P2X7 on mast cells participates in peripheral pain and serves as a potential target for salicylic acid and aspirin analgesia

Background Extracellular ATP signaling through excitatory and calcium-permeable P2X receptor channels is considered as a critical player in pain generation and maintenance. P2X7 has attracted much attention over the past decade because of its prominent role in driving in�ammatory processes. The role of P2X7 in mast cells in peripheral pain remains unclear. Methods P2X expression in mouse peritoneal mast cells was detected by RT-PCR. The subtypes of P2X receptors in mouse peritoneal mast cells were determined with a series of blockers by using calcium imaging and electrophysiology. The regulation of in�ammatory factors mediated by different P2X subtypes were detected by ELISA and real-time PCR. The role of mast cells and P2X7 receptor in peripheral pain was explored by behavioral assays, pathological analysis and real-time PCR. Several anti-inammatory small molecules were screened based on P2X7 in mast cells by using calcium imaging, electrophysiology and molecular docking. Results We found that ATP was signi�cantly increased in in�ammatory pain. Mouse peritoneal mast cells expressed P2X1, P2X3, P2X4 and P2X7 and could be activated by different concentrations of extracellular ATP, which could be blocked by speci�c ion channel antagonists. In particularly, high concentration of ATP could induce mast cells to release in�ammatory mediators such as histamine, IL-1β and CCL3 through P2X7 receptor. Furthermore, peripheral pain induced by high concentration of ATP could be alleviated by P2X7 blockers or mast cell defects. Interestingly, salicylic acid and aspirin could attenuate the inward current, the release of in�ammatory factors and peripheral pain induced by ATP with high concentration. Furthermore, salicylic acid and aspirin also inhibited the inward current evoked by P2X7 agonist BZATP. Molecular docking results showed that salicylic acid and aspirin had a�nity to the cytoplasmic GDP-binding region of P2X7. Conclusions We concluded that P2X7 on mast cells involved in peripheral pain. Salicylic acid and aspirin could inhibit the activity of


Background
Extracellular ATP is a "signal of danger" mediated by P2 purinergic receptors.P2 receptors have two types, P2X (P2X1-7) and P2Y receptors [1].P2X receptors carry out many important functions in the central and peripheral nervous system [2].Compelling evidences have shown that P2X3, P2X4 and P2X7 receptors are involved in the pathogenesis of chronic pain [3].P2X7 exists in neurons and glial cells of nervous, but is mainly expressed in cells of immune origin [4].The absence of P2X7 receptor can completely eliminate the in ammatory and neuropathic hypersensitivity to both mechanical and temperature stimulation [5,6].P2X7 receptor of microglia also plays an important role in chronic neuropathy and in ammatory pain by releasing IL-1β [7].Besides IL-1β, cytokines such as IL-6, CCL2, TNFα and CCL3 are also mediated by P2X7 receptor in neutrophil and monocyte [8][9][10][11][12].Hence, P2X7 receptor represents a promising target for pain intervention.
Mast cells are in close proximity to afferents and establishing dynamic interactions with pain-activating nociceptors [13].Performed and newly synthesized mediators released from mast cells could contribute to pain via the nervous system or other immune cells [14].Therefore, mast cells play important roles in the pathological process of pain [15].The classic pathway of mast cell activation is mediated by IgE receptor (FcεRI).Mast cells are also activated by a wide variety of triggers, such as substance P, lipid mediators and interleukins [16].Extracellular ATP also can stimulate mast cells and induce different inward currents through P2X receptors [17].Although activation of P2X4 can augment the degranulation mediated by the FcεRI in mouse bone marrow-derived mast cells, only P2X7 contributes to degranulation in human mast cell line LAD2 [18,19].As yet, the relationship between P2X7 receptor on mast cell and pain remains unclear.
Chronic pain is a global problem affecting more than two-thirds of the population.Extensive research has been conducted to nd appropriate methods of relieving pain and improving the quality of life.Salicylic acid, an extract of willow bark, has been used for pain management as early as 4,000 years ago.Aspirin, derived from salicylic acid, is a common peripheral analgesic drug via inhibiting cyclooxygenase-1 (COX-1).The analgesic effect of salicylates is apparently, however, the targets are still not entirely clear.
Previous literatures indicate that salicylates are associated with mast cells.Salicylic acid can signi cantly inhibit histamine release from rat peritoneal mast cells activated by compound 48/80 or anti-DNP IgE [20].The relationship between aspirin and mast cells remains controversial.There are some evidences show that the anaphylaxis triggered by aspirin is due to eosinophils and mast cells [21].High concentrations of aspirin such as 10 mM can activate mast cells directly [22], but the effect of normal concentrations of aspirin on mast cells remains unclear.
In this study, we evaluated the roles of several P2X receptor subtypes on mast cells, especially the P2X7 receptor.In addition, we also explored the relationship between salicylates and P2X7 channel.
Mouse peritoneal mast cells were established from C57BL/6 mice as Dong's Lab described [23].Brie y, 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.As supplementary Fig. S1 shown, the extracted mouse peritoneal mast cell was identi ed by toluidine blue staining, and the purity was about 90%.

P2X purinoceptors RT-PCR screen
Trizol method was used to isolate total RNA from mouse peritoneal mast cells or the mouse mastocytoma cells.10-500 ng RNA was used for reverse transcription reaction by using HiScript II Q RT SuperMix for qPCR Kit according to the manufacturer's instructions.Polymerase chain reaction conditions were as follows: 95°C for 5 min, 40 cycles of 15 s at 95°C, 30 s at 60°C, and 1 min at 72°C, and 10 min at 72°C. 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.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 ampli er.
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 ltered 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 10 5 -10 6 cells) stimulated by different concentration of ATP for 4 or 0.5 hours (5 mM ATP), tissues (10-50 mg) isolated from paw treated with 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. Histamine ELISA prepared according to the manufacturer's protocol.Brie y, mouse peritoneal mast cells were stimulated with different concentration of ATP (1 µM, 100 µM, 1 mM and 5mM), and the supernatants were harvested at time point of 0.5 h and stored at 80℃ until used for ELISA.

Behavioral assays
The Von Frey behavioral assays were performed in a blinded manner.In brie y, different groups of mice were put in a transparent plastic box, which was placed on a metal mesh for about 30min.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 at least 10 times at a speci c force (0.16g) manually.Histological The paw skin was isolated and washed, and then xed with 4% paraformaldehyde for 24 h, and then treated with 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 magni cation of 200.

Molecular docking
The discovery Studio 2016 4.0 software was used to verify the molecular docking.We downloaded the three-dimensional structure of salicylic acid (PubChem CID: 338) and aspirin (PubChem CID: 2244) from the NCBI PubChem Compound database (http://www.ncbi.nlm.nih.gov/pccompound) and the monomeric crystal structure of P2X7 (PDB ID: 6U9V) from the Research Collaboratory for Structure Bioinformatics (RCSB) Protein Data Bank (http://www.rcsb.org/pdb).

P2X receptors expression and ATP-induced calcium response in mouse peritoneal mast cells
Upon for instance tissue stress, hypoxia or in ammation, ATP is released from cells into the extracellular environment leading to the increase in extracellular ATP.Our results also showed that ATP was signi cantly increased in in ammatory pain such as CFA (0.4913 ± 0.04 µmol/g vs 1.116 ± 0.09 µmol/g, *** p < 0.001, Supplementary Fig. S2).Because mast cells play important roles in the pathological process of pain, we speculate that P2X receptors on mast cells involved in this function.
First, to identify P2X receptors on mast cell, we explored the P2X expression in mouse peritoneal mast cells by RT-PCR screen.As shown in Fig. 1A, we found that mouse peritoneal mast cells expressed several different ionotropic P2X receptors including P2X1, P2X3, P2X4 and P2X7.P2X receptor is a non-selective cation channel, the most obvious permeability to Ca 2+ .Hence, we examined the calcium in ux in mouse peritoneal mast cells induced by ATP.The results showed that there were transient increases of intracellular calcium in mast cells treated by different concentrations of ATP (Fig. 1B, C).The ratios of uorescence intensity were varied with different ATP concentrations, and EC50 was about 6.5 µM (Fig. 1C).In addition to uorescence intensity, the reaction durations also existed differences (Fig. 1B), which indicated that different concentrations of ATP could activate mast cells through different P2X ionotropic receptors.
To con rm that, we used special P2X channel antagonists.As shown in Fig. 1D-H, the calcium in ux caused by different concentrations of ATP could be partially blocked by a non-selective P2 purinergic receptor antagonist PPADS (20 µM, pre-incubation for 5 minutes).In addition, calcium in ux caused by 1 µM ATP was inhibited by P2X1 receptor antagonist NF449 (1 µM, pre-incubation for 5 minutes) (Fig. 1D).AF-353 (P2X3 receptor antagonist, 0.1 µM, pre-incubation for 5 minutes) could reduce the calcium in ux caused by 10 µM ATP (Fig. 1E).And the transient increase of the intracellular calcium induced by 100 µM ATP was blocked by 5-BDBD (1 µM, pre-incubation for 5 minutes) (Fig. 1F), which is a speci c P2X4 receptor antagonist.Furthermore, the speci c P2X7 receptor antagonist AZ10606120 (1 µM, preincubation for 5 minutes) had the ability of reducing the calcium in ux caused by high concentration ATP such as 1 mM and 5 mM (Fig. 1G, H).These results indicated that P2X1, P2X3, P2X4 and P2X7 might contribute to the activation of mouse peritoneal mast cells.

Electrophysiological characteristics of mouse peritoneal mast cells induced by extracellular ATP
According to previously published literature, human mast cells are sensitive to ATP in a concentrationdependent manner [17].Our experimental results proved that mouse peritoneal mast cells had the same characteristics.About 85% of mast cells were sensitive to 1 µM ATP (Fig. 2A, n = 17).The inward current could also be induced by 100 µM ATP (Fig. 2B, n = 21).When we increased the concentration of extracellular ATP to a high level, we found that both 1mM and 5 mM ATP had the ability to induce the inward currents repeatedly (Fig. 2C, n = 9; Fig. 2D, n = 8).As Fig. 2E, F shown, the current characteristics evoked by various concentrations of extracellular ATP were different from each other, including the amplitude of the inward current and the current durations.Although the current amplitude as well as the duration were different between 1mM ATP and 5 mM ATP, the inward currents had some similar characteristics such as "run-up" tendency (Fig. 2G, H).The current growth rate of second ATP application had no difference as Fig. 2I shown.The current growth rate is de ned as.Furthermore, the inward current evoked by 1mM ATP was voltage-dependent (Fig. 2J-L).The activate curves and inactivate curves induced by 5 mM ATP were shown in Fig. 2M.According these curves, the conductance curve was calculated as shown in Fig. 2N, which indicated that the current induced by 5 mM ATP had characteristics with faster activation and slower inactivation.
Inward currents induced by extracellular ATP with different concentrations could be blocked by P2X1, P2X3, P2X4 and P2X7 antagonist Electrophysiological results showed that there were different currents corresponding to ATP with different concentrations.Therefore, we speculated that several P2X receptor subtypes contributed to the activation progress.As Wareham described, P2X1,P2X4 and P2X7 receptors in LAD2 were activated by 1 µM ATP, 100 µM ATP and high concentrations of ATP, respectively [17].In our study, we also found that 1 µM ATP hardly induced inward current when 20 µM PPADS (a non-selective P2 antagonist) (Fig. 3A, G, n = 9) or 1µM NF449 (the blocker of P2X1 receptor) (Fig. 3A, G, n = 13) was applied, which indicated that P2X1 was activated by 1 µM ATP.As Fig. 3B, H shown, 20 µM PPADS (n = 9) or 0.1 µM AF-353 (the blocker of P2X3 receptor, n = 10) could reduce the current evoked by 10 µM ATP, which indicated that P2X3 was activated by 10 µM ATP.In addition, P2X4 was involved in the current induced by 100 µM ATP, which was blocked by 1 µM 5-BDBD (Fig. 3C, I, n = 10) or 20 µM PPADS (Fig. 3C, I, n = 13).The current caused by 1mM ATP were inhibited by using 1 µM AZ 10606120 (the blocker of P2X7 receptor) (Fig. 3D, J, n = 5).The current induced by 5 mM ATP could also be inhibited by AZ 10606120 (Fig. 3E, K, n = 6).These results demonstrated that P2X7 receptor was activated by high concentrations of ATP such as 1 mM and 5 mM.Intrestingly, we found that 20 µM PPADS could partially inhibit the current induced by 1 mM ATP (Supplementary Fig. S3A, S3C), but had no effect on the current evoked by 5 mM ATP (Supplementary Fig. S3B, S3D), which indicated that PPADS might have limited inhibitory effect on P2X7.Furthermore, P2X7 receptor was sensitive to divalent cation as North described [24].Consequently, our results illustrated that the inward current induced by 5mM ATP in the low divalent cation was greater than that in normal external solution (Fig. 3F, L), which con rmed that the existence of P2X7 in mouse peritoneal mast cells.Taken together, results con rmed that P2X1, P2X3, P2X4 and P2X7 expressed in mouse peritoneal mast cells and involved in the progress activation induced by extracellular ATP.
Activation of P2X7 receptor on mouse-derived mast cell could lead to degranulation and de novo synthesis of cytokines Degranulation is one of important indicators of mast cell activation.Consistent with the research by Wareham et al in human mast cell line LAD2 [18], our results indicated that there is no detectable histamine release at lower concentrations of ATP such as 1 µM ATP and 100 µM ATP.However, histamine released from mouse peritoneal mast cells was signi cantly increased at higher concentrations of ATP (Fig. 4A).Besides histamine, mast cells are effective producers of in ammatory cytokines in response to various stimuli.According to previous literatures, cytokines secretion especially IL-1β induced by the extracellular ATP has been widely studied in different immune cells [8][9][10][11][12].However, the release of cytokines from mast cells induced by extracellular ATP remains unclear.To examine the potential mediator release in mast cells, we detected a serious of mediators such as IL-6, IL-1β, CCL2 and CCL3.Due to the mouse peritoneal mast cells are too few to detect, we turned to mouse mastocytoma cells P815, which also expressed P2X1, P2X3, P2X4 and P2X7 receptors (Supplementary Fig. S4).Data in Fig. 4B-H demonstrated that the regulation of in ammatory mediators was related to the concentration as well.There was no signi cant change or up-regulated slightly in the expression of cytokines when treated with low concentration of ATP (Fig. 4B-D).ATP with high concentrations could signi cantly upregulate the expression of a variety of in ammatory cytokines, such as IL-1β and CCL3 (Fig. 4E, G).In addition, we found that cytokines were regulated slightly after treatment with 5mM ATP for 4 hours, but IL-1β and CCL3 were signi cantly up-regulated after treatment for 0.5 hours, which might be related with the negative feedback caused by high ATP concentration (Fig. 4F, G).AZ10606120, a speci c P2X7 receptor antagonist (5 µM, pre-incubation for 5 minutes), almost completely inhibited the upregulation of IL-1β and CCL3 induced by 1mM ATP (Fig. 4H).Therefore, we concluded that P2X7 receptor on mast cells could mediate mast cell degranulation and de novo synthesis of in ammatory factors such as IL-1β and CCL3.

High concentration of ATP could induce peripheral pain in mice by activating P2X7 channel on mast cells
Our experimental results indicated that P2X7 receptor on mast cell had the function of releasing in ammatory mediators, which might contribute to pain via neuro-immune interactions.We assumed that mast cells and P2X7 receptor promoted the peripheral pain induced by high concentration of ATP.In order to prove this hypothesis, we utilized the mast cell-de cient Kit (W-sh) Sash mutant mice and P2X7 receptor antagonist.Firstly, results showed that high concentration of ATP (100 mM, 20 µL, iH) did induce paw swelling (Fig. 5A, C), in ammatory cells in ltration (Fig. 5B), mast cells degranulation (Fig. 5D) and mechanical hyperalgesia (Fig. 5E).As our expected, mast cell-de cient mice could alleviate ATP-induced pain including paw swelling, mechanical withdrawal threshold and the in ltration of in ammatory cells (Fig. 5A-E).To further explore the mechanism, we also tested the in ammatory mediators.The RT-PCR data showed that the mRNA expression levels of IL-6, IL-1β, CCL2 and CCL3 were signi cantly upregulated for the C57/BL mice.However, only IL-6 and CCL3 were slightly upregulated for the Sash mice.In comparison with the C57/BL mice, the degree of upregulation of IL-1β, CCL2 and CCL3 for the Sash mice was reduced (Fig. 5F).At the same time, we also studied the function of P2X7 receptor in high concentration of ATP-induced peripheral pain.Our experimental results showed that AZ10606120 (speci c P2X7 receptor antagonist, 2 mg/kg, ip, pre-administration 1h) could signi cantly reduce the pain behavior and paw thickness at 4h after ATP treatment (Fig. 5G, H).P2X7 on mast cells is a potential target for salicylic acid and aspirin analgesia P2X7 is an appealing target for anti-in ammatory therapy, so we used P2X7 as an analgesic target to screen several anti-in ammatory substances monomers.We found that Matrine, Higenamine, Dictamnine, Prim-O-glucosylcimifugin, Liquiritin, Menthol, Ferulic Acid, 3-Hydroxy-4-methoxycinnamic acid, Isoginkgetin, Vanillic acid, Luteolin, Isoliquiritigenin or Aloeemodin had no inhibitory effect on the current evoked by high concentration of ATP (Supplementary Table 1).Interestingly, salicylic acid and aspirin could inhibit the inward current generated by high concentration of ATP as Fig. 6A shown.Compared with the current amplitude induced by rst ATP application, 300 µM, 500 µM or 1 mM salicylic acid could slightly inhibit the current amplitude induced by second ATP application (Fig. 6B-D, n = 21, n = 14 and n = 16 respectively).It is worth noting that the current induced by 5 mM ATP had a "run-up" tendency, which indicated that the current growth rate should be studied.Data showed that the current growth rate of second ATP application was signi cantly inhibited by salicylic acid as Fig. 6E shown.500 µM or 1 mM aspirin could also inhibit the current amplitude induced by 5mM ATP (Fig. 6G-H, n = 6, n = 8 and n = 20 respectively).The current growth rate was signi cantly inhibited by 500 µM or 1 mM aspirin (Fig. 6I).The intercellular Ca 2+ concentration assay results also showed that 300 µM salicylic acid or 1 mM aspirin could also inhibit 5mM ATP-induced calcium in ux (Supplementary Fig. S5).At the same time, we also explored the effects of drugs on in ammatory mediators.As our expected, 300 µM salicylic acid or 1 mM aspirin could attenuate the up-regulation of IL-1β and CCL3 mediated by high concentration of ATP, especially IL-1β (Fig. 6J).Results from behavioral test indicated that salicylic acid (50 mg/kg, ig) or aspirin (50 mg/kg, ig) could also alleviate the peripheral pain induced by high concentration of ATP (Fig. 6K).To further clarify the relationship between salicylates and P2X7 receptor, we used the P2X7 receptor agonist BzATP.Results showed that 300 µM salicylic acid (n = 18) or 300 µM aspirin (n = 7) signi cantly inhibited the current growth rate (Fig. 6L) and the calcium in ux (Supplementary Fig. S6) evoked by BzATP.These experimental results suggested that P2X7 on mast cells might be a potential target for salicylic acid and aspirin analgesia.

GDP binding region is the critical for the combination of salicylic acid and aspirin with P2X7
Our experimental results have shown that the analgesic effect of salicylic acid and aspirin may be achieved by inhibiting P2X7 channel.Next, we want to know how salicylic acid and aspirin work in combination with P2X7.To uncover this mystery, molecular docking was used to analyze the interaction between salicylic acid or aspirin and P2X7 by Discovery Studio software.As shown in Fig. 7, salicylic acid (Fig. 7G-L) and its derivative aspirin (Fig. 7A-F) had a nity to the GDP-binding region of P2X7, including A: GDP 703, B: GDP703 and C: GDP704 ligands.Among these, aspirin has the highest a nity with A: GDP 703 ligand as Fig. 7A, B shown.The -COOH of aspirin formed two Electrostatic-bonds with Arg546 (R546) and Arg578 (R578) of A: GDP 703 ligand, and van der Waals-bonds with Ser589 (S589).Consistent with the electrophysiological and the intercellular Ca 2+ concentration assay results, we considered that salicylic acid and aspirin could inhibit P2X7 activation by directly binding the receptor.

Discussion
It is well-known that P2X3 and P2X4 in the nervous system were involved in the pathogenesis of neuropathic pain [25].A more recent player in chronic pain is the P2X7 receptor in immune cells.The high threshold activation gives P2X7 a role in damage-sensing, only triggering downstream effects when ATP concentration is pathologically elevated [26].In neuroin ammatory and neurodegenerative diseases, P2X7 upregulation and function appears to contribute to disease progression [26].P2X7 receptor mediates NLRP3 in ammasome activation, cytokine and chemokine release, T lymphocyte survival and differentiation, transcription factor activation, and cell death [27].P2X7 promotes release of proin ammatory factors, such as IL-1β, IL-6, CCL2, TNFα and CCL3 [8][9][10][11][12].Hence, P2X7 is an appealing target for anti-in ammatory therapy.However, the relationship between P2X7 on mast cells and the pathogenesis of pain is still not illustrated.Our study aimed to explore the roles of P2X receptor subtypes on mast cells in in ammatory pain, especially the P2X7 receptor.At the same time, we also used P2X7 as an analgesic target to screen several anti-in ammatory small molecule compounds.
Firstly, we found that several functional P2X receptors including ionotropic P2X1, P2X3, P2X4 and P2X7 existed in mouse peritoneal mast cells.The expression pro le was similar to that previously observed in mouse bone marrow mast cells, LAD2 and human lung mast cells which express P2X1, P2X4 and P2X7.Furthermore, calcium image results demonstrated that ATP could also activate mouse peritoneal mast cells in a concentration-dependent manner.The blocker results indicated that P2X1, P2X3, P2X4 and P2X7 were involved in the calcium in ux caused by 1 µM ATP, 10 µM ATP, 100 µM ATP and high concentration ATP (1 mM and 5 mM) respectively.Notably, speci c P2X inhibitors only partially inhibited the increase of concentration of the intracellular calcium.It indicated that the process also be associated with other receptors.For example, previous literatures have shown that besides P2X4, P2Y2 could be stimulated by ATP with a concentration of 100 µM [19].
Just as other cell models, extracellular ATP provoked distinct inward currents in mouse peritoneal mast cells as well.P2X1 receptor was characterized by fast activation and desensitization [24].Our results demonstrated the current induced by 1 µM ATP was closely resemble for P2X1-like current, which could be eliminated by non-selective P2 antagonists PPADS as well as P2X1-selective antagonist NF449.
PPADS or P2X3-selective antagonist AF-353 could block the inward current induced by 10 µM ATP, which suggested that P2X3 participated in this activation process.In addition, the current evoked by 100 µM ATP could be blocked by PPADS or 5-BDBD, indicating that P2X4 receptor was involved.Unlike LAD2 and HLMC [17], the P2X1-like and P2X4-like current in mouse peritoneal mast cells did not exhibit 'run-down' phenomenon upon repeated application.The reason for this may be due to the heterogeneous of mast cells [28].Our results showed that both 1mM ATP and 5 mM ATP could induce P2X7-like currents with 'run-up' characteristic, which is in line with the conclusion reported by other literatures [17,24].Although the current evoked by 1mM ATP and 5 mM ATP existed some differences, AZ10606120 could block both of them.Hence, we con rmed that P2X7 was involved in the activation caused by 1 mM ATP and 5 mM ATP.
Compelling of researches pointed out several substances including histamine, tryptase, cytokines and chemokines released from mast cells could contribute to pain either directly or indirectly [15].We also explored the mediators released from mast cells induced by ATP.The data showed that ATP with high concentration could induce histamine release, which was consistent with previous report [15,19].In addition to degranulation, high concentration of ATP also promoted the upregulation and secretion of in ammatory mediators.Our study showed that ATP with high concentration could signi cantly upregulate IL-1β and CCL3, which could be blocked by AZ10606120.These results indicated that P2X7 receptor was involved in mast cells degranulation and release of in ammatory mediators.Neuronimmune crosstalk plays important roles in many in ammatory diseases, we suggested P2X7 on mast cells could also induce peripheral pain indirectly as demonstrated in Fig. 8. Histamine plays a critical role in neurogenic in ammation and pain transmission via speci c receptors in a bidirectional manner [15,29].IL-1β can modulate neuronal activity directly, in addition, IL-1β also mediate pain sensitization via IL-1 receptor.Blocking spinal IL-1 signaling could alleviate neuropathic pain [30,31].Furthermore, the roles of in ammatory chemokines could regulate synaptic transmission, especially CCL2/CCR2 and CCL3/CCR1 signaling [32,33].Consistent with the hypothesis, our results also demonstrated mast cell and P2X7 participated in the peripheral pain.Chronic pain is a serious global health issue and a huge clinical challenge without available effective treatment.The commonly used pain-relieving agents are often associated with harmful side effects such as addiction.Therefore, the discovery of novel therapeutic alternatives with superior effectiveness and minimal adverse effects would be bene cial.Hence, we screened several anti-in ammatory molecule compounds based on P2X7 receptor.Interestingly, we found that salicylic acid and aspirin could inhibit the inward current and calcium in ux induced by high concentration ATP or P2X7 agonist BZATP.Salicylic acid and aspirin could also attenuate the release of in ammatory factors and peripheral pain induced by high concentration ATP.Molecular docking results showed that salicylic acid and aspirin had a nity to the cytoplasmic GDP-binding region of P2X7.Therefore, the analgesic mechanism salicylic acid and aspirin may be the inhibition of P2X7 activity by binding to GDP-binding region.needs further exploration.The binding patterns and the binding sites of salicylic acid and aspirin to the P2X7 receptor still need to be further veri ed.

Conclusion
In summary, we found that mouse-derived mast cells could be activated by extracellular ATP via P2X1, P2X3, P2X4 and P2X7 receptors.Mast cells and P2X7 receptor on mast cells played important roles in peripheral pain via in ammatory mediators.In addition, we also found that P2X7 receptor may be a potential target for the analgesic drugs salicylate acid and aspirin.

Figures
Table S1 The effects of anti-in ammatory drugs on the current growth rate evoked by 5 mM ATP.

Figure 2 The
Figure 2

Figure 3 The
Figure 3