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. 2024 Jun 26;44(4):660–669. doi: 10.19852/j.cnki.jtcm.20240626.003

Adenosine triphosphate mediates the pain tolerance effect of manual acupuncture at Zusanli (ST36) in mice

Zhongzheng LI 1,2, Yadan ZHAO 3, Weigang Ma 1,2, Yonglong Zhang 1,2, Zhifang XU 1,2, Qiang XI 5, Yanqi LI 5, Siru QIN 1,2, Zichen ZHANG 1,2, Songtao WANG 1,2, Xue ZHAO 4, Yangyang LIU 4, Yi GUO 1,2,✉, Yongming GUO 4,✉
PMCID: PMC11337262  PMID: 39066526

Abstract

OBJECTIVE:

To investigate the mechanisms behind the effects of acupuncture in Traditional Chinese Medicine, we delved into the adenosine triphosphate/peripheral purinergic P2X receptor 3 (ATP/P2X3) receptor signaling system as an indicator of the body's energy state, commonly referred to as "Qi".

METHODS:

The tail-flick test was utilized to explore the impact of acupuncture on pain tolerance threshold (PTT) in mice, while also assessing adenosine (ADO) levels and adenylate energy charge (EC) at Zusanli (ST36). The study further investigated the dose-dependent effects of acupuncture on PTT and ADO levels at Zusanli (ST36). To shed light on the underlying mechanisms of acupuncture's effects, the study examined the impact of ATP, a P2X3 receptor antagonist, and adenosine disodium on PTT following acupuncture administration.

RESULTS:

Acupuncture at Zusanli (ST36) led to significant improvements in PTT in mice, with the most effective interventions being twirling for 2 min and needle retention for 28 min. These interventions also resulted in significant increases in ATP levels. The effects of acupuncture were further augmented by administration of different doses of ATP at Zusanli (ST36), and pretreatment with a P2X3 receptor antagonist decreased PTT. Adenylate EC peaked at 30 min after intraperitoneal injection of ATP, and pretreatment with various doses of i.p. ATP 30 min prior to acupuncture increased PTT in a dose-dependent manner. Additionally, pretreatment with an i.p. or intramuscular injection of adenosine disodium enhanced the effects of acupuncture.

CONCLUSION:

This research provides compelling evidence that ATP is involved in the regulation of PTT through acupuncture, revealing new avenues for achieving enhanced clinical outcomes.

Keywords: acupuncture; point ST36 (Zusanli); adenosine triphosphate; receptors, purinergic P2X3; energy state

1. INTRODUCTION

Acupuncture, a traditional Chinese medical practice, has gained widespread use in the treatment of diverse disorders.1,2 In 2002, the World Health Organization identified 107 specific indications that are potentially amenable to acupuncture therapy.3 While the efficacy and mechanisms of acupuncture in treating certain pathologies have been investigated in previous studies,4,5 there is currently a lack of data regarding the factors that impact the effects of acupuncture and their corresponding dose-response relationships. The effects of acupuncture are closely associated with various chemical mediators at acupoints and metabolic processes in the body. Current evidence indicates that the positive effects of acupuncture on bodily functions and disease treatment are based on the transmission and integration of acupuncture signals through the nervous system, which is achieved through the activation of specific types of peripheral afferent nerve fibers.6,7 Manual acupuncture (MA) is a widely utilized Traditional Chinese Medicine (TCM) technique, which aims to restore organic homeostasis by precisely inserting acupuncture needles into specific acupoints along the meridians.8 Acupuncture effects are directly influenced by various factors, including the physical sensory stimulus, such as the depth of insertion, intensity, frequency, and duration of the operation. These parameters are modulated by the acupuncturist, and the physical energy state of the patient also plays a critical role in determining the outcomes of acupuncture.9,10

Acupuncture has been traditionally used to balance and harmonize the flow of Qi and blood in the body. In TCM, the term Qi encompasses the vital and nourishing substances that circulate through the body to sustain and regulate various physical functions.11 The physical manifestation of Qi is reflected in the functional state of cells, which is influenced by the intricate interplay of neuroendocrine regulation and energy conversion processes within the body.12 The mitochondria serve as the main locations for intracellular oxidative phosphorylation and the synthesis of adenosine triphosphate (ATP), which is the primary source of energy for various cellular activities.11 When it comes to regulating bioenergetics, the mitochondrion serves as the fundamental unit of energy flow, with ATP production providing a vital boost to organic energy status. This "Qi-invigorating" action is essential for maintaining optimal cellular function.13,14 ATP plays a crucial role in various biological processes such as energy metabolism, DNA replication, and cell transcription, and also serves as a signaling molecule in both intracellular and extracellular signaling transduction pathways within the nervous system. Notably, the application of acupuncture at specific acupoints triggers the release of ATP from cells into the extracellular matrix, which subsequently under-goes degradation into adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine (ADO).15

Research indicates that purinergic signaling plays a significant role in the cellular mechanisms underlying the effects of acupuncture, as supported by evidence.16 The peripheral purinergic P2X receptor serves as a gated ion channel for ATP and is present on the cell membranes of neurons, immune cells, and cancer cells. Its pivotal regulatory functions include modulation of neuronal excitability, neurotransmitter release, and transduction of pain signals.17,18 Out of all the P2X phenotypes, the purinergic adenosine triphosphate/peripheral purinergic P2X receptor 3 (P2X3) receptor is exclusively found in small and medium-sized sensory neurons that are involved in processing nociceptive information. The expression of the P2X3 receptor is remarkably specific to nociception and is intensified in the pain transmission mechanisms of central sensory neurons.19,20 Research indicates that acupuncture stimulates the release of ATP into the extracellular space, which then breaks down into ADO, a neurotransmitter that activates neurons through the A1 receptor. However, there is still limited understanding of the exact roles of ATP and the P2X3 receptor in the mechanisms behind the effects of acupuncture.16,21

Our hypothesis suggests that ATP plays a synergistic role in the initiation of acupuncture. To explore this idea, our study aimed to investigate how ATP and the P2X3 receptor modulate pain tolerance threshold (PTT), which serves as an observational index for the effects of acupuncture. Specifically, we examined the dose-effect relationships between acupuncture, ATP, and its metabolites. Moreover, we explored the interdependent functions of ATP and its receptor, P2X3, at the Zusanli (ST36) acupoint. We assessed the impact of ATP-mediated activity on both the overall body and local acupoints to determine how energy levels affect the effectiveness of acupuncture. Our investigation establishes a scientific foundation for understanding the mechanisms underlying acupuncture's effects on energy metabolism and signal transmission.

2. MATERIALS AND METHODS

2.1. Animals

The animal experimentation was conducted in compliance with the Ministry of Science and Technology of China's guidelines for the care and use of laboratory animals. The Animal Care and Use Committee of Tianjin University of Traditional Chinese Medicine approved the protocol (Permit Number: TCM-LAEC2012009). Healthy Kunming adult male mice (28-32 g, 6 to 8 weeks old, specific pathogen free grade, n = 700) were procured from the Animal Experiment Center, Institute of Environmental Medicine, Academy of Military Medical Sciences, People's Liberation Army. The animals were handled according to the Tianjin University of Traditional Chinese Medicine's guidelines for the care and use of laboratory animals. They were group-housed in the Laboratory Animal Centre of Tianjin University of Traditional Chinese Medicine, acclimatized to the housing conditions for 1 week, and given ad libitum access to food and water in a humidity-controlled (55% ± 5%), temperature-controlled (22 ± 2) ℃, 12 h light/dark cycle environment. Prior to tissue collection, all mice were anesthetized using 4% isoflurane with oxygen as the carrier (Shenzhen RWD Life Technology Co., Ltd., Shenzhen, China).

2.2. Determination of pain tolerance threshold

The tails of each mouse were stimulated using TF2-photothermal pain detectors from the Institute of Medicine at the Chinese Academy of Medical Sciences. Tail withdrawal latency was measured as a proxy for photothermal pain (PTT). Prior to the experiment, the mice were acclimatized to the environment by being kept in movable transparent chambers for 20 min. The measurements were taken three times, with a 10-minute interval between each measurement, and the average was calculated. To prevent burns, the upper limit of the instrument was set to 20 s. Mice with a stable baseline PTT (between 6.5 and 7.5 s) were selected for further analysis, resulting in a 90% rejection rate. To ensure consistency, behavioral tests were evaluated independently by unbiased observers who were blinded to group treatments. The observers underwent training to ensure uniformity in scoring criteria. The subsequent analyses employed the behavioral scores obtained.

2.3. Experimental design

In this study, we conducted a series of five experiments to investigate the effects of acupuncture on various parameters. In Experiment 1, we utilized PTT as an effect index to compare the acupuncture effects of different MA and determine the optimal acupuncture parameters. These parameters were then applied in the subsequent experiments. Experiment 2 involved observing the changes in local adenosine (ATP, ADP, AMP) in acupoints after different MA, and analyzing the correlation between local ATP content and acupuncture effect (PTT). To investigate the role of ATP in the initiation of acupuncture effect, Experiment 3 involved injecting different concentrations of ATP solution and P2X3 receptor blocker into the acupoint area. Experiment 4 focused on the effects of the body's energy state on acupuncture by administering i.p. injections of ATP in mice and observing changes in adenosine energy charge in plasma at different time points. Finally, Experiment 5 explored the effects of an energy supplement mixture containing ATP, Adenosine Disodium, on acupuncture by observing the changes in acupuncture effect after different injection methods of energy mixture pretreatment.

2.4. Acupoint selection and Acupuncture intervention

The mice were immobilized using fixators, and the area of Zusanli (ST36) on their right hindlimb was exposed. Zusanli (ST36) in mice is located 5 mm inferior to the capitulum fibulae and posterior-lateral knee joint of the hindlimb. An acupuncture needle (Φ 0.28 mm × 25 mm, Huatuo, China) was inserted into Zusanli (ST36) to a depth of 3 mm and secured with tape to ensure consistent depth. After obtaining Qi, the needle handle is held between the thumb and index finger, and rotated equally in both directions with a twisting angle between 90° to 180°. The needle was then twisted for 2 min. To examine the effect of MA on PTT in mice and determine the optimal intervention parameters in experiment 1, and explore the relationship between adenylate content at Zusanli (ST36) and effects of MA in experiment 2, the mice were divided into different groups using a random number generator. The groups were as follows: Group A: the needle was twisted for 2 min, Group B: the needle was twisted for 2 min and retained for 8 min, Group C: the needle was twisted for 2 min and retained for 18 min, Group D: the needle was twisted for 2 min and retained for 28 min, Group E: the needle was twisted for 2 min and retained for 28 min, followed by a 10 min resting period. To investigate the involvement of ATP, P2X3 receptor, and adenosine disodium in the mechanisms underlying acupuncture, experiments 3, 4, and 5 were conducted. The acupuncture protocol used in the Acu group was refined based on the findings from the initial experiment, and the optimal intervention parameters were selected for subsequent experiments.

2.5. Experimental treatments

The ATP disodium was dissolved in normal saline to create solutions of 10, 20, and 40 µg/µL for the ATP-1, ATP-2, and ATP-3 groups, respectively. Each acupoint was injected with 2.6 µL of the corresponding ATP solution. The A-317491 compound, dissolved in sterile saline and diluted to a concentration of 5 nmol/μL, was injected with a standard microinjector (5 μL) at a volume of 3 μL per acupoint for the Sal group. The ATP and A-317491/ATP/saline injections are illustrated. ATP disodium was meticulously administered to every mouse in the ATP group via i.p injection at dosages of 2 (ATP-4), 4 (ATP-5), or 8 (ATP-6) µg/g. In mice without blood withdrawal, a syringe was inserted into either the right Zusanli (ST36) acupoint or abdomen, and the contents were meticulously injected from deep to shallow layers, ensuring that the tissue was adequately filled from the skin to muscle layers. We utilized a mixture of 2.1 mL normal saline solution containing 50 U coenzyme A (100 U, 20111005, Tianjin Biochemical Pharmaceutical Co., Ltd., Tianjin, China), 20 mg ATP disodium (2 mL: 20 mg, 12040911, Jiangsu Shenlong Pharmaceutical Co., Ltd., Jiangsu, China), and 4 U insulin (10 mL: 400 U, 1206205, Tianjin Biochemical Pharmaceutical Co., Ltd., Tianjin, China) to synthesize adenosine disodium. To eliminate any potential impact of the injection process on the effectiveness of acupuncture, we included a control (CTL) group where the mice were immobilized in the same manner as the other groups, but did not receive any intervention. In the AD group and Sal group, a 1-mL medical syringe was used to administer 0.1-mL injections of adenosine disodium and normal saline, respectively, via both intraperitoneal (i.p.) and intramuscular (i.m.) routes. The i.m. injection was performed in the left tibialis anterior muscle, as shown in Figure4A.

Figure 4. Pretreatment with adenosine disodium ameliorated the effects of acupuncture.

Figure 4

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A: flow chart of experimental intervention. Intervention stages are represented by different symbols: ∆: intraperitoneal (i.p.) injection of saline; ▲: i.p. injection of adenosine disodium (represented by AD); ▲: i.m. injection of saline; ♡: i.m. injection of AD; ●: determination of PTT after intervention; ▬: MA intervention; B: comparison of PTT in each group. PTT of mice in the Control (CTL) group was measured to indicate the basal level. PTT of mice in the Sal (i.p.), Sal (i.m.), AD (i.p.), and AD (i.m.) groups was measured after i.p. or i.m. injections of saline or AD. PTT of mice in the Acu group was measured after MA intervention (needle was twisted for 2 min and retained for 28 min). Mice in the Acu + AD (i.p.) and Acu + AD (i.m.) groups were subjected to MA (the intervention method was the same as that in the Acu group) after i.p. or i.m. injections of AD, respectively, followed by measurement of PTT. MA: Manual acupuncture; PTT: pain tolerance threshold; AD: adenosine disodium. Data are presented as mean ± standard deviation (n = 10 per group). The single factor analysis of variance of completely randomized design was used for parameter comparison between groups. Compared with group Acu, aP < 0.05; compared with group Acu +AD (i.p.), cP < 0.05; compared with group AD (i.m.), bP < 0.05.

2.6. High-performance liquid chromatography

To measure the plasma energy charge (EC) levels, blood samples were collected from mice that had inhaled 4% isoflurane with oxygen (0.7 mL/100 g of mouse weight) through their eyeballs. After centrifugation (13 000 rpm at 4 ℃ for 10 min), the plasma was separated. Similarly, to determine the adenylate content at Zusanli (ST36), a tissue sample (2 mm3) was taken from the right side of the body, precisely from Zusanli (ST36), and homogenized quickly on ice with 200 μL of perchloric acid. The homogenate was then centrifuged at 13 000 rpm at 4 ℃ for 10 min. After being stored at 4 ℃ for 1 h, the supernatant (pH 7.0) underwent a second round of centrifugation at 13 000 rpm for 5 min. Subsequently, the resulting supernatant was directly subjected to analysis using a CoMetro 6000 high-performance liquid chromatography (HPLC) system (CoMetro Inc., South-plainfield, NJ, USA). Chromatographic separation was accomplished using a Kiomasn C18 column (5 μm, 250.0 mm ×  Φ 4.6 mm) which was connected in series to a UV absorbance diode array detector. The samples were measured using a mobile phase consisting of 60 mmol/L K2HPO4 (5 : 1, CH3OH 1%) and HPLC grade water (pH 5.2), with 40-μL samples injected from an autosampler at a rate of 1.0 mL/min. The detection wavelength used was 259 nm, and daily calibration curves were prepared using 3-point standards for ATP, ADP, and AMP (0.2, 2, and 50 μg/mL, respectively). To quantitatively evaluate the samples, we compared the retention times and UV spectra of the standard preparation and sample peaks. The concentration of the samples was determined by calculating the peak areas relative to the standard preparation, and the concentration of the standard preparation was also taken into consideration. Standard curves of the samples were used to calculate the concentrations of ATP, ADP, and AMP per gram of tissue.

2.7. Calculation of the rate of change in PTT, adenylate EC, and total adenylate pool

To assess the impact of acupuncture on PTT, we determined the rate of change in PTT using the formula: rate of change in PTT = ([PTT after acupuncture-PTT before acupuncture]/PTT before acupuncture) ×100%. To evaluate the adenylate EC, which represents the balance between energy supply and demand, we employed the formula:22 adenylate EC = (ATP + 0.5 × ADP)/(ATP + ADP + AMP). Additionally, we determined the total adenylate pool by using the formula: total adenylate pool = ATP + ADP + AMP.

2.8. Statistical analysis

The data were presented as mean ± standard deviation ($\bar{x}±s$), and statistical analyses were conducted using analysis of variance for independent samples to compare differences between two groups when the data followed a normal distribution. For homogenous variances, the least significant difference method was utilized; otherwise, Dunnett’s T3 method was used. For non-normal distributions, a Kruskal Wallis nonparametric test was performed with SPSS 11.5 (SPSS Inc., Chicago, IL, USA) Statistical significance was set at P < 0.05.

3. RESULTS

3.1. PTT was increased after MA in different acupuncture parameters

To investigate the impact of MA on PTT in mice and identify the optimal intervention parameters, we conducted a study where we twirled the needle for 2 min followed by retention for different durations in each group (n = 9 per group). PTT was assessed before and after MA intervention, and the rate of change in PTT was determined. In healthy mice, we observed an increase in PTT following various MA interventions in each group (P < 0.05, after MA intervention vs before MA intervention) (Table 1). Notably, in group D (MA twirling for 2 min and needle retention for 28 min), PTT was significantly improved compared to baseline. Comparing the rate of change in PTT before and after MA in each group, we found that the rate of change in PTT increased by 20%-50% following the different MA interventions. The rate of change in PTT was the highest in group D (46.18% ± 13.25%) compared to groups A, B, C, and E (P < 0.01). Thus, we selected these intervention parameters (MA twirling for 2 min and needle retention for 28 min) as the acupuncture protocol for subsequent experiments.

Table 1.

Comparison of pain tolerance threshold before and after acupuncture in mice of every group (s, $\bar{x}±s$)

Group n PTT before MA PTT after MA
A 9 7.4±1.7 9.8±2.3a
B 9 6.5±1.3 8.2±2.2a
C 9 6.4±0.7 7.9±2.2a
D 9 7.0±1.2 10.1±2.0a
E 9 7.0±1.1 8.4±1.3a
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Notes: group A: needle twisted immediately for 2 min. Group B: needle twisted immediately for 2 min and retained for 8 min. Group C: needle twisted immediately for 2 min and retained for 18 min. Group D: needle twisted immediately for 2 min and retained for 28 min. Group E: needle twisted immediately for 2 min and retained for 28 min before removal followed by a 10 min rest. PTT: pain tolerance threshold; MA: manual acupuncture The single factor analysis of variance of completely randomized design was used for parameter comparison between groups. Compared with the before MA, aP < 0.05.

3.2. Adenylate content at Zusanli (ST36) was increased in a dose-dependent manner after MA

To investigate the link between ATP content at Zusanli (ST36) and the effects of acupuncture, we analyzed changes in adenylate (ATP, ADP, and AMP) content at Zusanli (ST36) following different MA protocols (n = 10 per group, Figure 1A). Our results indicate that the greatest increase in ATP content was observed after a 30-minute MA intervention (twisting for 2 min and needle retention for 28 min), which caused ATP levels to rise from (2.16 ± 0.82) to (4.15 ± 1.54) μg/g (P < 0.01, group D vs CTL group; P < 0.05, group D vs group A, P < 0.05, group D vs group E) (Figure 1B). Additionally, univariate regression analysis of the data for PTT and ATP content at Zusanli (ST36) following a 30-minute MA intervention (twirling for 2 min and needle retention for 28 min) showed that PTT was positively correlated with ATP content at Zusanli (ST36) after MA (R = 0.957) (Figure 1C). There were no significant differences in ADP content at Zusanli (ST36) between the different acupuncture intervention groups or relative to basal ADP content (P > 0.05, groups A, B, C, D, and E vs CTL group) (Figure 1D). However, relative to basal levels, AMP content was significantly increased after a 10-min MA intervention (twisting for 2 min and retention for 8 min), resulting in a rise from (57 ± 18) to (108 ± 37) μg/g (P < 0.05, group B vs groups A, E, and CTL group).

Figure 1. Adenylate content at Zusanli (ST36) was increased after MA in a dose-dependent manner.

Figure 1

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A: flow chart of experimental intervention. Intervention stages are represented by different symbols: ○: determination of basal PTT; ■: detection of adenylate at Zusanli (ST36); ▬: MA intervention; ▲: needle removal. After measurements of basal PTT, different MA interventions were performed in mice. B: ATP content at Zusanli (ST36) in each group; C: Scatter plot depicting the correlation between PTT and ATP concentration at Zusanli (ST36); D: ADP content at Zusanli (ST36) in each group; E: AMP content at Zusanli (ST36) in each group. F: Adenylate EC content at Zusanli (ST36) in each group. G: Total adenylate pool content at Zusanli (ST36) in each group. Control (CTL) group: direct detection of adenylate content at Zusanli (ST36). Group A: needle twisted immediately for 2 min. Group B: needle twisted immediately for 2 min and retained for 8 min. Group C: needle twisted immediately for 2 min and retained for 18 min. Group D: needle twisted immediately for 2 min and retained for 28 min. Group E: needle twisted immediately for 2 min and retained for 28 min before removal followed by a 10-minute rest. Adenylate content at Zusanli (ST36) was measured after MA intervention. MA: manual acupuncture; PTT: pain tolerance threshold; ATP: adenosine triphosphate; ADP: adenosine diphosphate; AMP: adenosine monophosphate; EC: energy charge. Data are presented as mean ± standard deviation (n = 10 per group). The single factor analysis of variance of completely randomized design was used for parameter comparison between groups. Compared with group D, aP < 0.01 and bP < 0.05; Compared with group B, cP < 0.05.

The adenylate pool, consisting of ATP, ADP, and AMP, is subject to interconversion, resulting in changes in metabolism. The energy status can be reflected by calculating EC based on the content of these molecules. In Figure 1F, it is shown that adenosine EC significantly increased following a 30 min MA intervention (P < 0.05, group D vs groups B and E). Moreover, after a 10-minute MA intervention, the total adenylate pool at Zusanli (ST36) was found to be significantly higher (P < 0.05, group B vs CTL group and group A). Although there was a trend towards an increase in the total adenylate pool at Zusanli (ST36) after a 30-minute MA intervention, it did not reach statistical significance (P = 0.057, group D vs CTL group).

3.3. Effect of acupuncture on PTT were mediated by ATP and the P2X3 receptor at Zusanli (ST36)

After investigating the role of ATP and the P2X3 receptor in mediating the effects of acupuncture on PTT in mice (n = 10 per group, Figure 2A; n = 15 per group, supplementary Figure 1A), we found that acupoint injections of saline, ATP, or A-317491 (P2X3 receptor antagonist) had no significant effect on PTT compared to the control group (P > 0.05, Sal, ATP, and A-317491 vs CTL group) (Figures 2B, supplementary Figure 2A). These results suggest that the acupoint injection itself has no impact on PTT and that the injection of ATP alone does not produce an acupuncture-like effect.

Figure 2. Effects of acupuncture on PTT were mediated by ATP and the P2X3 receptor at Zusanli (ST36).

Figure 2

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A: flow chart of experimental intervention. Intervention stages are represented by different symbols; ∆: Acupoint injection of saline; ▲: Acupoint injection of ATP; ●: Determination of PTT after intervention; ▬: MA intervention; B: comparison of PTT in each group. PTT of mice in the Control (CTL) group was measured to indicate the basal level. PTT of mice in the Sal, ATP-1, ATP-2, and ATP-3 groups was measured after acupoint injection of saline, ATP-1 (26 µg/2.6 µL), ATP-2 (52 µg/2.6 µL), and ATP-3 (104 µg/2.6 µL), respectively. PTT of mice in the Acu group was measured after MA intervention (needle was twisted for 2 min and retained for 28 min). Mice in the Acu + Sal, Acu + ATP-1, Acu + ATP-2, and Acu + ATP-3 groups were administered MA (intervention method was the same as that in the Acu group) after acupoint injection of saline, ATP-1 (26 µg/2.6 µL), ATP-2 (52 µg/2.6 µL), and ATP-3 (104 µg/2.6 µL), respectively, followed by measurement of PTT. ATP: adenosine triphosphate; PTT: pain tolerance threshold; P2X3: peripheral purinergic P2X receptor 3; MA: manual acupuncture. Data are presented as mean ± standard deviation (n = 10 per group). The single factor analysis of variance of completely randomized design was used for parameter comparison between groups. Compared with group Acu, aP < 0.05.

However, PTT significantly increased in mice treated with acupuncture (P < 0.05, Acu vs CTL group) (Figures 2B, 3D). Furthermore, the effect of acupuncture was further enhanced by acupoint injections of ATP at three different doses, from low to high (P < 0.05, Acu + ATP-1, Acu + ATP-2, and Acu + ATP-3 vs Acu group) (Figures 2B, supplementary Figure 2A). These results suggest that pretreatment of acupoint ATP injection before acupuncture can improve the acupuncture effect. Specifically, the PTT increased from (6.7 ± 0.7) s (Acu group) to (9.1 ± 0.6) s (Acu + ATP-1 group), (9.4 ± 0.9) s (Acu + ATP-2 group), and (10.4 ± 0.6) s (Acu + ATP-3 group).

Figure 3. Systemici pre-treatment wth ATP ameliorated PTT in mice.

Figure 3

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A: dynamic adenylate EC content of mice after intraperitoneal (i.p.) injections of ATP within 70 min (n = 5 per group). B: flow chart of experimental intervention. Intervention stages are represented by different symbols: ▲: i.p. injection of ATP; ∆: i.p. injection of saline; ▬: MA intervention; ●: determination of PTT after intervention; C: comparison of PTT in each group (n = 15 per group). PTT of mice in the Control (CTL) group was measured after 30 min. PTT of mice in the Sal and ATP-5(4 µg/g) groups was measured 30 min after i.p. administration of saline and ATP-5, respectively. PTT of mice in the Acu group was measured 30 min after MA intervention (needle was twisted for 2 min and retained for 28 min). Mice in the Acu + Sal and Acu + ATP-5 groups received i.p. injections of saline and ATP, respectively. Mice were subjected to MA 30 min later (the intervention method was the same as that in the Acu group) followed by measurement of PTT. ATP: adenosine triphosphate; PTT: pain tolerance threshold; MA: Manual acupuncture; EC: energy charge. Data are presented as mean ± standard deviation. The single factor analysis of variance of completely randomized design was used for parameter comparison between groups. Compared with group Acu, aP < 0.05.

There were no significant differences observed in PTT among mice that received acupoint injections of different doses of ATP in combination with MA (P > 0.05, Acu+ATP-1 and Acu+ATP-2 vs Acu+ATP-3). These findings indicate that the dosage of ATP did not affect the effects of MA (Figure 2B). Furthermore, PTT was significantly reduced after MA was combined with acupoint injection of A-317491, as compared to MA alone (P < 0.05, F = 2.48, Acu + A-317491 vs Acu group) (supplementary Figure 1B). In contrast, PTT was significantly increased after MA was combined with acupoint injection of A-317491, compared to basal values (P < 0.05, F = 2.48, Acu + A-317491 vs CTL group) (supplementary Figure 1B). These results suggest that the acupuncture effect decreased after the injection of the P2X3 receptor antagonist at the acupoint.

3.4. PTT was augmented after systemic pre-treatment with ATP

We proceeded to investigate the impact of intraperitoneal (i.p.) ATP injections on acupuncture outcomes. To determine the optimal time to conduct our interventions, we measured plasma levels of ATP, ADP, and AMP at various time intervals (0, 15, 30, 45, 60, and 70 min) after i.p. injection of ATP, and calculated the respective EC values. Notably, the highest EC value of mouse plasma was observed at the 30 min mark (Figure 3A). Hence, we performed MA interventions 30 min following i.p. administration of ATP, at which point the ATP-consuming systems were most strongly activated and the utilization efficiency of ATP was highest (Figure 3B). This enabled us to assess PTT under different MA protocols. There were no significant effects of either saline or ATP injections on PTT (P > 0.05, Sal and ATP vs CTL group) as shown in Figure 3C. However, PTT did increase from (7.2 ± 1.1) s at baseline to (8.6 ± 1.1) s after MA and further increased to (10.6 ± 1.7) s after MA in combination with i.p. ATP administration. Moreover, PTT increased after MA at Zusanli (ST36) (P < 0.05, Acu vs CTL group) and was further amplified in mice that received MA after i.p. administration of ATP (P < 0.05, Acu + ATP vs Acu group), indicating that the combination of MA and i.p. ATP administration had a synergistic effect on acupuncture.

Based on previous experiments, a dose of 4 µg/g of ATP (ATP-5) was used, and three additional doses were administered: 2 µg/g (ATP-4), 4 µg/g (ATP-5), and 8 µg/g (ATP-6) (supplementary Figure 2A). Administering different doses of ATP through i.p. pretreatment, 30 min prior to MA, resulted in the amplification of acupuncture effects (P < 0.05, Acu + ATP-4, Acu + ATP-5, and Acu + ATP-6 vs Acu group). The most significant increase in PTT was observed at 8 µg/g (P < 0.05, Acu + ATP-4 vs Acu + ATP-6 group; Acu + ATP-5 vs Acu + ATP-6 group) (supplementary Figure 2B).

3.5. Pretreatment with adenosine disodium ameliorated the effects of acupuncture

We conducted a study to examine the effects of adenosine disodium on MA outcomes. Adenosine disodium is a clinical supplement that contains coenzyme A, ATP, and insulin, which help increase energy, promote glucose metabolism, and improve the functional state of the body and diseased organs. To evaluate the impact of adenosine disodium on PTT in mice, we combined MA with i.p. and i.m. injections of adenosine disodium (represented by "AD" in Figure 4A). Our results showed that the addition of adenosine disodium significantly increased PTT from (8.6 ± 2.7) s after MA alone to (10.8 ± 3.0) s (MA combined with i.p. injection of AD) and (10.3 ± 3.4) s (MA combined with i.m. injection of AD) (P < 0.05, F = 4.03, Acu+AD [i.p.] vs Acu group; Acu+AD [i.m.] vs Acu group) (Figure 4B).

4. DISCUSSION

Acupuncture have been extensively studied in modern research on TCM, with a particular emphasis on understanding the mechanisms behind acupuncture's analgesic effects. Among the numerous acupoints that have been identified, Zusanli (ST36) stands out as a crucial location that can be stimulated to prevent or treat a wide range of ailments. The efficacy of Zusanli (ST36) in acupuncture therapy has been supported by evidence from both ancient and modern medical practices. In fact, Zusanli (ST36) has been found to be highly effective in relieving various types of pain, including visceral, cancer-related, inflammatory, and neuropathic pain, making it a preferred acupoint for pain management.23,26 Our findings indicate that Zusanli (ST36) acupuncture increased PTT in mice, which is in line with a previous study that showed the effectiveness of this technique in improving PTT for hot water tail-flick in mice. These results underscore the value of acupuncture at Zusanli (ST36) as a promising experimental model for future research.27

The precise manipulation and regulation of acupuncture techniques and associated parameters play a crucial role in achieving optimal therapeutic outcomes and ensuring consistent clinical practice. Our study focused on investigating the impact of varying retention times during acupuncture at Zusanli (ST36) on the PTT response to thermal stimulation in mice. Specifically, we utilized a needle-twisting approach and observed significant differences in the effects of different retention times on PTT. Our findings indicate that acupuncture led to an increase in PTT in mice, with the most pronounced benefits observed with a needle-twisting duration of 2 min and a retention time of 28 min. This protocol was selected for further investigation in our study, with the effects lasting up to 10 min after needle removal, consistent with previous reports on the temporal characteristics of acupuncture's effects on PTT.28 Barlas et al 29 examined the overall time-course of electro-acupuncture (EA) on pulse transit time (PTT) in healthy adults and found that PTT reached its maximum at 30 min of EA stimulation and persisted for 10 min after needle removal. The impact of acupuncture treatment can be segmented into three distinct phases: the optimal induction period, the half-life period, and the residual period, which all exhibit an augmentation in response to increased intervention time.

The observed effects align with the neural response pattern in the brain that typically occurs during acupuncture interventions.30 Considering that the correct application of acupuncture is crucial for achieving optimal therapeutic effects, our findings can aid in determining the most suitable needle retention times for single-manipulation acupuncture. While our study primarily examined the duration of acupuncture, it is important to note that MA involves various factors, such as frequency, amplitude, angle, and duration. Thus, further investigations are necessary to gain a better understanding of how these other factors impact the therapeutic outcomes of acupuncture.

Initially, ATP was solely regarded as a crucial energy source in organisms. However, in 1929, the biological implications of purine compounds in signal transduction were discovered, and subsequent research indicated that ATP could function as a neurotransmitter. In the 1970s, Burnstock of the Institute of Autonomous Neuroscience at the University of London, England, postulated the "purinergic nerve theory". Using the research framework of acupuncture at Zusanli (ST36) to enhance the PTT of mice, we investigated the impact of ATP at both local acupoints and the entire body in the effectiveness of acupuncture.

ATP acts as an autocrine or paracrine extracellular messenger, contributing to various biological processes in cells. Being both an energy source and a neurotransmitter, ATP potentially plays a role in transmitting acupuncture information and generating acupuncture effects. Therefore, during acupuncture therapy, could the implementation of specific techniques stimulate the release of ATP by local tissue cells at the acupoints, resulting in alterations in the local ATP concentration? Our findings suggest that acupuncture had a positive impact on the tail-flick time of healthy mice and led to an initial increase and subsequent decrease in ATP content at Zusanli (ST36). This increase in ATP release due to mechanical pressure and cell rupture resulting from acupuncture may trigger cellular responses. This perspective aligns with earlier findings that suggest acupuncture can affect adenosine and ATP levels at specific acupoints.21,31 The ATP that is released undergoes degradation into essential active metabolites that play a crucial role in transmitting and regulating both peripheral pain signals and central nervous system impulses.32 Furthermore, our findings indicate a positive correlation between the efficacy of acupuncture and increased ATP concentration at Zusanli (ST36), which was induced by a 30-minute acupuncture treatment. This suggests that ATP may serve as a mediator, transmitting biological signals that underlie the therapeutic effects of acupuncture.

Can the rapid and significant release of ATP trigger the activation of purinergic receptors in effector cells located in the vicinity of acupuncture points, including immune cells and nerve endings, thus facilitating the conversion and initial transmission of acupuncture signals? Additionally, our investigation delved into the functions of acupoint ATP injections and a P2X3 receptor antagonist in modulating the effects of acupuncture. We noted that the benefits of acupuncture were enhanced when combined with acupoint injections of varying doses of ATP during MA. However, although there were no noticeable distinctions between the effects of different ATP doses, it remains unclear whether the lack of effect is due to the insignificance of the ATP dose or the inadequate range of ATP doses tested in the study. Furthermore, inhibition of the P2X3 receptor appeared to mitigate the effects of acupuncture, indicating that this receptor plays a crucial role in the transmission of nociceptive information elicited by acupuncture. The P2X3 receptor is prevalent in neural structures implicated in nociception and is closely linked to pain modulation mediated by P2X receptors.33 The P2X3 receptor detects the presence of ATP that is leaked from damaged tissues or released from inflammatory cells at peripheral terminals.34 Acupuncture exerts its effects on a variety of cells, including skin keratinocytes and fibroblasts, through a range of stimulating mechanisms that trigger ATP release, activate P2X3 receptors, and excite sensory nerve endings. These impulses are transmitted to the spinal cord via intermediate nerves and the dorsal root ganglion, eventually reaching the brainstem through interneurons and culminating in their arrival at the cerebral cortex.35,36 ATP is an endogenous agonist of the P2X3 receptor.37 Our findings suggest a positive correlation between the increase in PTT and ATP content at Zusanli (ST36), while no such correlation was observed with AMP and ADP content. These results emphasize the role of the ATP/P2X3 receptor in acupuncture-induced nociception. However, it is important to note that the effects of acupuncture on PTT are the result of complex neural processes that involve interactions among the nervous, endocrine, and immune systems. Therefore, further investigation is necessary to fully understand the underlying mechanisms of these interactions.

The metabolic pathways involved in both producing and using ATP serve as a fundamental indicator of the cellular energy status. When the energy charge (EC) within cells is high, there is a reduction in ATP generation and an increase in its consumption. Conversely, when the EC is low, there is an elevation in ATP production and a decrease in its utilization.22 Our observation revealed that the concentration of EC in the blood reached its peak 30 min after the injection of ATP. This suggests that the ATP-consuming system was at its maximum activation level, and the efficiency of ATP utilization was at its highest. To maximize the effects of acupuncture, we conducted the procedure at the time point that corresponds to the highest EC value, and we observed a significant enhancement of the treatment's effects. Building on these findings, we further investigated the optimal dose for ATP pretreatment to provide scientific evidence for enhancing body energy, metabolism, and the effects of acupuncture. Although different doses of ATP were tested, they did not produce significant effects on PTT. This could be due to the limited range of ATP doses used. It's important to note that the therapeutic benefits of acupuncture are influenced by various factors within the acupoint microenvironment, and while ATP may play a necessary role, it alone may not be sufficient to mediate the process.38

Patients with poor constitution (low energy state) exhibit poor sensitivity to acupuncture.39 Based on our hypothesis, we proposed that a combination of acupuncture and energy supplements could potentially enhance the effectiveness of acupuncture and improve its therapeutic outcomes in clinical settings. To test this, we administered adenosine disodium injections to mice prior to acupuncture treatment. Our results showed that when adenosine disodium was administered via i.p. or i.m. route 30 min before acupuncture, it significantly improved the efficacy of acupuncture in mice compared to the control group. Based on the research results, it can be inferred that the effectiveness of acupuncture is closely related to the overall functional state of the body. Specifically, when the body is in excellent condition, it appears to be more receptive to the benefits of acupuncture. In acupuncture therapy, achieving the sensation known as "De Qi" is crucial to achieving a positive therapeutic outcome. As stated in the ancient text Ling Shu · Jiu Zhen Shi Er Yuan,39 needling is a necessary component of the therapy, but it is the mobilization of Qi that ultimately produces the desired effects. The sensation of De Qi during acupuncture is strongly influenced by a patient's mental state and physical condition, as well as the balance of their body's Yin and Yang. This experiment's findings have valuable implications for acupuncture and moxibustion clinics. Specifically, for patients who exhibit a weak response to needles due to poor physical condition, the use of energy substances such as energy mixture or disodium adenosine triphosphate injection can enhance the acupuncture's effectiveness.

After conducting our observations, it was found that the infusion of ATP increased the concentration of extracellular ATP and enhanced the impact of acupuncture. It is possible that multiple pathways contribute to these effects. Firstly, exogenous ATP has the potential to directly stimulate P2X receptors, enhancing ion transport and action potential generation at nerve endings. This, in turn, can facilitate nerve impulse transmission. Notably, P2X receptors are abundantly expressed in capillary endothelial cells and diverse immune cell populations, which aligns with the role of regulatory networks formed by the nervous, endocrine, and immune systems in the context of acupuncture signaling. The convergence of these systems serves to augment the therapeutic effects of acupuncture.40 Secondly, ATP serves as a precursor to extracellular ADO, as it is rapidly broken down by extracellular nucleotide enzymes into ADO, which cannot be directly taken up by cells. Acupuncture triggers the release of ADO, which binds to A1 receptors and enhances neurotransmission. This mechanism may be responsible for the observed increase in PTT following acupuncture.41,42

In summary, our research investigated the correlation between ATP and P2X3 receptors, and its influence on the efficacy of acupuncture at local acupoints. We also examined the role of ATP as an energy substrate in acupuncture, exploring its impact on overall energy metabolism and local signal transduction to gain a comprehensive understanding of how ATP contributes to the production of acupuncture effects. We observed a dose-dependent increase in PTT and ATP content at Zusanli (ST36) in mice, which peaked simultaneously under specific conditions when acupuncture was applied. Our pharmacological manipulations indicated that the antinociceptive effects of acupuncture are mediated, at least partly, by ATP/P2X3 receptor signaling, and that local and systemic ATP levels enhance the effects of acupuncture via this receptor. Our findings may guide the selection of optimal parameters for therapeutic acupuncture in clinical practice. Further research should explore the mechanistic roles of ATP to gain deeper insights into the biological mechanisms underlying the effects of acupuncture.

5. ACKNOWLEDGMENTS

The authors thank the institute and other collaborative teams for conducting the experiments and institutional funding which supported necessary research facilities.

6. SUPPORTING INFORMATION

Supporting data to this article can be found online at http://journaltcm.cn.

Footnotes

Supported by The National Key R&D Program of China: Biological Mechanisms of Acupoint Function-Effect Associations (No. 2019YFC1709003); National Natural Science Foundation of China (NSFC) Top-level Project: Study on the Neuroimmunological Mechanism of Macrophage Phenotypic Polarisation for Antiinflammation Regulated by Acupuncture (No. 81873369); National Natural Science Foundation of China Young Science Fund Project: Study on the Neuromodulation Mechanism of Electroacupuncture to Improve Neutropenia after Chemotherapy for Lung Cancer (No. 81704146); National Natural Science Foundation of China Key Project: Research on the Initial Kinetic Regulation Mechanism of Acupuncture Effect Based on the Physicochemical Coupling Network of Acupuncture Point Microenvironment (No. 82030125)

Contributor Information

Yi GUO, Email: guoyi_168@163.com.

Yongming GUO, Email: guoymxr@163.com.

REFERENCES

  • 1. Moudgil KD, Berman BM. Traditional Chinese Medicine: potential for clinical treatment of rheumatoid arthritis. Expert Rev Clin Immunol 2014; 10: 819-22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2. Goh YL, Ho CE, Zhao B. Acupuncture and depth: future direction for acupuncture research. Evid Based Complement Alternat Med 2014; 2014: 871217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Li NC, Li MY, Chen B, Guo Y. A new perspective of acupuncture: the interaction among three networks leads to neutralization. Evid Based Complement Alternat Med 2019; 2019: 2326867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Madsen MV, Gøtzsche PC, Hróbjartsson A. Acupuncture treatment for pain: systematic review of randomised clinical trials with acupuncture, placebo acupuncture, and no acupuncture groups. BMJ 2009; 338: a3115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Lee IS, Cheon S, Park JY. Central and peripheral mechanism of acupuncture analgesia on visceral pain: a systematic review. Evid Based Complement Alternat Med 2019; 2019: 1304152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Zhao ZQ. Neural mechanism underlying acupuncture analgesia. Prog Neurobiol 2008; 85: 355-75. [DOI] [PubMed] [Google Scholar]
  • 7. Kagitani F, Uchida S, Hotta H. Afferent nerve fibers and acupuncture. Auton Neurosci 2010; 157: 2-8. [DOI] [PubMed] [Google Scholar]
  • 8. Chang KH, Bai SJ, Lee H, Lee BH. Effects of acupuncture stimulation at different acupoints on formalin-induced pain in rats. Korean J Physiol Pharmacol 2014; 18: 121-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Vixner L, Schytt E, Stener-Victorin E, Waldenström U, Pettersson H, Mårtensson LB. Acupuncture with manual and electrical stimulation for labour pain: a longitudinal randomised controlled trial. BMC Complement Altern Med 2014; 14: 187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Kong J, Gollub R, Huang T, et al. Acupuncture De Qi, from qualitative history to quantitative measurement. J Altern Complement Med 2007; 13: 1059-70. [DOI] [PubMed] [Google Scholar]
  • 11. Leong PK, Wong HS, Chen J, Ko KM. Yang/Qi invigoration: an herbal therapy for chronic fatigue syndrome with Yang deficiency. Evid Based Complement Alternat Med 2015; 945901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. Ko KM, Chiu PY. Biochemical basis of the "Qi-invigorating" action of Schisandra berry (wu-wei-zi) in Chinese medicine. Am J Chin Med 2006; 34: 171-6. [DOI] [PubMed] [Google Scholar]
  • 13. Wallace DC. Mitochondria as chi. Genetics 2008; 179: 727-35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Huang Y, Kwan K, Leung KW, et al. The extracts and major compounds derived from Astragali Radix alter mitochondrial bioenergetics in cultured cardiomyocytes: comparison of various polar solvents and compounds. Int J Mol Sci 2018; 19: 1574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15. Yegutkin GG. Enzymes involved in metabolism of extracellular nucleotides and nucleosides: functional implications and measurement of activities. Crit Rev Biochem Mol Biol 2014; 49: 473-97. [DOI] [PubMed] [Google Scholar]
  • 16. Tang Y, Yin HY, Liu J, Rubini P, Illes P. P2X receptors and acupuncture analgesia. Brain Res Bull 2019; 151: 144-52. [DOI] [PubMed] [Google Scholar]
  • 17. Surprenant A, North RA. Signaling at purinergic P2X receptors. Annu Rev Physiol 2009; 71: 333-59. [DOI] [PubMed] [Google Scholar]
  • 18. Kasuya G, Fujiwara Y, Tsukamoto H, et al. Structural insights into the nucleotide base specificity of P2X receptors. Sci Rep 2017; 7: 45208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Shi L, Zhang HH, Hu J, Jiang XH, Xu GY. Purinergic P2X receptors and diabetic neuropathic pain. Sheng Li Xue Bao 2012; 64: 531-42. [PubMed] [Google Scholar]
  • 20. Xiao Z, Ou S, He WJ, Zhao YD, Liu XH, Ruan HZ. Role of midbrain periaqueductal gray P2X3 receptors in electroacupuncture-mediated endogenous pain modulatory systems. Brain Res 2010; 1330: 31-44. [DOI] [PubMed] [Google Scholar]
  • 21. Goldman N, Chen M, Fujita T, et al. Adenosine A1 receptors mediate local anti-nociceptive effects of acupuncture. Nat Neurosci 2010; 13: 883-88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. De la Fuente IM, Cortés JM, Valero E, et al. On the dynamics of the adenylate energy system: homeorhesis vs homeostasis. PLoS One 2014; 9: e108676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. Choi JW, Kang SY, Choi JG, et al. Analgesic effect of electroacupuncture on paclitaxel-induced neuropathic pain via spinal opioidergic and adrenergic mechanisms in mice. Am J Chin Med 2015; 43: 57-70. [DOI] [PubMed] [Google Scholar]
  • 24. Lu KW, Hsu CK, Hsieh CL, Yang J, Lin YW. Probing the effects and mechanisms of electroacupuncture at ipsilateral or contralateral ST36-ST37 Acupoints on CFA-induced inflammatory pain. Sci Rep 2016; 6: 22123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. You X, Wang Y, Wu J, et al. Zusanli (ST36) Acupoint injection with neostigmine for paralytic postoperative ileus following radical gastrectomy for gastric cancer: a randomized clinical trial. J Cancer 2018; 9: 2266-74. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Lorenzini L, Giuliani A, Giardino L, Calzà L. Laser acupuncture for acute inflammatory, visceral and neuropathic pain relief: an experimental study in the laboratory rat. Res Vet Sci 2010; 88: 159-65. [DOI] [PubMed] [Google Scholar]
  • 27. Park JH, Kim SK, Kim HN, et al. Spinal cholinergic mechanism of the relieving effects of electroacupuncture on cold and warm allodynia in a rat model of neuropathic pain. J Physiol Sci 2009; 59: 291-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Yang X, Li X, Fu N, et al. Effect of acupuncture for pain threshold among the groups of different constitutions. Zhong Guo Zhen Jiu 2016; 36: 491-5. [PubMed] [Google Scholar]
  • 29. Barlas P, Ting SL, Chesterton LS, Jones PW, Sim J. Effects of intensity of electroacupuncture upon experimental pain in healthy human volunteers: a randomized, double-blind, placebo-controlled study. Pain 2006; 122: 81-9. [DOI] [PubMed] [Google Scholar]
  • 30. Bai L, Qin W, Tian J, et al. Time-varied characteristics of acupuncture effects in fMRI studies. Hum Brain Mapp 2009; 30: 3445-460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. Bodin P, Burnstock G. Purinergic signalling: ATP release. Neurochem Res 2001; 26: 959-69. [DOI] [PubMed] [Google Scholar]
  • 32. Katsuragi T, Migita K. The mechanism of ATP release as an autocrine/paracrine molecule. Nihon Yakurigaku Zasshi 2004; 123: 382-388. [DOI] [PubMed] [Google Scholar]
  • 33. Kuan YH, Shyu BC. Nociceptive transmission and modulation via P2X receptors in central pain syndrome. Mol Brain 2016; 9: 58. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34. Brederson JD, Jarvis MF. Homomeric and heteromeric P2X3 receptors in peripheral sensory neurons. Curr Opin Investig Drugs 2008; 9: 716-25. [PubMed] [Google Scholar]
  • 35. Zhang ZJ, Wang XM, McAlonan GM. Neural acupuncture unit: a new concept for interpreting effects and mechanisms of acupuncture. Evid Based Complement Alternat Med 2012; 2012: 429412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36. Burnstock G. Purinergic signalling: from discovery to current developments. Exp Physiol 2014; 99: 16-34. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. Lambertucci C, Dal Ben D, Buccioni M, Marucci G, Thomas A, Volpini R. Medicinal chemistry of P2X receptors: agonists and orthosteric antagonists. Curr Med Chem 2015; 22: 915-28. [DOI] [PubMed] [Google Scholar]
  • 38. Chen B, Li MY, Guo Y, Zhao X, Liu YY. Mast cell-derived exosome participates in acupoint-stimulation initiated local network activities. Zhen Ci Yan Jiu 2015; 40: 82-5. [PubMed] [Google Scholar]
  • 39. Song dynasty. Ling Shu Jing. Taiyuan: Shanxi Science and Technology Press, 1992: 4. [Google Scholar]
  • 40. Yin CS, Chae Y, Kang OS, et al. De Qi is double-faced: the acupuncture practitioner's and the subject's perspective. Evid Based Complement Alternat Med 2015; 2015: 635089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41. Bo C, Ming-yue LI, Sha-sha D, et al. Research progress on regulations on nerve-endocrine-immune network by acupuncture. World J Acupunct Moxibustion 2014; 24: 49-53. [Google Scholar]
  • 42. Illes P, Ribeiro JA. Neuronal P2 receptors of the central nervous system. Curr Top Med Chem 2004; 4: 831-8. [DOI] [PubMed] [Google Scholar]
  • 43. Khakh BS, North RA. P2X receptors as cell-surface ATP sensors in health and disease. Nature 2006; 442: 527-32. [DOI] [PubMed] [Google Scholar]

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