparisons and Student’s t test for single comparisons. A p < 0.05 was considered significant in statistics.