Honokiol (lot number M8P0236) and magnolol (lot number M8F3374) (≥98% pure based on HPLC) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). These compounds were dissolved in dimethyl sulfoxide (DMSO) as stock solutions. DMEM, penicillin, streptomycin, 0.05% (w/v) trypsin/EDTA, and PBS were obtained from GIBCO (Gaithersburg, MD). Interferon-γ (IFNγ) was purchased from R&D Systems (Minneapolis, MN). Lipopolysaccharide (LPS) (rough strains) from Escherichia coli F583 (Rd mutant) and methylthiazolyldiphenyl-tetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO). The AlamarBlue™ kit was from Invitrogen (Carlsbad, CA). Fetal bovine serum was from Atlanta Biologicals (Lawrenceville, GA). Antibodies used for Western blotting include: goat anti-rabbit IgG-horseradish peroxidase, goat anti-mouse IgG-horseradish peroxidase, and iNOS polyclonal (Santa Cruz Biotechnology, Santa Cruz, CA); monoclonal anti-β-actin peroxidase (Sigma-Aldrich, St. Louis, MO); ERK1/2, phospho-ERK1/2, (Cell Signaling, Beverly, MA). Antibodies used for immunocytochemical staining include rabbit anti-p47phox antibodies (Calbiochem, Billerica, MA), mouse anti-gp91phox (Thermo Fisher, Waltham, MA), goat-anti-rabbit Alexa fluor 488 (Jackson Immunoresearch, West Grove, PA), and goat-anti-mouse Alexa fluor 549 (Jackson Immunoresearch, West Grove, PA).
For ROS detection, CM-H2DCFDA (DCF) was obtained from Invitrogen, Inc. (Carlsbad, CA), and dihydroethidium (DHE) from Sigma-Aldrich (St. Louis, MO). Inhibitors used in this study include: MEK inhibitor U0126 (Cell Signaling, Beverly, MA), 4-(2-aminoethyl)-benzenesulfonylfluoride (AEBSF, Calbiochem, San Diego, CA), diphenyleneiodonium (DPI) and apocynin (Sigma-Aldrich, St. Louis, MO).
Preparations of primary cortical neuron cells involved pregnant E17 Sprague–Dawley rats (Harlan, IN, USA). All animal care and experimental protocols were carried out in accordance with National Institutes of Health (NIH) guidelines and with permission from the University of Missouri Animal Care and Use Committee (protocol #6728). Primary cortical neurons were prepared from the cerebral cortices of E17 Sprague–Dawley rat embryos as described . Briefly, cerebral cortices were dissected and meninges removed. The tissues were suspended in 3 ml 0.05% (w/v) trypsin/EDTA and incubated for 30 min at 37°C. The cell suspension was triturated through a fine-burned-tip glass pipette until tissues were homogenized. The filtrate was centrifuged at 1000g for 1 min and resuspended in 10% FBS in DMEM containing 100 units/ml penicillin and streptomycin (100 μg/ml). Finally, cells were plated in 24-well plates for MTT analysis and 35 mm dishes for ROS detection. The plates were precoated with poly-L-lysine (Sigma-Aldrich, St. Louis, MO) the day before plating and incubated overnight. Four hours after plating, culture medium was completely changed to B27 supplemented Neurobasal medium containing 100 units/ml penicillin, 100 μg/ml streptomycin, and glutamine. Culture was maintained by changing 1/2 volume of B27 medium in each well every 4 days. Experiments were conducted 8 days after plating, to ensure adequate culture maturation.
The immortalized mouse BV-2 and rat HAPI microglial cells were cultured as described previously . Briefly, cells were cultured in 75 cm2 flasks with DMEM (high glucose) supplemented with 10% FBS containing 100 units/ml penicillin and 100 μg/ml streptomycin, and maintained in a 5% CO2 incubator at 37°C. For subculture, cells were removed from the culture flask with a scraper, resuspended in the culture medium and subcultured in 12, 24, or 96-well plates for experiments.
Assessing cell viability
Two assay protocols were used to assess neuron viability after exposure to Hon and Mag. Since mitochondrial dysfunction is an initial step in apoptotic pathways that lead to neuronal cell death, the MTT assay was used to determine mitochondrial dysfunction. In this assay, neurons were cultured in 24-well plates and treated with Hon or Mag (1 to 10 μM) at 37°C for 24 h. After treatment, culture medium was removed and 1 ml of MTT reagent (0.5 mg/ml) dissolved in serum-free DMEM was added to each well. The plates were incubated for 3 h at 37°C, and the formazan particles in each well were dissolved in 500 μl of DMSO. After shaking the plates at room temperature for 5 min, absorbance was read at 540 nm using a Synergy4 Plate Reader (BioTek Instruments, Inc., Fisher Scientific, St. Louis, MO, USA.).
AlamarBlue™ is a cell-permeable nonfluorescent dye, which can be converted into red fluorescence on reductive reactions within live cells. This assay was used to determine the extent of neuronal viability after exposure to Hon and Mag (1 to 10 μM). In this assay, neurons were cultured in 24-well plates and then treated with Hon or Mag (1 to 10 μM) at 37°C for 24 h. After treatments, 100 μl of AlamarBlueTM was added to each well and neurons were further incubated at 37°C for 3 h. Absorbance was read at 570 nm using the Synergy4 Plate Reader (BioTek) with measurement at 600 nm as a reference.
Measurement of superoxide in neurons and ROS production in microglial cells
Dihydroethidium (DHE) is oxidized by superoxide anions to produce fluorescent ethidium, which is intercalated into DNA, and can be quantified by summing the fluorescence within the cell . This protocol has been successfully used to measure superoxide production in neurons [16, 21]. Neurons were cultured on 35 mm dishes precoated with poly-L-lysine. Neurons were then treated with Hon or Mag (10 μM) for 30 min, and exposed to NMDA (100 μM) for 30 min in phenol red free Neurobasal medium with 0.5 mg/ml BSA. At 30 min prior to image acquisition, cells were loaded with DHE (10 μM) and incubated at 37°C. Fluorescence images were acquired using a Nikon TE-2000 U inverted microscope with a 20× NA 0.95 objective and a cooled charge-coupled device (CCD) camera controlled with a computer running MetaView imaging software (Universal Imaging, West Chester, PA). The fluorescence excitation source was controlled with a Uni-Blitz mechanical shutter. For image acquisition, a short exposure time (200 ms) and low-intensity excitation light were applied to minimize photobleaching. Digital images were analyzed using the MetaView software with automatic background subtraction. Threshold fluorescence levels were obtained for each image prior to the quantification. For each field, the total fluorescence was measured and expressed as the average fluorescence normalized by the total number of cells. For each treatment group, at least three random images from the same dish were captured and analyzed, and each treatment was repeated three times independently for statistical analysis.
For measurement of ROS production in microglial cells, we adopted a protocol using CM-H2DCF-DA, a compound that becomes fluorescent upon interacting with ROS including H2O2. In this study, microglial cells were seeded in 96-well plates, and when they became 90% confluent, they were serum-starved for 4 h. Cells were treated with cytokines or LPS, or both, for different times and CM-H2DCF-DA (10 μM) was added 1 h before measurement. In some experiments, cells were pretreated with U0126 or Hon or Mag for 1 h prior to stimulating with IFNγ for 11 h, the CM-H2DCF-DA (10 μM) was then added and the cells were further incubated for 1 h. The fluorescent intensity of DCF was measured using the Synergy4 microplate reader with an excitation wavelength of 490 nm and an emission wavelength of 520 nm.
Measurement of NO
Cells were serum-starved in phenol red free DMEM for 3 h, followed by pretreatment of compounds of interest for 1 h. Cells were then treated with IFNγ and LPS or IFNγ alone and incubated at 37°C for 16 h. Nitric oxide released from cells was converted to nitrite in the culture medium, which was determined using the Griess reagent protocol. Aliquots (50 μl) of culture medium were transferred to a 96-well plate and incubated with 50 μl of Reagent A (1% sulfanilamide in 5% phosphoric acid) per well for 10 min at room temperature, covered and in the dark. This was followed by incubation with 50 μl of reagent B (0.1%, w/v, N-1-napthylethylenediamine dihydrochloride, Sigma-Aldrich) per well for 10 min at room temperature, covered and in the dark. Serial dilutions of sodium nitrite (0 to 100 μM) were used to generate the nitrite standard reference curve. Following the incubation period, absorbance at 543 nm was measured using the Synergy4 microplate reader.
Western blot analysis
Cells were harvested in lysis radioimmunoprecipitation assay (RIPA) buffer containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS). The extract was centrifuged at 10,000g for 15 min at 4°C to remove cell debris. Protein concentration was determined with the BCA protein assay kit (Pierce Biotechnology, Rockford, IL). For each sample, 5 μg of protein was loaded and resolved in 8% SDS-PAGE and run at 100 V. After electrophoresis, proteins were transferred to 0.45 μm nitrocellulose membranes at 300 mA for 3 h. Membranes were blocked in Tris-buffered saline (TBS), pH 7.4, with 0.1% Tween 20 (TBS-T) containing 5% nonfat milk for 1 h at room temperature. For different experiments, the blots were incubated with ERK1/2 (1:2000), phospho-ERK1/2 (1:2000), iNOS polyclonal (1:1000) antibodies overnight at 4°C. After repeated washing with TBS-T, blots were incubated with goat anti-rabbit IgG- horseradish peroxidase (1:4000) or goat anti-mouse IgG- horseradish peroxidase (1:2000) for 1 h at room temperature. The blots were then washed three times with TBS-T. Immunolabeling was detected by chemiluminescence ECL/WestPico/femto. For loading control, blots were incubated with monoclonal anti-β-actin peroxidase (1:30,000). For quantification, blots were scanned and the intensity of protein bands was measured as optical density using the QuantityOne program (BioRad, Hercules, CA).
Immunostaining of p47phox and gp91phox in microglial cells
BV-2 microglia cells were serum-starved for 4 h, followed by stimulation with IFNγ (10 ng/ml) for 12 h. The cells were fixed in 4% paraformaldehyde for 15 min and then permeabilized with 0.1 % Triton X-100 in PBS for 30 min. Cells were incubated with 10% normal goat serum in 0.005% Triton X-100 in PBS for 60 min. Cells were then incubated overnight in 0.5% normal goat serum in 0.005% Triton X-100 in PBS containing primary antibodies: rabbit anti-p47phox antibodies (1:500; Calbiochem) and mouse anti-gp91phox antibodies (1:500; Thermo Fisher). The next day, cells were incubated in 0.005% Triton X-100 in PBS containing secondary antibodies, goat-anti-rabbit Alexa fluor 488 (Jackson Immunoresearch) and goat-anti-mouse Alexa fluor 549 (Jackson Immunoresearch) for 60 min, followed by nuclear counterstaining in PBS containing 1 μg/ml of 4,6-diamidine-2-phenylindole dihydrochloride (Pierce) for 10 min. The coverslips were then mounted on fluoromount (Sigma-Aldrich) and sealed with nail polish. Fluorescence photomicrographs were captured using a Leica DMI 6000B fully automated epifluorescence microscope (Leica Microsystems Inc., Buffalo Grove, IL) as serial z-stack images and processed for deconvolution with AF6000 applications.
Data are presented as mean ± standard error of the mean (SEM). Results were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison tests or two-way ANOVA with Bonferroni posttests (V4.00; GraphPad Prism Software Inc., San Diego, CA). Statistical significance was considered for P < 0.05.