Animals
All procedures were performed under protocols approved by the Animal Care and Use Committee of Nanjing University of Chinese Medicine (Approval no. A190401). Adult male mice used were 22–26 g males in a C57BL/6 background (Qinglongshan, China). Animals were housed at constant humidity (40–60%) and temperature (22 ± 2℃) on a 12 h light/dark cycle and allowed free access to food and water. C-kit mutant genetically mast cell-deficient Kit (W-sh) "sash" mice were donated by Johns Hopkins.
Chemicals
ATP (Sigma-Aldrich, St. Louis, MO, United States), PPADS (10 µM, Tocris Bioscience, Missouri, USA, a non-selective P2 purinergic receptor antagonist), NF449 (1 µM, Cayman, Ann Arbor, Michigan, USA, P2 × 1 receptor antagonist), 5-BDBD (1 µM, Sigma-Aldrich, St. Louis, MO, United States, P2 × 4 receptor antagonist), AZ10606120 (1 µM, Tocris Bioscience, Missouri, USA, P2 × 7 receptor antagonist), recombinant mouse SCF protein (10 ng/mL, R&D Systems, Minneapolis, MN, USA), Penicillin and streptomycin (100 µg/mL; Gibco, Waltham, MA, USA), fibronectin (30 µg/mL; Sigma-Aldrich, St. Louis, MO, United States), Fluo 4-AM (Solarbio, Beijing, China, calcium indicator), Histamine ELISA Kit (Yifeixue, Nanjing, China), Trizol (Vazyme Biotech, Nanjing, China), HiScript II Q RT SuperMix for qPCR (Vazyme Biotech, Nanjing, China), Taq MasterMix (Vazyme Biotech, Nanjing, China), AceQ qPCR SYBR Green Master Mix (Vazyme Biotech, Nanjing, China), salicylic acid (300 µM, Yuanye Biotech, China), aspirin (1 Mm, Tocris Bioscience, Missouri, USA).
P815 cell culture and mouse peritoneal mast cell purification
Mouse Mastocytoma Cells (P815) was cultured in 1640 complete medium (90% 1640 medium, 10% fetal bovine serum, 100 µg/ml penicillin and 100 µg/ml streptomycin). Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2 incubator.
Mouse peritoneal mast cells were established from C57BL/6 mice as Xinzhong Dong described [23]. Briefly, the mouse peritoneal cells were collected with mast cell dissociation media MCDM (HBSS with 10 mM HEPES and 3% fetal bovine serum, pH 7.2), and then centrifuged at 200 g for 5 min. The pellet was resuspended and layered over 70% percoll suspension, and then centrifuged at 500 g for 20 min. Pipetted off supernatant carefully and the mast cells were washed with fresh MCDM. Mast cells were resuspended in DMEM containing 10% fetal bovine serum and 10 ng/mL recombinant mouse stem cell factor.
P2X purinoceptors RT-PCR screen
Trizol method was employed to isolate total RNA from mouse peritoneal cells. 10–500 ng RNA was used for RT reactions by using HiScript II Q RT SuperMix for qPCR Kit according to the manufacturer’s instructions, and then PCR screen explored by Taq MasterMix. 10 µL of the PCR reaction product was used for agarose gel electrophoresis and stained with Gold View and then observed by ChemiDoc MP (Bio-rad, California, USA). All of the PCR primers were synthesized by Genescipt Biotechnology (Nanjing, China). The primer sequence and product size are shown in Table 1.
Table 1
Gene | Primer sequence (5’-3’) | Product size |
P2 × 1 | Forward: GCCCAAGGTATTCGCACAGG | 496 bp |
Reverse: GACGACGGTTTGTCCCATTCT |
P2 × 2 | Forward: ACCTGCCATTTAGATGACGACTG | 241 bp |
Reverse: TGTTGCCCTTGGAGAACTTGA |
P2 × 3 | Forward: GCTTCGGACGCTATGCCAACA | 490 bp |
Reverse: AAATCCTGCCCAGCAAACTTAA |
P2 × 4 | Forward: GTGCTCGGGTCCTTCCTGTTC | 154 bp |
Reverse: CCGTTTCCTGGTAGCCCTTTT |
P2 × 5 | Forward: TGTAGCGGGACACGGACTGA | 209 bp |
Reverse: TTTCTAGCACATTGGCTTTGGA |
P2 × 6 | Forward: GGTACAACTTCAGGACAGCCAATC | 207 bp |
Reverse: CATACAGTAGCAGCAGGTCACAGAG |
P2 × 7 | Forward: AACATCTTGCCAACTATGAACGG | 132 bp |
Reverse: TCCTCCCTGAACTGCCACCT |
Intracellular calcium measurement
The mouse peritoneal mast cells were isolated and plated on the glass cover slips, which coated by 30 µg/mL fibronectin for two hours of incubation at 37 °C, 5% CO2. After that, mast cells were incubated by 1µL/mL Fluo-4 calcium ion indicator along with 0.02% Pluronic F-127 for 30 minutes at room temperature. Then cells immediately washed for 3 times by calcium imaging buffer ( 125 mM NaCl, 3 mM KCl, 2.5 mM CaCl2, 0.6 mM MgCl2, 20 mM glucose, 10 mM HEPES, 20 mM sucrose, 1.2 mM NaHCO3, pH 7.4). Finally, cells were used for imaging within two hours.
Electrophysiology
The whole cell recording of the patch-clamp technique was used. Patch pipettes typically had a resistance of 6–8 MΩ. Osmolality was adjusted to 300–310 mOsM (adjusted by sucrose as necessary). The standard pipette solution contained 135 mM CsCl, 8 mM NaCl, 10 mM EGTA, 3.6 mM CaCl2, 10 mM HEPES, PH 7.3 (adjusted by CsOH). The standard external solution contained 147 mM NaCl, 2 mM KCl, 2 mM CaCl2, 1 mM MgCl2.6H2O, 10 mM HEPES, 16 mM glucose, pH 7.3 (adjusted by NaOH). In addition, low divalent external solution contained 147 mM NaCl, 10 mM HEPES, 13 mM glucose, 0.2 mM CaCl2, 2 mM KCl, pH 7.3 (adjusted by NaOH).
All the experiments were performed at room temperature. Whole-cell currents were record by using Multiclamp 700 B and Digidata 1440 A (Molecular Devices, Inc., San Jose, USA), capacitance transients and series resistance were minimized by using the capacitance neutralization circuits on the amplifier. Experiments were performed with a perfusion system, and drugs were directly added to the recording chamber with a pipette. The cells were usually evoked by holding the membrane potential, and applied voltage commands to a range of potentials with 10 mV steps from − 130 mV to + 130 mV in 100 ms. In addition, currents were evoked by ramping the membrane potential from − 90 mV to + 100 mV in 300 ms. The currents were digitized (sampled at a frequency of 10 kHz and filtered at 0.1 kHz for analysis), stored and subsequently analyzed by using Clampex 10.3 (Molecular Devices, Inc., San Jose, USA).
Quantitative Real-time PCR
Total RNA from Mouse Mastocytoma Cells (about 105-106 cells) stimulated by different concentration of ATP for 4 hours, tissues (10–50 mg) isolated from paw treated by 100 mM ATP or saline were prepared by using Trizol reagent. cDNA was generated by HiScript II Q RT SuperMix for qPCR Kit according to the manufacturer’s instructions. Real-time qPCR was performed using AceQ qPCR SYBR Green Master Mix and GAPDH were used as internal controls. The primer sequences are shown in Table 2.
Table 2
Gene | Primer sequence (5’-3’) |
IL-6 | Forward: GTTGCCTTCTTGGGACTGAT |
Reverse: CTGGCTTTGTCTTTCTTGTTAT |
IL-1β | Forward: AAATCTCGCAGCAGCACATC |
Reverse: AGCAGGTTATCATCATCATCCC |
CCL2 | Forward: GGCCTGCTGTTCACAGTTGC |
Reverse: CAGAAGTGCTTGAGGTGGTTG |
CCL3 | Forward: GGCCTGCTGTTCACAGTTGC |
Reverse: CAGGCATTCAGTTCCAGGTCAG |
GAPDH | Forward: GCACAGTCAAGGCCGAGAAT |
Reverse: GCCTTCTCCATGGTGGTGAA |
Histamine ELISA
Histamine ELISA prepared according to the manufacturer’s protocol. Briefly, mouse peritoneal mast cells were stimulated with different concentration of ATP (1 µM, 100 µM, 1 mM and 5 mM), and the supernatants were harvested at time point of 0.5 h and stored at 80℃ until used for ELISA.
Behavioral assays
As references, the Von Frey behavioral assays were performed in a blinded manner. In briefly, different groups of mice were put in a transparent plastic box, which was placed on a metal mesh for about 30 min. Then the value of threshold was measured with a time interval of 1 h, 3 h, 5 h after 100 mM ATP treatment. Each mouse was tested by more than 10 times at a specific force manually. At last, we determined the threshold by the lowest force, which elicited responses with more than 50% of the time.
Histological
The paw skin was isolated and washed with PBS, then fixed with 4% paraformaldehyde for 24 h and 30% sucrose for 48 h. The tissue was embedded in OCT and sliced to a thickness of 10 microns, followed by hematoxylin–eosin (HE) staining and toluidine blue staining. Images of each section were obtained by using the Nikon ECLIPSE 80i microscope (Nikon, Tokyo, Japan) with a magnification of 200.
Quantification and statistical analysis
The data were analyzed by GraphPad 6.0 and presented as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05, ##p < 0.01, ###p < 0.001 was considered statistically significant. Statistical analysis of the results was performed by two-tailed, unpaired Student’s T-test or ANOVA analysis.