Reagents and materials
Modified Eagle’s medium (MEM) was purchased from Gibco (Grand Island, NY). Fetal bovine serum (FBS) was obtained from ScienCell (Carlsbad, CA). Compound 48/80 (C48/80), p-nitrophenyl-N-acetyl-β-D-glucosami- nide and poly-D-lysine hydrobromide (PDL) were purchased from Sigma Aldrich (Saint Louis, MO). Triton X-100 was obtained from Amresco (Solon, OH). Penicillin and streptomycin were purchased from Beyotime Biotechnology (Shanghai, China). Reference substances were purchased from Duan Li Bio-technology (Nanjing, China). Fluo-4 AM was purchased from Beyotime Biotechnology. ELISA kits for histamine and TNF-α were purchased from Jiangsu Meimian Industrial Co., Ltd (Yancheng, China). The Annexin V-FITC fluos staining kit was purchased from KeyGEN BioTECH (Nanjing, China).
Animals
ICR male mice (18–22 g) were obtained from Yangzhou University (Yangzhou, China). Animals were housed under a 12-h light–dark cycle at 22 °C and relative humidity of 55 ± 5%, and had free access to food and water. Protocols involving animals were carried out according to the guidelines set by the Institutional Animal Care and Use Committee of Jiangsu Province Academy (Jiangsu, China).
Cells
RBL-2H3 cells were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in MEM supplemented with 15% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin and 0.11 g/L sodium pyruvate in a humidified atmosphere of 5% CO2 at 37 °C.
Isolation of MPMCs
Adult male mice were killed. A total of 10 mL ice-cold phosphate-buffered saline (PBS) was used to perform two sequential peritoneal lavages, which were combined and centrifuged at 800 g for 10 min at 4 °C. The supernatant was decanted to leave a small mast cell pellet (≈ 2 mL), and 2 mL of 30% Percoll and 80% Percoll were added successively to form an interface (with a final volume ratio: 1:1:1). After centrifugation at 600 g for 15 min at 4 °C, cells at the junction interface were collected and washed with PBS twice (200 g, 10 min, 4 °C). MPMCs were resuspended in DMEM with 10% FBS. Purity and viability were determined by toluidine blue staining and trypan blue exclusion.
Preparation of LE
Licorice was ultrasonically extracted for 30 min, using 70% ethanol as the solvate. The filtrate was evaporated under reduced pressure at 50 °C, and the residue was suspended in H2O and freeze-dried to obtain LE.
RBL-2H3 cell extraction
LE was dissolved in MEM without FBS and filtered through a membrane (pore size, 0.22 μm) to create the sample solution. After incubation of RBL-2H3 cells with the filtrate at 37 °C and 5% CO2 for 1 h, the supernatant was discarded. The deposited cells were washed six times with 3 mL of PBS each time. The eluates were discarded except the last one, which was collected and used as a control for UPLC-DAD-Q-TOF-MS/MS analysis. Then, the deposited cells were denatured and extracted with 3 mL of 80% methanol. After centrifugation at 9000 g for 10 min, the supernatant was collected and dried. The residue was dissolved in methanol again and filtered through a membrane (pore size, 0.45 μm) for UPLC-DAD-Q-TOF-MS/MS analyses [11]. Cells incubated with MEM without FBS were prepared as the control sample using the same procedures described above.
UPLC-DAD-Q-TOF-MS/MS analysis for identification of components combined with RBL-2H3 cells
UPLC-DAD-Q-TOF-MS/MS analysis was carried out using 1290 Infinity UPLC instrument (Agilent Technologies, Santa Clara, CA) coupled with a 6538 Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA) equipped with a dual electrospray ionization (ESI) source. The mobile phase consisted of water-0.05% formic acid (A) and acetonitrile (B). Gradient elution conditions were as follows: 0–2 min, 10%–25% B; 2–4 min, 25%–35% B; 4–14 min, 35%–70% B; 14-22 min, 70%-82%; 22-24 min, 82%-95%, followed by a return to the initial condition. The flow rate was 0.3 mL/min. Chromatographic separation was carried out at 30 °C on an Ultimate UHPLC LP-C18 (1.8 μm, 2.1×100 mm). Samples were detected at 237 nm. Parameters used for MS detection were optimized as follows: drying gas temperature, 350 °C; flow rate of drying gas (N2), 8.0 L/min; fragmentor voltage, 100 V; nebulizer, 40 psi; capillary, 3500 V; skimmer, 65 V; Oct RFV, 750 V. The sample collision energy was set at 35 V. All data acquisitions and analyses were controlled by Mass Hunter software (Agilent Technologies). Mass spectra were recorded in the range m/z 100–2000 with accurate mass measurement of all peaks. Each sample was analyzed in negative mode.
Measurement of cell viability
Cell viability was determined by the MTT assay. RBL-2H3 cells were seeded in a 96-well plate at 1 × 104 cells/well. After 24 h of incubation at 37 °C in an atmosphere of 5% CO2, cells were treated with different concentrations of test compounds for 30 min. Then, the supernatant was discarded and cells were incubated with MEM containing 0.5 mg/mL MTT at 37 °C and 5% CO2. After 3 h, the medium was removed and 150 μL dimethyl sulfoxide added to each well. Absorbance was measured at 570 and 650 nm.
MPMCs were seeded in a 96-well plate at 3 × 104 cells/well. After 12 h of incubation at 37 °C in an atmosphere of 5% CO2, cells were treated with different concentrations of test compounds for 30 min. Then the method was the same as above.
Measurement of β-hexosaminidase release
β-Hexosaminidase release from RBL-2H3 cells was examined as described previously with some modifications [12].
RBL-2H3 cells (1×104 cells/well) or MPMCs (3×104 cells/well) were cultured for 24 h in a 96-well plate, washed with MEM/ DMEM without FBS, and incubated with MEM/DMEM containing different concentrations of the sample solutions for 30 min at 37 °C in an atmosphere of 5% CO2. Cells in the blank control and positive control were treated with MEM/DMEM alone under the same incubation conditions. Then, the cells in sample solutions and positive control were stimulated with C48/80 (30 μg/mL) for 30 min at 37 °C in an atmosphere of 5% CO2. Cells in the blank control were incubated with MEM/DMEM without FBS.
β-Hexosaminidase released into the supernatant and cell lysate was quantified by hydrolysis of p-nitrophenyl-N-acetyl-β-D-glucosamide in 0.1 M sodium citrate buffer (pH 4.5) for 60 min at 37 °C. The absorbance of each well was measured at 405 nm. Percentage of release of β-hexosaminidase was calculated as a percentage of the total content, using the following formula:
Measurement of histamine and TNF-α release
RBL-2H3 cells (5×104 cells /well) were cultured for 24 h in a 24-well plate, and washed with MEM without FBS. The administration method was the same as used for the measurement of β-hexosaminidase release. The supernatants were collected and centrifuged at 845 g for 10 min. Histamine content in the supernatant was determined using an ELISA histamine kit according to the manufacturer’s instructions.
Mice were randomly divided into eight groups (n = 8), including the blank control group, positive control C48/80 group and treatment groups. The treatment groups were orally administered different doses of the target components. The blank control group and positive control group were administered saline. After 30 min, the positive control group and treatment groups were challenged via an intravenous injection with C48/80 (2.5 mg/kg). The blank control group was intravenously injected with saline. Blood was collected after 10 min, and serum was obtained through centrifugation (825 g, 10 min, room temperature). Histamine content and TNF-α in the serum were determined using ELISA histamine kits according to the manufacturer's instructions.
Fluorescence microscopy
According to the instructions provided with the Annexin V-FITC detection kit, RBL-2H3 cells were incubated with different sample solutions for 30 min and treated with C48/80 (30 μg/mL). After 30 min, the cells were washed twice with cold PBS and then binding buffer containing Annexin V-FITC (5 μL) was added. After 5 min in the dark, cells were examined and photographed at 630× magnification under a laser scanning confocal microscope (Zeiss LSM700, Jena, Germany).
Measurement of intracellular Ca2+ concentration ([Ca2+]i)
[Ca2+]i was measured using the fluorescence indicator Fluo-4 AM. RBL-2H3 cells were plated onto a 96-well plate coated with 50 μg/mL PDL and balanced at 37 °C in an atmosphere of 5% CO2. After 24 h, the cells were treated with different sample solutions for 30 min, and loaded with Fluo-4 AM for 40 min at 37 °C in an atmosphere of 5% CO2. Then the cells were washed five times with Locke’s buffer (pH 7.4, containing 2.144 g/L HEPES, 0.417 g/L potassium chloride, 9 g/L sodium chloride, 1.009 g/L glucose, 0.096 g/L magnesium chloride, 0.256 g/L calcium chloride and 500 μL 0.1 mM glycine), to leave a final volume of 150 μL in each well. Then, the plate was transferred to Varioskan Multifunctional microplate reader 3001 (Thermofisher Scientific, Germany). Cells were excited at 488 nm, and emission detected at 520 nm. Fluorescence was continuously scanned for 1 min to establish the baseline. Then, 50 μL of C48/80 (120 μg/mL,4×) was added to the positive well and different sample wells, respectively, yielding a final volume of 200 μL/ well. Locke’s buffer was added to the control well. Fluorescence was continuously scanned for 5 min.
Molecular docking
To investigate the interactions between receptors and ligands, molecular docking studies were conducted using Auto Dock 4.2 (The Scripps Research Institute, USA). The docking model of MRGPRX2 used in this study was based on the homology model of MRGPRX2 reported by Dr. Bryan L Roth and Protein Data Bank [13].
Statistical analyses
All analyses were performed using GraphPad Prism v5.01 (GraphPad Software, La Jolla, CA). Data are presented as the mean ± SEM. One-way analysis of variance followed by Dunnett's test was used for multiple comparisons. p < 0.05 was considered significant. Each experiment was repeated at least three times.