P2X receptors and acupuncture analgesia

Accepted Manuscript Title: P2X receptors and acupuncture analgesia Authors: Yong Tang, Hai-yan Yin, Juan Liu, Patrizia Rubini, Peter Illes PII: DOI: Reference:

S0361-9230(18)30576-8 https://doi.org/10.1016/j.brainresbull.2018.10.015 BRB 9547

To appear in:

Brain Research Bulletin

Received date: Revised date: Accepted date:

6 August 2018 13 October 2018 18 October 2018

Please cite this article as: Tang Y, Yin H-yan, Liu J, Rubini P, Illes P, P2X receptors and acupuncture analgesia, Brain Research Bulletin (2018), https://doi.org/10.1016/j.brainresbull.2018.10.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

P2X receptors and acupuncture analgesia Yong Tang1,3, Hai-yan Yin3, Juan Liu3, Patrizia Rubini2, Peter Illes2,3 1Medical

& Nursing School, Chengdu University, 610106 Chengdu, China, 2Rudolf-

Boehm-Institut für Pharmakologie und Toxikologie, Universität Leipzig, 04107 Leipzig,

Medicine, 610075 Chengdu, China

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Send correspondence to: Dr. Yong Tang

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Germany, 3Acupuncture and Tuina School, Chengdu University of Traditional Chinese

Acupuncture and Tuina School, Chengdu

University of Traditional Chinese Medicine,

Tel.: (+86)28-87683962

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Fax.: (+86)28-61800105

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610075 Chengdu, China

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Dr. Peter Illes

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e-mail: [email protected]

Rudolf-Boehm-Institut für Pharmacology und Toxikologie

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University of Leipzig, Haertelstrasse 16-18 04107 Leipzig, Germany

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Tel.: (+49)341-9724614 Fax: (+49)341-9724609

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e-mail: [email protected]

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Highlights: 

Acupuncture analgesia is based on ATP release from (sub)cutaneous/muscular tissue

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ATP and its degradation product adenosine participate in acupuncture analgesia

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Peripheral and central mechanisms participate in the effect of acupuncture analgesia

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A1/P2X3,4,7 receptors appear to mediate acupuncture analgesia

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Abstract Purinergic signaling has recently been suggested to constitute the cellular mechanism underlying acupuncture-induced analgesia (AA). By extending the original hypothesis on endogenous opioids being released during AA, Geoffrey Burnstock and Maiken Nedergaard supplied evidence for the involvement of purinoceptors (P2 and P1/A1 receptors) in the beneficial effects of AA. In view of certain pain states (e.g. neuropathic pain) which respond only poorly to therapy with standard analgesics, as well as with respect to the numerous unwanted effects of opioids and non-steroidal anti-

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inflammatory drugs, it is of great significance to search for alternative therapeutic options. Because clinical studies on AA yielded sometimes heterogeneous results, it is

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of eminent importance to relay on experiments carried out on laboratory animals, by evaluating the data with stringent statistical methods including comparison with a sufficient number of control groups. In this review, we summarize the state of the art situation with respect to the participation of P2 receptors in AA and try to forecast how

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the field is likely to move forward in the future.

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Keywords: acupuncture analgesia, purinergic signaling, ATP, P2X receptors

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Introduction

Liu et al. (1994) were probably the first to report that weak electro (E)-acupuncture

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(AP) applied to the acupoints Yanglingquan (GB34) and Xuanzhong (GB39) prolonged the latency of nociceptive hind limb withdrawal reflex, and that the intraperitoneal administration of two adenosine receptor (R) antagonists, theophylline and caffeine,

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blocked the EAP-induced analgesia. Since that time recognition of the roles of purines in acupuncture analgesia (AA) has gradually increased. In 2009, Professor Geoffrey Burnstock, who presented previously the hypothesis of purinergic signaling (Burnstock,

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1972), proposed a crucial role for purines in AA (Burnstock, 2009b). He pointed out that sensory nerve activity initiated in the skin by AP is likely to have an inhibitory modulating effect on the spinoparabrachial and spinothalamic tracts to the brain pain centers by a mechanism which still remains to be clarified. The purinergic signaling system includes ATP, ADP, AMP and adenosine (ADO), and two families of purinoceptors called P1 and P2, for ADO and ATP/ADP (UTP/UDP), respectively (Burnstock, 1978). These receptors are expressed in most cell types in

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mammalians but also in invertebrates or more primitive creatures (Burnstock and Knight, 2004; Koles et al., 2007). P1Rs consist of four subtypes: A1, A2A, A2B and A3, all of which are coupled to G proteins. P2Rs are classified into two families: P2X and P2Y. P2XRs are ligand-gated cationic channels forming the P2X1-7 subtypes and P2YRs belong to G protein-coupled receptors consisting of the P2Y1,2,4,6,11-14 subtypes. The role of purinergic signaling in pain has been confirmed and a great number of candidate drugs for this therapeutic indications have been developed in this

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field (Burnstock, 2009a, 2016, 2017, 2018; Ford, 2012; Park and Kim, 2016)

Evidence for the roles of ADO and A1Rs in AA has been provided by supplying solid

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data in mice (Goldman et al., 2010) and healthy human volunteers (Takano et al., 2012). Further, a new concept of acupuncture therapy, PAPuncture has been described (Hurt and Zylka, 2012), by injecting prostatic acid phosphatase (PAP; an ectonucleotidase that dephosphorylates extracellular AMP to ADO) into the space

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behind the knee that encompasses the acupoint Weizhong (BL40). PAPuncture caused

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analgesia for an extended period of time considerably surmounting the duration of the

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effect of AP alone.

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ATP/ADP concentration and acupuncture stimulation

ATP and its metabolite ADP are the main purinergic ligands binding to P2 receptors.

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Under physiological conditions, intracellular concentrations of ATP are in the range of 3–5 mM and serve for the storage of energy; they are mobilized under anaerobic conditions. Noxious stimuli, shear stress, stretch, osmotic swelling or metabolic

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limitation cause the release of ATP/ADP into the extracellular space from the interior of the afflicted cells (Lazarowski, 2012). Typically, ATP concentration required for half maximal activation of purinergic receptors is 3–500 nM, which is 1,000-fold lower than

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those existing inside the cell and sufficient to initiate purinergic receptor-mediated signaling. After having been released into the extracellular space, ATP acts at P2Rs and is degraded into ADP, AMP and ADO by different ecto-nucleotidases (Yegutkin, 2008). The P2X7R-type has a uniquely low sensitivity to ATP in the range of 100-1,000 µM (Sperlagh et al., 2006; Sperlagh and Illes, 2014; Xing et al., 2016). Fig. 1 shows some AP family procedures leading to the local release of ATP and the generation of its functionally active metabolites, as well as the receptor types at which

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these purinergic agonists act. Currently, only few data are available on the changes of extracellular ATP concentration after acupuncture stimulation. In male mice, a small acupuncture needle was gently inserted into the Zusanli point (ST36) located 3–4 mm below and 1–2 mm lateral from the midline of the knee (Goldman et al., 2010). Gentle manual rotations of the acupuncture needle every 5 min for a total of 30 min sharply increased the extracellular concentrations of all detected purines (ATP, ADP, AMP and ADO) as measured in the muscle/subcutaneous tissue 0.4-0.6 mm apart from the

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acupoint by a microdialysis probe. ADO concentration increased ~24-fold during the 30min acupuncture session. ADP, AMP and ADO remained markedly elevated 60 min

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after AP. However, the extracellular concentration of ATP returned to baseline soon after needling (Goldman et al., 2010). In healthy volunteers, AP also caused a marked, long-lasting increase of ADO, but not ATP concentrations during acupuncture (Takano et al., 2012). Already these findings suggest that ADO, instead of its mother compound

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ATP was responsible for the analgesic effect of AP in mice and human volunteers. The

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below experimental data have lent further support to this notion. Deoxycoformycin blocks AMP deaminase and ADO deaminase which help to

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generate extracellular ADO from ATP (Goldman et al., 2010). The intraperitoneal

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application of deoxycoformycin to mice both sharply increased the concentration of ADO in muscle/subcutaneous tissue during and after AP as well as significantly

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prolonged the ensuing anti-nociception. Therefore it appeared most likely that extracellular ADO was generated enzymatically from the released ATP rather than by

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the operation of an outward transporter/release mechanism of ADO itself from the cell interior into the interstitial space. Experiments on Sprague-Dawley rats in vivo and mast cell cultures in vitro

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delivered data on changes in the concentration of ATP in response to different forms of AP stimulation. In rats, administration of sparrow-pecking manual acupuncture (MAP) technique on the right tibialis anterior muscle at a location 7–8 mm below the knee

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(without naming the acupoint) and at 30 repetitions per min for 1 min induced significant increase of the extracellular concentrations of ATP and ADP as well as ADO rather than AMP at the 30 min time-point. 60 min after MA, all four purine substances returned to baseline values. It was proposed that the increased ATP concentration induced by MAP could derive from skeletal muscle cells rather than vascular cells or sensory nerves (Nagaoka et al., 2016). Another experiment to measure ATP concentration was performed on rat neck-incision pain model (Gao et al., 2017). The neck incision induced

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an increase in ATP content in the dorsal part of the cervico-spinal cord 4, 24, and 48 h post-incision. One session of EAP intervention further elevated the ATP levels in all three EAP groups (LI18, LI4-PC6 and ST36-GB34). However, after two sessions of EAP intervention, the ATP levels decreased in both LI18 and LI4-PC6 groups and returned to the normal level after three sessions of EAP. EAP at ST36-GB34 did not cause significant changes 24 and 48 h post-incision. It seems that EAP only cause a sudden and short increase in ATP concentration which rapidly fades thereafter.

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In fact, treatment of the skin at the acupoint is a necessary measure in most therapies of AP family procedures. Accordingly, current data demonstrate that mast

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cells of acupoints participate in the mechanism of analgesia when stimulated during needling (Zhang et al., 2008), moxibustion (Shi et al., 2011) and laser acupuncture (Cheng et al., 2009). In in vitro experiments on cells of the human mast-cell line HMC-1, mechanical stress was induced by superfusion of the cells with hypotonic solution, heat

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was generated by incubation of the cells at 43 °C or 52°C, and red laser light of 657 nm

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wavelength was used for irradiation. It was found that all these stimuli induced ATP release from human HMC-1 cells, and this release was associated with an increase of

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the intracellular free Ca2+ concentration (Cheng et al., 2009; Zhang et al., 2008).

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On the basis of the above data showing that AP stimulation induces ATP release from cutaneous/subcutaneous tissue or muscle cells, and the fact that the subcutaneous or

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intraplantar application of ATP and α,β-meATP causes nociceptive responses and hyperalgesia/allodynia as characteristics of acute and neuropathic pain, respectively,

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we decided to put up the following questions: (1) Does the released ATP supports or alleviates AA? (2) Is the release of ATP only a preceding step to cause an increased local concentration of ADO which contributes to AA? (3) Current clinical trials

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demonstrated that deep acupuncture applied to muscular tissue or sham acupuncture applied to non-acupoints has effects similar to those of real acupuncture (Madsen et al., 2009; Moffet, 2009). Could the concentration increase of ATP in subcutaneous tissue or

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muscle cells explain the AA induced anti-nociception? (4) What is the role of other sources of ATP release from different stimulated cells at acupoints i.e., keratinocytes, fibroblasts, epithelial or endothelial cells, Merkel or Langerhans cells? The present concept on the participation of the AP-induced release of ATP itself via P2X/P2YR activation or after enzymatic degradation to ADO via A1R activation in the alleviation of pain is shown in Fig. 2.

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P2X3 and P2X2/3 receptors in acupuncture analgesia

In 1995, the P2X3R was cloned and shown to be localized predominantly at the terminals of Aδ and C fibres projecting from sensory neurons of dorsal root ganglia (DRG) to the innervated tissues in most tissues and organ systems, including skin, joints, and hollow organs, suggesting a high degree of specificity to the pain sensing system in the human body (Chen et al., 1995; Lewis et al., 1995). The homomeric

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P2X3Rs respond to the ATP structural analogue α,β-methylene ATP (α,β-meATP) and desensitize rapidly during agonist application. The heteromeric P2X2/3Rs are also

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sensitive to ATP/α,β-meATP, but do not exhibit desensitizing properties. Both P2X3 and P2X2/3Rs were identified to participate in acute, inflammatory, neuropathic and cancer pain in a variety of experimental pain models (Wirkner et al., 2007; Burnstock, 2006). On the one hand, subcutaneous or intraplantar application of ATP and α,β-meATP

neuropathic

pain,

respectively.

On

the

other

hand,

P2X3R-selective

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and

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causes nociceptive responses and hyperalgesia/allodynia as characteristics of acute

pharmacological antagonists (A317491, AF-010, AF-130, AF-353, AF-792, AF-219, etc.)

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and antisense oligonucleotides or non-selective antagonists (suramin) improve different

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pain conditions in animal models (Ford, 2012; Kennedy et al., 2003; Donnelly-Roberts et al., 2008). Additionally, in experimental pain states, mice with genetic deletion of

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P2X3Rs also display decreased nocifensive behavior in comparison with their wild-type backgrounds. Therefore, reducing the concentration of local ATP or inhibitory

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modulation of P2X3-containing receptors appears to be an exciting new approach in pain management (Hansen et al., 2012; Prado et al., 2013; Schiavuzzo et al., 2015; Mansoor et al., 2016).

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Most studies investigating the effects of purines (acupurines) released by AP

stimulation have dealt with the role of P2X3Rs or P2X2/3Rs in AP-mediated analgesia, including neuropathic pain, inflammatory pain and visceral pain using different acupoints

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for different pain modalities. In rat neuropathic pain induced by constricting one sciatic nerve with a ligature

(chronic constriction injury; CCI), EAP was employed to ipsilateral or contralateral acupoints (Zusanli, ST36; Yanglinquan, GB34) for 30 min daily and for 7 days in total starting 7 days post CCI (Wang et al., 2014; Tu et al., 2012; Cheng et al., 2013). Hyperalgesia and allodynia, as two symptoms of neuropathic pain were measured for 14 days in total. During the first 7 days of observation, the severity of these two

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symptoms gradually increased and then reached a maximum level. The mentioned AP treatment schedules caused a gradual and moderate reversal of neuropathy by about 40%; the recovery was almost identical for mechanical pain and temperature sensation, but was practically indistinguishable for the ipsi- and contralateral EAP. On the 10th day, intrathecal injection of the selective P2X3R antagonist A317491 immediately and dramatically facilitated the EAP-induced analgesia (Wang et al., 2014). In this experiment, A317491 was applied only in combination with EAP but never alone and

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thereby it is difficult to decide whether the two effects were additive or not. An additive interaction would indicate that the mechanism of analgesia is the same, mediated in

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both cases by the exclusion of P2X3R function. However, the equal efficiency of ipsiand contralateral EAP implied that the spinal or supraspinal level rather than the peripheral terminals of DRG neurons would be important to cause pain relief after EAP. Another series of experiments with EAP at the same acupoints in the CCI rat model,

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induced by sciatic nerve ligation, led to very similar results. However, a different time-

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schedule was used which appears to be sub-optimal. EAP at Zusanli-Yanglingquan acupoints 3 days after ligating the sciatic nerve induced moderate analgesia.

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Extracellular signal-regulated kinase 1/2/P2X3R signal pathway in the spinal cord was

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proposed to be a potential mechanism of AA (Yu et al., 2013). It may be criticized that the effect of EAP in this study could not be reliably evaluated, because neuropathic pain

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does not reach its maximum intensity at the early time-point of 3 days. Further, all studies reported an increase of the α,β-meATP-induced current amplitudes in acutely dissociated DRG neurons of CCI rats (Tu et al., 2012; Cheng et al., 2013; Wang et al.,

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2014). In rats, treated ipsi- or contralaterally with EAP, the α,β-meATP currents decreased, but still exceeded the current amplitudes measured in non-neuropathic EAP

also moderately reduced the

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DRGs.

increased

expression

of

P2X3R

mRNA/protein detected by quantitative RT-PCR, in situ hybridization, quantitative immunohistochemistry, and western blotting in DRG neurons and the dorsal horn of

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spinal cord after CCI to the unilateral sciatic nerve (Wang et al., 2014; Tu et al., 2012, Yu et al., 2013). In accordance with the increased expression of P2X3R mRNA/protein in DRG or spinal cord of neuropathic rats, crucial sites in the endogenous pain modulatory systems, in the midbrain periaqueductal gray (PAG) also showed upregulated P2X3R protein levels (Xiao et al., 2010). EAP at Zusanli and Sanyinjiao (SP6) ipsilateral to the side of the ligated nerve upregulated the P2X3R protein, whereas sham-acupuncture was ineffective. Interestingly, knock-down of the upregulated P2X3R

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expression in the PAG with an antisense oligonucleotide for the P2X3R gene significantly attenuated the antinociceptive effect of EAP. The positive effects of EAP were observed also in case of diabetic neuropathic pain (DNP) and inflammatory pain by stimulating acupoints Zusanli (ST36) and Kunlun (BL60) with 2 and 100 Hz frequencies. In DNP rats, thermal hyperalgesia measured by paw withdrawal latency was drastically reduced (He et al., 2017). EAP with 2 Hz exhibited stronger analgesic effect than that caused by 100 Hz. The expression of

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P2X3R protein in lumbar (L4–L6) DRGs was assessed by immunofluorescence. Interestingly, EAP with 2 Hz reduced the increased expression of P2X3Rs in L4–L6

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DRGs of DNP rats whereas EAP induced by 100 Hz stimulation reduced it only in L5 and L6 DRGs. It seems that low frequency EAP involved more spinal cord segments with the attached DRGs contributing to the stronger analgesic effect of 2 Hz over that of 100 Hz. In contrast, when inflammatory pain was induced by intraplantar injection of

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Freund's Complete Adjuvant (CFA) into the right hind paw, 100 Hz EAP generated

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stronger analgesic effect than EAP with 2 Hz (Fang et al., 2018). Then, the number of P2X3 immunopositive neurons was counted and P2X3R protein expression was

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determined by western blotting. The CFA-induced inflammatory pain raised the number

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of P2X3 positive neurons in L4, L5, and L6 DRGs and the expression of P2X3 protein in L6, but not in L4 or L5 DRGs. 100 Hz EAP markedly reduced the number of P2X3

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immunopositive neurons in L4-6 DRGs and down-regulated the P2X3 protein expression in the L6 DRG.

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Visceral pain also responded to AP. The painful irritable bowel syndrome was modelled in few days old rats by inflating a balloon in their terminal colon twice daily for 14 days in total (Weng et al., 2013, 2015). Within a further period of 6 weeks, these rats

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developed hypersensitivity to bowel distension. To determine the intensity of pain reaction caused by subsequent colorectal distension, an arbitrary withdrawal reflex score was used. In pressure-treated rats, the pain withdrawal score decreased after

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EAP (at acupoint ST37 and ST25) in a reversible manner. The levels of P2X2 and P2X3 mRNAs in DRGs also responded with increase and decrease to balloon distension and EAP, respectively. Similar changes of P2X3 mRNA were also found in colon myenteric plexus, spinal cord, prefrontal cortex and anterior cingulate cortex. Thus, it is assumed that P2X3Rs became upregulated along the neuronal pathways mediating pain to higher brain centers. Nonetheless, we are left with some justified doubt, because the criteria to prepare the spinal cord for RT-PCR were not reported in the discussed

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papers. Of course the spinal cord segments L4-L6 innervated by the appropriate DRGs should be prepared (optimally only their dorsal parts containing the dorsal horn) and processed for RT-PCR. The major deficit of these studies is that sham EAP is almost always missing from the experimental designs; electrical stimulation by AP needles inserted at non-acupoints is, however, an indispensable control procedure.

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P2X4 receptors and acupuncture analgesia

It was reported for the first time in 2003 that P2X4Rs are causally related to the

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generation of neuropathic pain (Tsuda et al., 2003). It was found that intrathecal injection of high doses of trinitrophenyl-ATP (TNP-ATP; an antagonist of P2X1-4Rs) but not PPADS (an antagonist of P2X1-3,5,7Rs) caused a rapid and transient reversal of mechanical allodynia induced by injury to the fifth lumbar (L5) spinal nerve in rats.

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Intrathecal administration of a P2X4R antisense oligodeoxynucleotide also reduced the

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increase in P2X4R protein expression accompanying neuropathic pain in the spinal cord and prevented the development of mechanical allodynia. It was concluded that P2X4Rs

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located at spinal microglia mediate neuropathic pain, and knock-down or knock-out in

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the spinal cord of P2X4Rs prevents the development of mechanical allodynia after peripheral nerve injury (Coull et al., 2005; Inoue et al., 2007; Ulmann et al., 2008; Tsuda

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et al., 2009). It appears that ATP-induced activation of microglial P2X4Rs leads to the synthesis and release of brain-derived neurotrophic factor (BDNF) (Ulmann et al., 2008;

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Trang et al., 2009). This BDNF causes an altered transmembrane anion gradient in a subpopulation of dorsal horn lamina I neurons presumably through the downregulation of the neuronal chloride transporter KCC2, which results in conversion of GABAA- and

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glycin-R-mediated inhibition to excitation (Coull et al., 2005). Thus, microglial P2X4Rs are central players in the pathogenesis of neuropathic pain (Tsuda et al., 2013). EAP (2 Hz frequency) started on the day after CCI surgery at acupoint Huantiao (GB

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30; Chen et al., 2015). Stimulation for 30 min daily and for 14 days in total significantly increased the paw withdrawal threshold in rats with CCI. The elevated expression of P2X4 as well as interferon-γ (IFN-γ) mRNA and protein in the spinal cord of CCI rats were suppressed by EAP. Intrathecal injection of IFN-γ also promoted the appearance of P2X4Rs on microglia. However, EAP did not exert the same analgesic effect after intrathecal IFN-γ injection. It was concluded that EAP ameliorated tactile allodynia after peripheral nerve injury by down-regulating excessive expression of IFN-γ in the spinal

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cord and subsequently reduced the expression of P2X4Rs. Another report focused on visceral hypersensitivity after colorectal distension in rats indicating that EAP at the Shangjuxu (ST37) and Tianshu (ST25) acupoints not only markedly decreased abdominal withdrawal reflex scores in rats with visceral hypersensitivity, but in addition P2X4R immunoreactivity in colon and spinal cord was decreased after EAP (Guo et al., 2013).

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P2X7 receptors and acupuncture analgesia

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The P2X7R is an unusual member in P2XR superfamily. First, the C-terminal domain is some 200 amino acids longer than that of the other superfamily members and has been implicated in regulating receptor function including activation of signaling pathways, cellular localization, protein-protein interactions, and post-translational

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modification (Costa-Junior et al., 2011). Secondly, the P2X7R can function as a

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bifunctional receptor, i.e., either as a non-selective cationic channel after activation by relatively low concentrations of ATP, which however may form on long-lasting activation

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by high ATP concentrations large diameter non-selective pores with permeability to

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molecules of molecular mass up to ~900Da (Surprenant et al.,1996; Pelegrin and Surprenant, 2006; Locovei et al., 2007). The first evidence for the involvement of

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P2X7Rs in chronic inflammatory and neuropathic pain was derived from a study with P2X7R-deficient mice (Chessell et al., 2005). Currently, the general belief is that

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P2X7Rs are also involved in bone cancer pain, extra-territorial facial pain, diabetic neuropathic pain, chronic postsurgical pain and chronic pain sensitivity associated with chronic inflammatory and neuropathic pain (Inoue et al., 2007; Carroll et al., 2009; Wu

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et al., 2017; Li et al., 2017; Liu et al., 2017; Falk et al., 2015;Ying et al., 2014; Murasaki et al., 2013;Sorge et al., 2012). Activation of the apoptotic caspase enzyme cascade and induction of a rapid maturation and release of the pro-inflammatory cytokine

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interleukin-1β (IL-1β) by P2X7Rs and their distribution at peripheral and central immunocytes (lymphocytes, monocytes/macrophages, microglia, astrocytes) enables them to become key players at the neuroimmune interface (Carroll et al., 2009; Burnstock et al., 2011). Systemic application of P2X7R antagonists such as A-438079 produced dose-dependent antinociceptive effects in models of neuropathic (Nelson et al., 2006) and inflammatory pain (Honore et al., 2006). Administration of TAT-P451, a peptide corresponding to the P2X7R C-terminal domain, which blocked pore formation

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but not cation channel activity, selectively reduced nerve injury and inflammatory allodynia only in mice with the pore-forming P2rx7 allele (Sorge et al., 2012). In the case of neuropathic pain, P2X7Rs located at satellite glial cells (astrocyte-like cells in sensory ganglia) initiate an inflammatory reaction (Villa et al., 2010). Accordingly, disruption of P2X7Rs not only altered inflammatory pain but also reduced pain associated with nerve injury (Chessell et al., 2005). The role of P2X7Rs in AA has been tested in three different types of pain models.

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Visceral hyperalgesia treated with moxibustion was firstly investigated. In this series of experiments, a new moxibustion manipulation technique, named heat-sensitive

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moxibustion (HSM) was used (Liu et al., 2015). That is, Dachangshu acupoint (BL25) or a nearby non-acupoint (1 cm outside of BL25) were heated by moxibustion using a moxibustion cigar produced from mugwort four times a day for 8 consecutive days. It was reported that HSM at BL25 significantly decreased the abdominal withdrawal reflex

also

prevented

by

moxibustion

as

documented

by

quantitative

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was

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scores and the up-regulation of P2X7Rs in L6-S1 DRGs caused by balloon distension

immunohistochemistry, real-time RT-PCR, and western blotting.

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The second study was performed in CCI-induced neuropathic pain with EAP at

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Huantiao acupoint (Xu et al., 2016). EAP was applied at a frequency of 2 Hz for 30 minutes per day. EAP treatment was initiated on the day after CCI surgery or dibenzoyl-

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ATP (BzATP) intrathecal injection and lasted for 14 days. EAP at Huantiao acupoint increased paw withdrawal threshold on day 5 and paw withdrawal latency on day 7. EAP also inhibited spinal P2X7 immunoreactive microglia activation induced by CCI or

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BzATP administration, which was accompanied by the suppression of spinal IL-1β and IL-18 overexpression.

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Finally, neck-incision pain model was also used to underline the role of P2X7Rs in AA

(Gao et al., 2017). Under isoflurane inhalational anesthesia, repeated blunt dissection stimulation along the bilateral sternohyoideus around the thyroid gland regions for 30

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min with a forceps was applied to induce neck-incision pain model. Thermal pain thresholds were tested before and after the neck-incision surgery (4 h after each EAP intervention). EAP stimulation (2 Hz/100 Hz frequency) under light anesthesia with isoflurane at bilateral LI18 or LI4-PC6 at bilateral Zusanli (ST36)-Yanglingquan (GB34) was applied during the process of neck-incision surgery. These experiments demonstrated that two sessions of EAP stimulation of both LI18 and LI4-PC6, but not ST36-GB34, attenuated the thermal hyperalgesia of the local neck incision region in this

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pain model. These results implied that, to maintain homeostasis at the early stage of neck incision, EAP may suppress fractalkine/CX3CR1/p38 MAPK-induced neuronmicroglia crosstalk through regulation of ATP release and P2X7R activity in microglia.

Acupuncture-induced analgesia: ATP versus adenosine effects

Acupurines are promising signaling molecules to explain the mechanism of

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acupuncture, especially in AA. This complements a theory according to which mainly central opioids are responsible for analgesia caused by AP, and in addition it provides

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an explanation for the observed effectiveness of AP in human patients. It is proposed that the mechanical, electrical or thermal stimulation of acupoints by AP needles, as well as moxibustion or cupping causes the release of ATP, which can be enzymatically degraded to a whole range of biologically active metabolites (ATP, ADP, AMP, ADO)

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(Tang et al., 2016). It is a remote possibility that the stimulation of any of the P2XR

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types releases ADO, but for this idea there is hitherto no experimental evidence. AP causes pain by acting at P2X3,4,7Rs, while ADP causes analgesia by acting at

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P2Y1,12Rs or pain by acting at P2Y1 or P2Y2Rs. Eventually, ADO has an analgesic

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activity especially via A1R stimulation, although under special conditions (inflammatory

(Tang et al., 2016).

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pain, A2ARs; neuropathic pain, A3Rs) other ADO receptor-types may also participate

In the course of the recent 10 years, it was unequivocally shown that A1Rs (P1Rs)

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mediate AA for inflammatory and neuropathic pain. However, further experiments demonstrated that the P2X family receptors (P2X2-4,7) have an increased expression and function in peripheral and central sensory neurons during inflammatory, neuropathic

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and visceral pain. It has also been shown that EAP or moxibustion at different acupoints on the one hand induced analgesia and on the other hand decreased the facilitated expression of P2X3,4,7R-types in DRG, spinal cord, pre-frontal cortex, nucleus

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accumbens and increased it in periaqueductal grey (Table 1). However, the modulation of P2XR-types might be the consequence rather than the

reason for anti-nociception. An increased local or central ATP concentration caused by any type of AP stimulation could result, because of enzymatic degradation of ATP, in an elevated tissue level of ADO. This ADO may then act at A1Rs and normalize pain sensation which, as a consequence, then leads to a reversal of P2X3R up-regulation. Another possibility is, however, that ATP stimulates via P2X2-4,7Rs other neuronal

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pathways than those directly involved in the mediation of painful stimuli and thereby causes analgesia. Of these receptor-types P2X3Rs are located at the DRG neuronal cell bodies or their peripheral and central terminals, while P2X7Rs occur i.e. on peripheral immunocytes or satellite glial cells in sensory ganglia. Additional P2XR-types located in the CNS may participate as well.

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Further relevant questions

A number of interesting questions have been pointed out in our previous review

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(Tang et al., 2016). Here we will update this previous list as follows:

Purines: It has been shown that different forms of AP stimulation release ATP from the cell interior to the extracellular space. What is the role of the raised local ATP levels in

U

AP-induced analgesia? Does local ATP functions only as a source of enzymatically

N

generated ADO, or does ATP/ADP participate in AA by stimulating P2X and probably

A

also P2YRs?

M

P2 receptors: Based on agonist efficacy and desensitization characteristics, P2X receptors were classified into three distinct groups (1) high affinity for ATP (P2X1,

ED

P2X3Rs); (2) lower affinity for ATP (P2X2-5Rs); (3) very low affinity for ATP (P2X7Rs). Whereas some P2XRs rapidly desensitize (P2X1,3Rs), others desensitize only slowly.

PT

Apart from a range of P2XRs also P2YRs might participate in AP-induced analgesia (Gerevich and Illes, 2004; Ando et al., 2010; Barragán-Iglesias et al., 2014, 2015, 2016). A further important point is that interactions between different purine receptors

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(e.g. P2X4 and P2X7, P2Y1 and P2X3) or between pain-sensitive purinoceptors and non-purinoceptors (e.g. TRPV1 and P2X3, ASIC3 and P2X3) can fine tune the analgesic effect of AP (Gerevich et al., 2007; Koles et al., 2008; Bernier et al., 2018;

A

Stephan et al., 2018). Thus, it should be clarified whether all these factors or only some of them are primarily involved in AA.

Pain conditions: In ICD-11 (International Classification of Diseases 11th Revision), 7 major categories of chronic pain were presented (Treede et al., 2015): chronic primary pain, chronic cancer pain, chronic postsurgical and posttraumatic pain, chronic neuropathic pain, chronic headache and orofacial pain, chronic visceral pain and chronic

14

musculoskeletal pain. Clinical trials with AP in other types of painful conditions (low back pain, cancer pain, migraine, knee osteoarthritis) have demonstrated that this manipulation is able to relieve pain (Berman et al., 2010; Hershman et al., 2018; Deng et al., 2018; Zhao et al., 2017; Scharf et al., 2006). The question is which type(s) of P2 receptors is/are particularly involved in AA for these pain conditions.

Multiple and new experimental tools: A real deficit of most studies elucidating the role of

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purines in AA is that a selective antagonist for P2X3Rs has been used only in some cases and P2X4 and P2X7R antagonists have not been utilized at all. It is an absolute

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necessity to use multiple and new experimental tools and methods in order to clarify the role of purinoceptors in AP-induced analgesia. These range from molecular biology to genetic

engineering

(knock-down,

knock-out,

gene

editing),

high-throughput

sequencing, optogenetics, calcium imaging, Designer Receptors Exclusively Activated

U

by Designer Drugs (DREADD), etc. (Latremoliere and Costigan, 2018; Moutal et al.,

N

2017;Li et al., 2018; Xie et al., 2018; Anderson et al., 2018; Grace et al., 2018).

A

AA in humans: ATP, as the ligand of P2 receptors has been tested in clinical trials and

M

showed analgesic effect on intravenous or intrathecal application (Hayashida et al., 2005), although on subcutaneous application it proved to induce pain (Chizh and Illes,

ED

2001). It appears likely that the analgesic effect was due to degradation to ADO and the subsequent activation of ADORs. There are no data available on the concentration of

PT

ATP in blood or preferably in the cerebrospinal fluid after AP. Moreover, an imminent question is whether single nucleotide polymorphisms of the involved P2 receptors (P2X7, P2Y12) (Sorge et al., 2012; Ide et al., 2014; Sumitani et al., 2018) might also

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modify the alleviation of pain by AP or contribute to distinguish the responders and non-

A

responders to AP with relation to analgesia.

Conclusions

Whereas there is unequivocal evidence that ATP is locally released by all forms of AP stimulation, and that this ATP serves as a precursor for ADO, which activates neuronal A1Rs, causing analgesia, the role of ATP itself is far from being understood in these processes. ATP is a sensory transmitter/modulator reinforcing nociception at all levels

15

of the neuronal pain pathways connecting peripheral sites with higher CNS centers. Three types of P2XRs are involved in these functions: P2X3Rs are located at the sensory neurons themselves, while P2X4Rs (microglia) and P2X7Rs (microglia, astrocytes, oligodendrocytes) are situated mostly at glial cells. Hence P2X3Rs modulate neuronal functions directly, while P2X4/P2X7Rs act via an indirect glia-neuron crosstalk. Painful stimuli invariably increase the quantity of P2X3,4,7R mRNA and protein in dorsal root ganglia, spinal cord ventral horn, periaqueductal grey, nucleus accumbens,

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and pre-frontal cortex. AP has been reported to counteract this increased receptor density and in consequence to restitute the pathologically low pain threshold. In spite of

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a large amount of experimental work it is not yet clear whether this effect of AP is due to P2X/P2YR activation in addition to the occupation of A1Rs by the enzymatic breakdown product ADO. The desensitization of P2X3Rs as well as the analgesic effect of P2Y1Rs has to be considered in this respect. However, it may be hypothesized that the

U

activation of P2X3,4,7Rs probably alters pain sensation by stimulating other neuronal

M

Declaration of conflicting interest

A

N

pathways than those directly involved in the mediation of painful stimuli.

Funding

PT

ED

The authors declare no competing financial interests.

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This work has been supported by grants from Deutsche Forschungsgemeinschaft (IL

20/21-1), Sino-German Centre (GZ919), Sichuan Provincial Administration of Foreign Affairs (SZD201846, SZD201731), Science &Technology Department of Sichuan (2018HH0123),

National

Natural

A

Province

(81373735,81774437,81704190,81873240) University of Sichuan Province (16TD0015).

and

Science Innovative

Foundation Research

of

China

Team

in

16

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Figure Legends

Fig. 1. Acupurine involvement in acupuncture therapy. (A) Acupuncture (AP) family procedures (e.g. needle insertion, cupping, moxibustion, chemical AP, etc.) stimulate by physical or chemical means the acupoints of the skin. This causes the release of acupurines from the cell interior into the extracellular space. (B) Acupurine structures

27

are ATP and its enzymatic metabolites ADP, AMP and adenosine. (C) These endogenous agonists stimulate P1 (A1) and P2 (P2X1-7 and P2Y1,2,4,6,11-14) receptors in the peripheral and central nervous systems to modulate pain sensation. The multitude of agonists and the abundance of their receptors causing sometimes opposing effects create a wide spectrum of neuronal responses participating in

A

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PT

ED

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A

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acupuncture analgesia to a variable extent.

A

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PT

ED

M

A

N

U

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28

Fig. 2.

Possible neuronal effects participating in acupuncture analgesia. (A)

Acupuncture family procedures (shown are needle insertion and moxibustion) stimulate the skin, subcutaneous tissue and muscle. (B) Acupuncture releases ATP from the cell interior into the extracellular space. ATP and its enzymatic degradation products stimulate a range of P1 (A1) and P2X/P2Y receptors located at the terminals of sensory nerves originating from dorsal root ganglia (DRG). (C) These receptors modulate

29

directly the propagation of action potentials to the DRG neurons themselves but indirectly also the chemical neurotransmission of the ascending neuronal pathways ending in higher pain centers. A segment of the spinal cord with the attached DRG neuronal cell bodies as well as the sensory inputs and motor outputs are shown in the

A

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PT

ED

M

A

N

U

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inset.

I N U SC R

Pain condition

P2 receptor

P2 response

rat

Neuropathic pain

P2X3

↑

rat

Neuropathic pain

P2X3

↑

rat

Neuropathic pain

P2X3

Visceral hypersensiticity

CC E

rat

Visceral hypersensiticity

A

rat

rat

rat

rat

P2X3

PT

rat

ED

Species

M

Table 1. P2 receptors involved in acupuncture (AP)-induced analgesia

Visceral hypersensiticity

P2X2

P2X3

↑

Immunofluorescence

A

Western blotting

Immunohistochemistry In situ hybridization

AP family procedures EAP EAP

Electrophysiological recordings

EAP

Immunohistochemistry

EAP

AP site ST 36 GB 34 ST 36

Effect of AP on P2 ↓

P2X3

Inflammatory pain

P2X3

Reference

DRG:L4-L5

Wang and others 2014

DRG:L4-L6 DRG:L4-L5

Tu and others 2012

↓

DRG:L4-L6

Cheng and others 2013

↓

DRG:L6-S3

Weng and others 2013

↓

DRG:L6-S3

Weng and others 2013

ST37

↓

Clonic myenteric plexus DRG & spinal cord (L6-S2) Prefrontal & anterior cingulate cortex

Weng and others 2015

ST36

2Hz: ↓ L4-6 100 Hz: ↓L5,L6

DRG:L4,5,6

He and others 2017

DRG:L4,5,6

Fang and others 2018

DRG:L4-6

Shin and others 2018

ST 36 GB 34 ST25 ST37

↑

Immunohistochemistry

EAP

ST25 ST37

Immunofluorescence ↑

Real-time PCR

ST25 EAP

↑

Immunofluorescence

EAP

BL60 Immunofluorescence

P2X3

Location

↓

GB 34

Immunohistochemistry

Diabetic neuropathic pain

Neuropathic pain

↑

P2 receptor measurement

↑

↑

Western blotting Reverse transcription PCR

ST36 EAP Needle insertion with rotation

2Hz: ↓ L6

BL60

100 Hz: ↓L4-L6

ST36

↑↑

I N U SC R 31

P2X2

-

rat

Neuropathic pain

P2X3

↑

rat

Neuropathic pain

P2X3

↑

rat

Visceral hypersensiticity

P2X4

ED

Visceral hypersensiticity

CC E

rat

Neuropathic pain

A

rat

rat

rat

P2X4

PT

rat

Neuropathic pain

Neck-incision pain

Neck-incision pain

P2X7

Immunofluorescence Western blotting Real-time PCR

A

Neuropathic pain

M

rat

Reverse transcription PCR

↑

↑

Western blotting Immunohistochemistry

Needle insertion with rotation EAP EAP

EAP

ST36 ST36 SP6

-

DRG:L4-6

Shin and others 2018rar

↑↑

Periaqueductal gray

Xiao and others 2010

Spinal cord

Yu and others 2013

ST36

2Hz: ↓↓

GB34

15Hz: ↓

ST37

↓

ST25

Colon Spinal cord

Guo and others 2013

Immunohistochemistry Real-time PCR

EAP

GB30

↓

Spinal cord: L4-5 microglia

Chen and others 2014

moxibustion

BL25

↓

DRG: L6-S1

Liu and others 2014

EAP

GB30

↓

Spinal cord: L4-5 microglia

Xu and others 2016

LI18

LI 18 : ↑ 4h

LI4-PC6

LI4-PC6: ↑ 4h Cervical spinal cord:C2-5

Gao and others 2017

Cervical spinal cord:C2-5

Gao and others 2017

Western blotting Immunohistochemistry ↑

Reverse transcriptional PCR Western blotting Immunofluorescence

P2X7

↑

Real-time PCR Western blotting

- 4h ↑ 24h P2X7

P2X1

Real-time PCR Western blotting EAP

↑ 48h

- 4h - 24h

Real-time PCR

EAP

ST36GB34

LI18

LI 18 : ↓ 24h,48h LI4-PC6: ↓ 24h,48h -4h -24h

I N U SC R 32

rat

Neck-incision pain

P2X3

rat

Neck-incision pain

P2X4

rat

Neck-incision pain

P2X5

rat

Neck-incision pain

P2X6

rat

Neck-incision pain

rat

Neck-incision pain

rat

Neck-incision pain

P2Y4

rat

Neck-incision pain

P2Y6

rat

Neck-incision pain

P2Y12

rat

Neck-incision pain

P2Y13

rat

Neck-incision pain

P2Y14

P2Y1 P2Y2

PT

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A

- 4h - 24h ↑4h ↑ 24h - 4h - 24h

Real-time PCR

EAP

LI18

Real-time PCR

EAP

LI18

Real-time PCR

EAP

LI18

Real-time PCR

EAP

LI18

Real-time PCR

EAP

LI18

Real-time PCR

EAP

LI18

Real-time PCR

EAP

LI18

Real-time PCR

EAP

LI18

Real-time PCR

EAP

LI18

Real-time PCR

EAP

LI18

Real-time PCR

EAP

LI18

Real-time PCR

EAP

LI18

A

P2X2

- 4h

- 24h

M

Neck-incision pain

ED

rat

- 4h

- 24h - 4h - 24h - 4h - 24h - 4h - 24h - 4h - 24h ↑ 4h ↑24h - 4h - 24h - 4h - 24h

↑, potentiation of pain; ↓, inhibition of pain; -, no relevance to pain

- 4h - 24h -4h ↓ 24h - 4h - 24h - 4h - 24h - 4h - 24h - 4h - 24h - 4h - 24h - 4h - 24h - 4h - 24h - 4h ↓ 24h - 4h - 24h - 4h - 24h

Cervical spinal cord:C2-5

Gao and others 2017

Cervical spinal cord:C2-5

Gao and others 2017

Cervical spinal cord:C2-5

Gao and others 2017

Cervical spinal cord:C2-5

Gao and others 2017

Cervical spinal cord:C2-5

Gao and others 2017

Cervical spinal cord:C2-5

Gao and others 2017

Cervical spinal cord:C2-5

Gao and others 2017

Cervical spinal cord:C2-5

Gao and others 2017

Cervical spinal cord:C2-5

Gao and others 2017

Cervical spinal cord:C2-5

Gao and others 2017

Cervical spinal cord:C2-5

Gao and others 2017

Cervical spinal cord:C2-5

Gao and others 2017