Mast Cell-Associated Serotonin in Acupoint Contributes to Acupuncture Analgesia in Arthritis Rats by Mediating ATP Release

Background : The activation of subcutaneous mast cells (MCs) helps to trigger the analgesic effect induced by acupuncture (AP), a traditional oriental therapy, that has been gradually accepted worldwide. This work aimed to reveal whether the serotonin (5-hydroxytryptamine, 5-HT) released from MCs plays an important role in this process, which has a controversial effect in the mechanism of pain. Methods : In vivo tests, a 20-min session of AP was applied at Zusanli acupuncture point (acupoint) of acute ankle arthritis rats. Pain thresholds of the injured hindpaw were assessed to reflect the pain state, and the targeting substances in the interstitial space of the treated acupoint were sampled by microdialysis. In vitro experiments, exogenous 5-HT (exo-5-HT) was introduced to mediate adenosine triphosphate (ATP) release from cultured MCs. Results : Needling promoted 5-HT accumulation at the Zusanli acupoint, which was prevented by sodium cromolyn. AP’s analgesic effect was suppressed by the inhibition of 5-HT receptors at the acupoint, especially 5-HT 1A subtype. In vitro tests, mechanical perturbation mimicking needling stimulation induced MCs to release 5-HT. 1 µ M and 10 µ M of exo-5-HT facilitated ATP release, which was restrained by blocking of 5-HT 1 receptors rather than 5-HT 3 receptors. As 5-HT, ATP and adenosine were also transiently accumulated in the treated acupoint during needling. Promoting ATP hydrolysis or activation adenosine A1 receptors duplicated AP analgesic effect. Finally, the inhibition of ATP receptors by suramin or pyridoxal phosphate-6-azo tetrasodium salt hydrate (PPADS) prevented AP analgesic effect. Conclusions : Our results suggest that MC-associated 5-HT release at acupoints contributes to AP analgesia, and the mediation of ATP secretion through 5-HT 1A receptors might be the underlying mechanism at play. ATP could facilitate adenosine production or the propagation of needling signals.


Introduction
Alongside subcutaneous tissue and skeletal muscles, the skin is an important structural layer of acupuncture points (acupoints), which are special sites on the body surface that receive a variety of physical stimuli to modulate functional disorders of inner organs.Our researches and some other related studies have shown that subcutaneous mast cells (MCs) are present at greater densities in acupoints, and their activation plays an initiating role in the mechanism of acupuncture (AP) [1], an ancient oriental therapeutic approach gradually becoming more accepted worldwide.During AP, fine needles are inserted into certain acupoints and subsequent manipulations generate mechanical stimulation on acupoint, 240-280 mN in force, and 10-15 mN × mm −1 in torque [2].These signals are transmitted to the wider and deeper space by subcutaneous collagen fibers twisting around the needles [3].Our studies in vitro reveal the mechanosensitivity of MCs and tests performed in vivo have uncovered such characteristic contributes to AP analgesia [4,5].Additionally, MCs can also be activated by biological agents.Substance P (SP) and calcitonin gene related peptide (CGRP) released from the nerve terminals due to needling stimulation can induce adject MCs to degranulate [6].
Our previous work in vivo had shown that MCsreleased histamine was involved in the trigger mechanism of AP analgesic effect via activating H1 receptors [4].In the current work, we wondered whether serotonin (5-hydroxytryptamine, 5-HT) was also involved in this process, which is another endogenous substance exported from subcutaneous MCs [7].In rat acupoints, 5-HTimmunopositive MCs are present in subcutis and dermis, and their degranulation occurs in response to AP [8,9].5-HT has a controversial effect in pain mechanism, in which pain or analgesia relies on which subtype of 5-HT receptors (5-HTRs) is activated [10].Considering the tissue components of acupoints, 5-HT 1A R and 5-HT 3 R might be expressed [11], and they mediate the sustained and transient pain processing on rat neurons, respectively [12].Therefore, the current work tried to address whether MCs-released 5-HT also contributes to AP-analgesia via binding to some 5-HT 1 Rs or 5-HT 3 Rs subtypes.
Our results verified that in acute adjuvant arthritis (AA) rat model, MCs-associated 5-HT accumulated in treated acupoint and contributed to AP-analgesia.The underlying mechanism might be that mechanosensitive 5-HT release promote ATP secretion and initiate the downstream signalings.
Although these considerable basic studies have been executed to explore the underlying mechanisms, it is still not fully understood.A better understanding of how a fine needle to trigger the anti-nociceptive signals at the acupoint would shed light on the initiation mechanism of AP and provide guidance for proper clinical operation.

Animals
SPF-grade male Sprague-Dawley (SD) rats weighing 200 ± 20 g were used.The rats were purchased from Zhejiang Viton Lihua Experimental Animal Technology Co., Ltd., and maintained at the Animal Experimental Center of Shanghai University of Traditional Chinese Medicine.We performed the animal experiments in accordance with the procedures approved by the Animal Experimental Center (Animal Ethics No.: PZSHUTCM200911014) and implemented the relevant provisions from the Guidance Suggestions for the Care and Use of Laboratory Animals formulated by the Ministry of Science and Technology of the People's Republic of China.

Rat Model of AA
We used the rat model of AA established as previously described [16].Briefly, 50 µL complete Freund's adjuvant (CFA) (Sigma-Aldrich, St. Louis, MO, USA) was injected into the left ankle joint cavity of rats anesthetized with 1.5% isoflurane.Local swelling and behavioral disability appeared within 24 h.

AP Treatments
A single course of AP treatment for 20 min was performed at Zusanli (ST 36) acupoint of the affected side of conscious rats.According to our researches and related studies [4], the ST 36 has a desirable anti-nociceptive effect on AA rats.ST 36 is located at the posterolateral side of knee joint, about 5 mm below the fibula capitulum.A stainless-steel needle (0.18 mm × 13 mm, Cloud Dragon Medical Equipment Company, Wujiang, China) was gently inserted to an approximate depth of 7 mm.AP procedure was dominated by twirling-rotating motion (~100 times/min), supplemented with lifting-thrusting motion (~80 times/min).

Behavioral Tests
Pain thresholds of the injured hind plantar were determined to reflect the pain levels.Mechanical allodynia (paw withdrawal threshold, PWT) and thermal hyperalgesia (paw withdrawal latency, PWL) were evaluated to determine the pain level.The timeline and methods for behavioral tests was referred to our published work [16,18].Briefly, CFA was administrated on day 0 and AP was performed 2 days later.Pain thresholds were determined for three times, just before CFA injection, just before AP intervention, and after 30 min-AP treatment.Thermal hyperalgesia was measured 2 h after the mechanical allodynia assessment.

Acupoint Injection
In order to intervene certain corresponding signals at the acupoints, 20-50 µL related reagents were pre-injected into the left ST 36 in layers 20 min prior to AP treatment.

Microdialysis
The performance of microdialysis technology referred to our published work [19].Briefly, after rat was anesthetized with 1.5% isoflurane, a linear microdialysis probe (CMA 31, CMA Microdialysis AB, Kista, Sweden) was implanted at a distance of 4-6 mm from ST 36.The dialysis solution, Hank's balanced salt solution (HBSS, Corning Cellgro, Manassas, VA, USA), was controlled by syringe pumps (NE-1000, New Era Pump Systems, Farmingdale, NY, USA) and continuously perfused at a rate of 1 µL/min.Subsequently, rat was restrained in the rat fixator and was gradually awakened from anesthesia.After a 2 h recovery period, microdialysis outflow was collected for 30 minutes per sample.

Determination of 5-HT, ATP and Adenosine in the Microdialysates
5-HT and ATP in the microdialysate were determined by ELISA kit (HZ-5-HT-RA, Zhen Shanghai and Shanghai Industrial Co., Ltd., CN) and luciferin-luciferase assay (L-L, Sigma-Aldrich, St. Louis, MO, USA), respectively.The procedures were performed according to the manufacturer's instructions.The light emission was measured by a microplate reader (Synergy Mx, BioTek, Winooski, VT, USA).Calibrations were executed before and after each dertermination.
Adenosine levels was performed by high-performance liquid chromatography (HPLC) as previously described [4].The adenosine standard was checked both before and after the measurement, and the concentrations were 3000 nM, 1000 nM, 300 nM, and 100 nM.The detection results of the animal samples were compared to the standard according the peak areas, and the concentration was calculated.

Tissue Sectioning and MC Staining
Tissue sectioning and MC Staining referenced to our previous article [4].Briefly, after the final behavioral test, a 5 × 5 × 5 mm specimen of ST 36 was taken and 5 µm paraffin sections were prepared.After staining with toluidine blue, the MCs were dark purple in color.MCs with a blurred boundary, or dispersed granules were counted as degranulated MCs.

Cell Origin and Culture
The human MC (HMC-1) cell line was kindly provided by Dr J. H. Butterfield (Mayo Clinic) and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium.Rat basophilic leukemia (RBL-2H3) cell line was purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, CN) and cultured in Minimum Essential Medium (MEM) medium.The culture medium was supplemented with 10% Fetal Bovine Serum (FBS), 100 units/mL penicillin and 100 units/mL streptomycin in a 5% CO 2 humidified atmosphere at 37 °C.

Mechanical Stimulation
Hypotonic shock was introduced to HMC-1 cells to induce degranulation, and tryptase or ATP release.The entire procedure referenced to our article [15].Briefly, 1-2 mL of HMC-1 cell suspension (0.5-1.5 × 10 6 cells/mL) was gently introduced into the polycarbonate filter chamber, and superfused with bath solution at 1.3 mL/min.After an equilibration period, hypotonic solution (200 mOsm/Kg H 2 O) was introduced to the cells and the perfusate was collected at 1 min.Hypotonic environment is a common method to stimulate cells with force [21].The cell becomes swollen and its cellular membrane is extended, then the corresponding downstream signals are initiated.In our previous work, we used hypotonic shock to activate mast cells in vitro to mimic manual AP [5,15,22].Tryptase or ATP were assessed by ELISA (#IMM001, Sigma-Aldrich, St. Louis, MO, USA) and luciferase-luciferin assay.For 5-HT release measurements, we stimulated the cells by medium displacement.HMC-1 cells grew in the 96-well plate containing 100 µL culture medium.Half of the medium was gently pipetted up and down ten times with a pipette gun.Subsequently, 5-HT in the cell suspension was qualified by ELISA assay.

Exo-5-HT Stimulation
For each experiment, RPMI 1640 and MEM medium was used to further dilute 10 mM 5-HT stock solutions to acquire various required concentrations of 5-HT.5-HTsensitive ATP released from HMC-1 cells in response to exogenous 5-HT (exo-5-HT, #H9523, Sigma-Aldrich, St. Louis, MO, USA) in the range of 1 nM-10 µM for 15 min.Released ATP levels in the fractions were quantified by L-L assay, as described in 2.8.Based on our preliminary results, when exo-5-HT concentration ≥50 µM, cell mortality tended to be above 80%, and the large increase of ATP at this time might be caused by cell death.

Statistical Analysis
The measurement data are expressed as the mean ± standard error (SE) values.The data were analyzed using SPSS 25.0 (IBM Co., Armonk, NY, USA).The figures were prepared by GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA).Differences among multiple groups were tested with one-way or two-way analysis of variance (ANOVA), followed by Fisher's least significant difference (LSD.)test.Where only two groups were compared, a two-sample t-test was used.When data were non-normally distributed, a Mann-Whitney U test was performed.We considered p < 0.05 to be statistically significant.

5-HT Induced ATP Release from MCs via Acivation of 5-HT 1A R
5-HT is reported to induced ATP release from type II cells of the rat carotid body [17].Mechanosensitive ATP release is discovered in various mammalian cells [21], including MCs [5].Needling-induced ATP transient accumulation in treated acupoint is supposed as one of the primary steps toward AP-analgesic mechanism by providing the precursor for adenosine [16,19].Besides primary mechanosensitive ATP release, secondary ATP secretion had been found in MCs in our previous work [15].Subsequently, we tried to verified whether 5-HT could mediate MCs to release ATP.Fig. 2A shows the dependency of ATP generation on the concentration of exogenous 5-HT (exo-5-HT) (1 nM-10 µM).When the concentration of exo-5-HT reached 1 µM and 10 µM, extracellular ATP level was significantly potentiated from baseline of 15.7 nM ± 2.5 nM to 43.6 nM ± 5.3 nM and 107.3 nM ± 3.4 nM, respectively (Fig. 2A).5-HT concentration greater than 10 µM was not determined because cells viability was affected (Supplementary Fig. 3).Pretreatment HMC-1 with Methiothepin (100 nM) significantly impaired ATP release induced by 1 µM-and 10 µM-exo-5-HT as well.In contract, Granisetron (100 nM) had null effect (Fig. 2B,C).Similar results were also obtained from rat MCs, RBL2H3 cell line (Supplementary Fig. 4).However, exogenous ATP had null effect on 5-HT release (Fig. 2D).These results suggest that 5-HT mediates ATP release from MCs via activating 5-HTRs, especially 5-HT 1 Rs.
Based on these findings, we returned to the test in vivo.Behavioral tests found that pre-injection of WAY.(2 mg/mL, 20 µL) in ST 36 canceled AP analgesic effect on PWT (n = 5-6, p = 8.1 × 10 −5 vs. AP) and PWL (n = 4-6, p = 0.0003 vs. AP) (Fig. 2G,H).Taken these results together, we infer that contribution of MC-associated 5-HT release to AP analgesia mechanism might be due to mediate ATP release from MCs or other adjacent cells by activating 5-HT 1A R.

Needling-Induced Inters. ATP Mobilization in Acupoint was Involved in AP Analgesia Possibly via Producing Adenosine or Propagating AP Signals
AP-induced adenosine accumulation and activation of local adenosine A 1 Rs contribute to initiate analgesia [14].Such adenosine mobilization might partially rely on the hydrolyzation of ATP.Our microdialysate determination revealed that both adenosine and ATP in the interstitial space of treated acupoint were significantly potentiated by needling (adenosine, from baseline of 58.4 nM ± 6.9 nM to 76.8 nM ± 9.1 nM, n = 9, p = 0.0250; ATP, from baseline of 3.4 nM ± 1.4 nM to 56.0 nM ± 12.5 nM, n = 4, p = 0.0269).More importantly, the presence of ARL (100 µM, 50 µL), a non-specific inhibitor of ecto-ATPase, in acupoint further potentiated AP-induced ATP accumulation (136.3 nM ± 22.7 nM, n = 4, p = 0.0258 vs. AP group) (Fig. 3A), indicating the activities of ecto-ATPase and hydrolyzation of inters.ATP existed in the treated acupoint.Further behavioral tests demonstrated that AP analgesic effect was impaired by ARL (100 µM, 50 µL) (n = 4, p = 0.0233), and was duplicated by CCPA (0.04 mg/mL, 20 µL), an agonist of adenosine A1Rs (n = 5, p = 0.9773) (Fig. 3B).Taken together, these findings suggest that mobilization of inters.ATP via primary or secondary release contributes to antinociceptive mechanism of AP by promoting adenosine production.
Calcium wave propagation (CWP) is supposed as the basis of ATP regeneration, which facilitates the localized propagation of biological signals [29].CWP has been uncovered in mouse treated acupoint [30].Our previous work had found a similar CWP and cascaded ATP release in HMC-1 cells when perturbed by hypotonic shock, in which primary mechanosensitive ATP released induced secondary ATP release via activating P2Y 13 or P2X 7 receptors [15].The present work found an additional 5-HT-induced secondary ATP release.We hypothesized that besides as the precursor of adenosine, such cascaded ATP release could amply and propagate the localized needling signals.Our WB determination revealed that some components for forming CWP/ATP regeneration, including Panx-1, connexin 43 and P2Y 1 were expressed in HMC-1 cells (Supplementary Fig. 5).Subsequently, further behavioral tests observed that inhibition of local purinergic receptors (P2 receptors) by Suramin (100 µM, 50 µL) (Relative PWT: reduced from 0.9 ± 0.0 to 0.7 ± 0.1, n = 4, p = 0.0057 vs. AP and Relative PWL: reduced from 0.9 ± 0.0 to 0.6 ± 0.0, n = 4, p = 0.0269 vs. AP) or PPADS (100 µM, 50 µL) (Relative PWT: reduced from 0.9 ± 0.0 to 0.7 ± 0.1, n = 4, p = 0.0009 vs. AP and Relative PWL: reduced from 0.9 ± 0.0 to 0.8 ± 0.1, n = 4, p = 0.0749 vs. AP) partially suppressed the AP-relieved tactile allodynia and thermal hyperalgesia (Fig. 3C,D).This finding implies that besides adenosine and A1Rs, ATP/ADP and related P2 receptors in acupoint also play a role to mediate AP analgesia and its underlying mechanism might be the ATP-related CWP.

Local MC-Associated 5-HT Reflected the State of Acupoints
5-HT-immunopositive MCs are present in acupoints and become more when the acupoints are sensitized [8,9].Acupoint sensitization means acupoints transformed dynamically from a "silent" state to an "activated" one when the body is suffering from some corresponding diseases [31].Neurogenic inflammation is one of the biological bases for acupoint sensitization due to dorsal root reflex or axon reflex [32,33].In the present work, we noticed that compared to normal rats, ST 36 in model group had higher inters.5-HT level at baseline (Supplementary Fig. 1).Here, the onset site and treated acupoint are innovated by tibial nerve and peroneal nerve, respectively, branches of sciatic nerve, suggesting that they are innovated by the same or adjacent peripheral ganglia and spinal segments.Similarly, in knee osteoarthritis rats, 5-HT together with tryptase and histamine are upregulated in the nearby acupoints, Yanglingquan acupoint and Heding acupoint rather than Weizhong acupoint [34].The greatest benefit of acupoint sensitization is to facilitate AP to exert better therapeutic effect.Our previous work had revealed that AP modulated higher ATP accumulated in AA rats than in normal ones [19].Systematic review demonstrates the comparative superiority of needle stimulation of sensitized points over non-sensitized points, especially for some pain syndromes [31].
Degranulation of local subcutaneous MCs induced by needling has been often reported [1].While the certain associated mediators are not well quantitatively studied.Beside adenosine assessed in our previous work [4], the current study clearly recorded MC-associated 5-HT mobilization (Fig. 1A,B).Based on the microdialysis recovery percentage [19] and considering dilution by diffusion and/or convection into the bulk extracellular volume [23], the real concentration of inters.5-HT at the cell surface was more than 500 ng/mL (2.8 µM).This concentration is within the range we used in our test in vitro (1 and 10 µM), which is sufficient to activate 5-HT 1A R (Fig. 2F).Additionally, this concentration range can activate 5-HT 1A R [24], 5-HT 2A R [17] and 5-HT 3 R [25], implying various functions are involved, for example, influence the differentiation, proliferation, migration, adherence and life-span of skin cells, regulate the tonus of blood vessels and influence nerve transmission [35].The potentiated inters.5-HT could last for more than 2.5 h (Fig. 1A,B).It might contribute to the maintenance of AP effect.

5-HT Induced ATP Release by Activating 5-HT 1A R
The activation of MCs can easily establish a positive feedback loop.Matrix metalloproteinase-9 [36] or ATP [15] secreted from MCs, in turn, activate MCs.In the present work, unidirectional regulation of 5-HT on ATP release via activation of 5-HT 1A R was another example of this self-activation mechanism (Fig. 2A).Other cells can also be activated, as long as they express 5-HT 1A R, for example, melanocytes [37].Similar finding has been reported in type II cells of rat's carotid body, in which exo-5-HT increases [Ca 2+ ] i partially via activating Panx-1 channels, implying 5-HT might facilitate ATP release [17].Although we demonstrated that MCs express Panx-1 channels (Supplementary Fig. 5), whether they assisted ATP re- Additionally, interaction between 5-HT mediated signaling and ATP-related P2X receptors have been characterized.While it is mainly inhibitory rather than facilitative.In submucosal neurons of guinea pigs, whole-cell recordings reveal that 5-HT 3 Rs and P2X channels negatively modulate each other when they are simultaneously activated [38].5-HT 3A R and P2X2 channels were found to co-immunoprecipitate constitutively and be associated in clusters in cultured myenteric neurons of guinea pig as well as expression cells [39].5-HT 3 Rs in pelvic afferent neurons is up-regulated in mice lacking P2X2 or P2X3 receptor genes [25].

Inters. ATP Contributed to AP-Analgesia as the Precursor of Adenosine
We demonstrated an upregulation of inters.ATP in the treated acupoint by needling (Fig. 3A), and prevention its hydrolysis by ARL impaired AP-analgesic effect (Fig. 3B).
These findings imply that ATP mobilization and subsequent hydrolysis are vital steps towards antinociception during needling.The aggregated inters.ATP originates from cell lysis and non-lytic release as well.Regarding the former, needle piercing and subsequent manipulation can cause tissue damage.As reported, damage-associated molecules, high mobility group box 1 protein (HMGB1) and toll-like receptor 4 (TLR4), increases in the treated acupoint of normal rats by 2-min needling [40].The non-lytic release of ATP results from the primary ATP secretion via activating mechanosensitive channels [1] and secondary ATP release induced by ATP or 5-HT, as we discussed above.But unlike 5-HT, mechanosensitive ATP release via non-degranulation approach (Supplementary Fig. 2A).
Adenosine, as the downstream product of ATP, aggregates in acupoints too [14,41].The current work and other similar study [14] observed that pharmacologic activation of adenosine A1R in acupoints had antinociceptive effect (Fig. 3B), in which A1R are supposed to be situated on lo-cal nerve terminals [14].Like ATP, adenosine might also release out via lytic and non-lytic approaches [42].

Inters. ATP may Facilite Localized Propagation of Needling Signals
Needling manipulations generate mechanical stimulation that is transmitted to the wider and deeper space by subcutaneous collagen fibers twisting around the needles [51,52].This is the "physical propagation" of needling signals [3].In addition, "biochemical propagation" also occurs.As reported, AP-generated acoustic shear waves can cause [Ca 2+ ] i shock and CWP in vitro and in vivo [30].CWP permits regenerative signal amplification.It has been proposed that non-neural cells respond to mechanical stimulation by ATP release and send signals to nerve terminals via CWP [30].CWP formation is commonly based on ATP regeneration [29].Compelling evidence indicates that ATPmediated ATP release involves P2YRs [53], among which P2Y1R is the most documented [54,55].Our previous work had observed a similar CWP phenomenon in cultured MCs in response to mechanical stimulation, which relied on the activation of P2Y 13 and P2X 7 receptors via "ATP-induced ATP release" mechanism [15].5-HT-mediated ATP release uncovered in this work is a good complement to ATP regeneration, then facilitate CWP formation.The necessary components for CWP, including P2Y1 receptors, Connexin 43 and Panx-1 channels are expressed in mice acupoint (ST 36) [20].In the present work, WB determined their presence in MCs (Supplementary Fig. 5).Furthermore, Panx-1 [56] and connexin 43 [57] are been reported to express in skeletal muscle and fibroblasts that are also main tissues contained in acupoints.Hence, besides as the precursor of adenosine, inters.ATP in acupoint might exert function of transmitting the needling signals.This hypothesis was partially confirmed by the inhibitory effect of Suramin and PPADS on ATP regeneration in MCs in our previous work [15] and AP-analgesic effect in the present study (Fig. 3C,D).

Contribution of 5-HT to AP Analgesia is Might via Direct Activating 5-HT 1A R
We found that 5-HTRs, especially 5-HT 1A R, in acupoint played role in the initiation mechanism of APanalgesia (Fig. 1E,F and Fig. 2G,H).Beside facilitating needling signals as we discussed above, 5-HT might contribute to AP analgesic effect by activating 5-HT 1A R on the adjacent peripheral nerve endings.As we investigated previously, AP-activated MCs were eventually involved in the analgesic effect via transmitting the biological signals to the nearby nerve endings, then ascending to central nerve system [58].5-HT 1 Rs class represents the most complex subtype families of 5-HTRs, which comprise of five receptor subtypes, 5-HT 1A , 5-HT 1B , 5-HT 1D , 5-HT 1E and 5-HT 1F .Although 5-HT 1A R activation is reported to elicit hyperalgesia [59,60], later researches reveal its antinociceptive effect.Peripheral sensory nerve endings are the main location of 5-HT 1A subtype [61].Administered intrathecally with (±)-8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT), a 5-HT 1A R agonist, was to suppressed the second phase of formalin-induced aversive responses in the 5,7-dihydroxytryptamine treated rats [62].Another research using 8-OH-DPAT also suggested an analgesic action is exerted by 5-HT 1A R in the formalin model of tonic nociceptive pain in rats [63].It still needs further work to explore whether 5-HT mediates AP analgesia by activating 5-HT 1A R situated on nearby nerve endings.

Conclusions
The present study supports the critical role of MCassociated 5-HT in the initiation mechanism of AP analgesia.The underlying mechanisms involve 5-HT 1A Rmediated ATP release and its downstream events, including adenosine generation and CWP formation.Fig. 4 demonstrates our conclusion in detail.

Fig. 4 .
Fig. 4. Schematic representation of how MC-associated 5-HT in acupoints participates in AP analgesia.Collagen fibers winding within acupoints generated by AP activates MCs to release 5-HT and ATP in the manner of degranulation and non-degranulation, respectively.5-HT facilitates ATP release by activating 5-HT1ARs in the adjacent cells.The released ATP hydrolyzes into Adenosine with the aid of ecto-nucleotidases, which contributes to AP analgesia through acting A1 receptors on the local nerve endings; or activates P2 receptors in the adjacent cells to forming CWP via opening ATP channels to amplify AP signals.Eventually, these signals are sensed by peripheral sensory ganglia and further ascend to central nerve system.