Cell cultures and cell viability
RAW 264.7 cells cultured in DMEM medium containing 10% FBS, and incubated at 37 °C and 5% CO2. Isorhapontigenin (≥98% purity)(Sigma, St. Louis, MO, USA) dissolved in DMSO solution, and the final DMSO concentration was ≤0.1% in the culture medium. Cell viability detected using CCK-8 assay kit (Sigma). Briefly, RAW 264.7 cells treated with (0-120μM) isorhapontigenin for 24 h. Next, cells added CCK-8 solution and cell viability was determined using a microplate spectrophotometer (Multiskan FC, Thermo Scientific, MA, USA).
Proinflammatory cytokine assay
RAW 264.7 cells treated with (0–20 μM) isorhapontigenin for 1 h. Next, 100 ng/mL LPS (Escherichia coli 055:B5, Sigma) stimulated the cells, and cultured for 8 h to detect IL-6 and TNF-α expression, and cultured 24 h to detect PGE2 and IL-1β production by ELISA kits (R&D Systems, Minneapolis, MN, USA). SP600125 (JNK inhibitor), SB203580 (p38 inhibitor), and PD98059 (ERK inhibitor) (Sigma) treated with or without 10 μM isorhapontigenin to assay IL-6 and TNF-α production. Specific cytokine or PGE2 were determined using a microplate spectrophotometer (Thermo Scientific).
Cells treated with isorhapontigenin (0–20 μM) for 1 h. Next, cells stimulated with 100 ng/mL LPS (Sigma) for 24h. The supernatant treated with the Griess reagent (Sigma) for 15min, and nitrite levels was detected at 570 nm, a microplate spectrophotometer (Thermo Scientific).
Preparation of total and nuclear protein
Cells treated with isorhapontigenin for 1 h. Next, cells stimulated with or without 100 ng/mL LPS for 30 min to detect protein phosphorylation, or for 24 h to investigate total protein expression. Briefly, cells collected and lysed using protein lysis buffer containing phosphatase inhibitor cocktail (Thermo Scientific). Furthermore, nuclear proteins isolated using NE-PER cell fractionation kits (Thermo Scientific).
Protein separated using SDS-PAGE gel and the protein bands transferred to PVDF membranes. The membranes incubated with specific antibodies, including ERK1/2, JNK, p38, ICAM-1, inducible nitric oxide synthase (iNOS), phosphorylated-ERK 1/2, phosphorylated-JNK, and phosphorylated p38 (Cell Signaling, MA, USA), cyclooxygenase-2 (COX-2), and β-actin (Sigma), IκB-α, lamin B1, HO-1, phosphorylated-IκB-α, and Nrf2 (Santa Cruz, CA, USA). Following washing, membranes incubated with secondary antibodies, and finally proteins were presented using enhanced chemiluminescence solution (Thermo Scientific) and the BioSpectrum imaging system (UVP, Upland, CA, USA).
Cells treated with isorhapontigenin for 1 h, then cells stimulated with LPS for 24 h. Cells washed and added with DCFH-DA as described previously . ROS expressions observed using fluorescence microscopy (Olympus). Cells also detected ROS levels using the multimode plate readers (BioTek synergy HT). Furthermore, MAPK inhibitors (Enzo Life Sciences) were treated with or without 10 μM isorhapontigenin to detect ROS expression.
Cells were used to seed microscope slides in culture plates and incubated with isorhapontigenin for 1 h, then LPS stimulated cells for 24 h. The slides treated with a specific P65 antibody (1:100), followed by a fluorescent secondary antibody. Finally, the slides treated with DAPI solution for nuclear staining and presented images using fluorescence microscope (Olympus).
Experimental results performed at least three independent experiments. Data are presented as the mean ± SD. Statistical analysis of experimental data performed using one-way ANOVA followed by Dunnett’s test and post hoc analysis. Statistical significance defined as p < 0.05.