Neuropathic pain (NP), characterized by spontaneous pain, hyperalgesia, and allodynia, is often caused by nerve injury or other diseases.1 Because conventional analgesics, such as opiates and nonsteroidal antiinflammatory drugs, do not have good efficacy against NP, the choice of treatment has been largely limited. Anticonvulsants and antidepressants are prescribed for the management of NP, but their effects are limited.2,3
P2X3, a subtype of P2X receptors, which is a nonselective cationic channel, is expressed mostly in sensory neurons and is involved in transduction of pain stimuli.4 Adenosine triphosphate (ATP), a fast neurotransmitter, by activating P2 receptors especially P2X3 receptor, can modulate pain transmission under NP conditions.5–7 Rats receiving P2X3 gene knockout or P2X3 antisense receptor gene disruption have been shown to be insensitive to noxious stimulus. The acute administration of a highly selective nonnucleotide P2X3 antagonist, A317491, can fully block NP. These findings support the significant role for P2X3 receptors in the expression of pain.
Both animal experiments and clinical pain investigations have shown that electroacupuncture (EA) has an effective analgesic effect on chronic pain.8–12 In addition, studies have shown that different frequencies of EA may exhibit different degrees of analgesia.13–15 Although EA has been shown to stimulate the release of endorphins and other neurotransmitters,16,17 little is known about its effect on the P2X3 receptor.
Extracellular signal-regulated kinase 1/2(ERK1/2), a member of the mitogen-activated protein kinase (MAPK) family, is implicated in neural plasticity, which contributes to development of chronic pain.18,19 ERK1/2 activation in the spinal cord induced by nociceptive stimulation plays a critical role in central sensitization.20 The inhibition of ERK1/2 phosphorylation (p-ERK1/2) by EA in the spinal cord dorsal horn, the activation of which contributes to the induction and maintenance of chronic constriction injury (CCI)–induced NP, is possibly involved in acupuncture’s analgesic effect.21 On the other hand, a previous study showed that the enhanced fast-inactivating current induced by α,β-methylene ATP in chronic compression of dorsal root ganglion (DRG)–operated DRG neurons was significantly suppressed by a specific inhibitor of the ERK1/2, U0126.22 In addition, in peripheral inflammation and sciatic nerve transaction rat model, p-ERK1/2 labeling occurs through the P2X3 receptors in the terminals of DRG neurons after the mechanical stimuli into the inflamed tissue.23 However, whether ERK1/2 is involved in mediating EA’s effect on P2X3 receptors in the spinal cord after nerve injury has not been fully demonstrated.
In the present study, we sought to evaluate the effects and mechanisms of EA on P2X3 receptor expression in spinal cords of rats with nerve injury–induced NP and investigated the involvement of the ERK1/2 signaling pathway in regulating the effect of EA on P2X3 receptor for better understanding of EA on persistent pain.
All experiments were approved by the local animal ethics committee of the Tianjin Medical University and conducted in accordance with the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health and the International Association for the Study of Pain. Male Sprague Dawley rats weighing 200 ± 20 g, supplied by Tianjin Medical University (Tianjin, China), were given free access to food and water.
The study consisted of the following 2 experiments: Experiment 1: Effects of EA on P2X3 receptor expression—CCI rats were divided into 6 groups (n = 10 each): sham-CCI group, CCI group, low-frequency EA (LEA) group (CCI + 2 Hz EA at Zusanli-Yanglingquan acupoints [ST36-GB34]), high-frequency EA (HEA; CCI + 15 Hz EA at ST36-GB34), NA-LEA (CCI + 2 Hz EA at nonacupoints), and NA-HEA (CCI + 15 Hz EA at nonacupoints); Experiment 2: Involvement of ERK1/2 signal pathway in EA regulating P2X3 receptor expression—CCI rats with an indwelling catheter placed in the subarachnoid space were divided into 5 groups (n = 10 each): sham-CCI group, CCI group, EA group (CCI + 2 Hz EA at ST36-GB34), NA-EA group (CCI + 2 Hz EA at nonacupoints), and U0126 group (CCI + U0126 intrathecal [IT] injection). U0126 dissolved in 5% dimethyl sulfoxide (DMSO) was administered to the U0126 group; the other 4 groups received the same volume of the vehicle (5% DMSO). The administration of U1026 (5 µg/10 µL IT) or DMSO (5%/10 µL IT) was started 4 days after CCI and continued twice a day until day 9. Thirty-minute EA treatment was given on days 4 to 9 after CCI. Thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) were determined at baseline and on days 3, 5, 7, and 9 after CCI. Lumbar 4 and lumbar 5 spinal cord was removed under deep anesthesia on day 10 after CCI. P2X3 receptor protein and messenger RNA (mRNA) levels were measured by Western blotting and real-time polymerase chain reaction, respectively. Another total ERK1/2 and ERK1/2 phosphorylation were assayed by Western blotting in experiment 2.
Chronic Constriction Injury
CCI, as described by Bennett and Xie,24 was performed on anesthetized rats. Briefly, after skin incision and blunt dissection of subcutaneous tissue and muscle in the right midthigh, the right common sciatic nerve was exposed, and 4 ligatures were loosely placed (4-0 silk thread) with 1-mm intervals to cause CCI. In sham-CCI rats, the right sciatic nerve was exposed but not ligated.
Intrathecal Drug Delivery
As described by Yaksh and Rudy,25 catheterization of the rat spinal subarachnoid space was performed on anesthetized rats 2 days before CCI. Briefly, rats were implanted with a PE-10 polyethylene catheter (8 cm) in the lumbar subarachnoid space. Rats with no postoperative neurologic deficits after surgery were kept for experiment 2. In experiment 2, rats in the U0126 group were injected intrathecally twice daily at 12-hour intervals (days 4–9) with selective ERK1/2 inhibitor U0126 (5 µg; Sigma, St. Louis, MO) dissolved in 5% DMSO (10 µL; Sigma), and rats in sham-CCI, CCI, EA, and NA-EA groups were injected intrathecally with 10 µL of 5% DMSO as a vehicle, followed by 10 µL of normal saline flush.
Rats received EA stimulation for 30 minutes once a day on days 4 to 9 after CCI. Rats without any anesthetic remained quiet and awake in tubular acrylic holders during EA treatment. Two stainless acupuncture needles of 0.3 mm diameter and 50 mm long connected to a pair of electrodes were inserted perpendicularly approximately 6 mm into the ipsilateral acupoints on the leg of the rat corresponding to the Zusanli (ST36) and Yanglingquan (GB34) acupoints in humans. In humans, ST 36 is located on the anterior aspect of the lower leg, 4 finger breadths (middle finger) below the knee joint, and 1 finger breadth lateral to the anterior crest of the tibia, while GB34 is located in the depression anterior and inferior to the head of the fibula. Two nonacupoints are located 0.5 cm horizontal and lateral to ST36 and GB34 acupoints, respectively, at nonmeridian points.26,27 The other ends of electrodes were connected to the output channel of the electrostimulator (Electronic pulse therapeutic apparatus G6805-1A; Huayi Medical Instruments, Shanghai, China). The constant frequency (2 Hz or 15 Hz) and the intensity of less than 1.5 mA to induce moderate muscle contraction of the hind limb was applied to perform EA stimulation. Rats of no-EA groups were kept in holders for 30 minutes as control.
Paw withdrawal to thermal and mechanical stimulation was tested on the day before CCI surgery and on days 3, 5, 7, and 9 after CCI. The behavioral test was performed 20 minutes after EA on days 5, 7, and 9. Animals were placed on an elevated wire grid for acclimation for 30 minutes before testing. Mechanical allodynia was determined by the 50% withdrawal threshold for the up–down method using a set of von Frey filaments (Stoelting, North Coast).28 Thermal hyperalgesia was assessed by the method described previously,27 using a hotplate with Plexiglas walls (model YLS-6B; Zhenghua Bio-instruments, Anhui, China). Rats were placed on a hotplate with a stable temperature of 52°C ± 0.2°C. The average value of 3 sessions at 10-minute intervals for the latency of ipsilateral paw withdrawal testing was used as TWL.
The right lumbar enlargement (lumbar 4 and lumbar 5) was quickly dissected under deep anesthesia on day 10 after CCI and immediately frozen in liquid nitrogen, then stored at –80°C until use.
Tissues were homogenized in lysis buffer that contained proteinase inhibitor mixture (Santa Cruz Biotechnology, Santa Cruz, CA) and centrifuged at 12,000g for 15 minutes at 4°C. Protein concentration was determined by BCA assay (Pierce, Rockford, IL), and 60-µg protein samples were separated using SDS-–PAGE gel (5%–10% gradient gel) then transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). Membranes were blocked in 5% low-fat milk with buffer containing 0.1% Tween-20 for 2 hours at room temperature, then incubated overnight at 4°C with anti-P2X3 antibody (antirabbit, 1:800; Merck Millipore, Germany) or anti-ERK1/2 antibody (1:1000; Cell Signaling Technology, Beverly, MA), p-ERK1/2 (1:1000; Cell Signaling Technology), or anti-β-actin (1:5000; Sigma), followed by incubation with horseradish peroxidase–conjugated secondary antibody (1:2000; Santa Cruz Biotechnology) for 2 hours. Protein-antibody complexes conjugated with enhanced chemiluminescence reagent (Millipore) were visualized on the Chemidoc XRS System (Bio-Rad).
Quantitative Real-Time Polymerase Chain Reaction
Trizol reagent (Invitrogen, Carlsbad, CA) was used to extract total RNA. One microgram of total RNA was reverse-transcribed to complementary DNA by the protocol of reverse transcription reaction kit (Invitrogen).The P2X3 receptor sequence-specific primers (5-TGGCGTTCTGGGTATTAAGATCGG-3/5-CAGTGGCCTGGTCACTGGCGA-3) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sequence-specific primers (5-GACAACTTTGGCATCGTGGA-3/5-ATGCAGGGATGATGTTCTGG-3) were designed and synthesized in the Tianjin Institute of Biochemistry (China). The melting temperature for GAPDH was 56°C and 61°C for P2X3. Quantitative reverse RT-PCR amplification was performed on a DNA Engine thermal cycler (Bio-Rad) using SYBR Green I (Invitrogen). Each sample was run in triplicate for each gene. The P2X3 relative mRNA levels were calculated by the method described previously.29
Data are expressed as the mean ± SEM and statistically analyzed by the Statistical Package for the Social Sciences (SPSS) (Version 16.0; SPSS Inc., Chicago, IL). Data from all the measurements were analyzed using a one-way analysis of variance followed by post hoc analysis. P < 0.05 was considered statistically significant.
EA at ST36-GB34 Acupoints, but Not at Nonacupoints, Attenuated CCI-Induced Mechanical Allodynia and Thermal Hyperalgesia
Compared with the sham-CCI group, TWL and MWT of ipsilateral hindpaws decreased significantly on day 3 after CCI in rats treated with CCI (P < 0.001; Fig. 1, A and B), which indicated that CCI induced a prominent mechanical allodynia and thermal hyperalgesia. EA at SB36-GB34 acupoints significantly increased TWL and MWT from day 5 (P = 0.017 LEA versus CCI for MWT, P = 0.020 HEA versus CCI for MWT; P = 0.033 LEA versus CCI for TWL, P = 0.026 HEA versus CCI for TWL) to day 9 (P < 0.001, LEA and HEA versus CCI). Compared with the HEA group, TWL and MWT tested on day 9 in the LEA group increased more significantly (P = 0.023 for MWT; P = 0.041 for TWL). The value of TWL and MWT in rats from nonacupoint EA groups after CCI did not show a significant change when compared with CCI groups (P > 0.05).
EA Reduced P2X3 Receptor Synthesis at both mRNA and Protein Level in the Spinal Cord of Rats After CCI with Acupoint Specificity
The P2X3 protein level in the spinal cord is shown in Figure 2A. CCI nerve injury significantly increased P2X3 receptor protein levels in the spinal cord (P < 0.001 compared with sham-CCI), and this increase was significantly attenuated by EA stimulation of ST36-GB34 acupoints (P < 0.001, CCI versus LEA and HEA). Figure 2B shows that a CCI-induced increase in mRNA synthesis of P2X3 receptors in the spinal cord was significantly attenuated in EA (at acupoints) treatment groups (P < 0.001, CCI versus LEA and HEA). Furthermore, the suppressive effects of EA stimulation on the P2X3 mRNA and protein levels were more remarkable in the LEA group (P = 0.045 for protein level, P = 0.047 for mRNA level, LEA versus HEA). However, the P2X3 receptor protein and mRNA levels in rats of nonacupoint EA groups did not change significantly when compared with CCI groups (P > 0.05).
These data demonstrated that EA at SB36-GB34 acupoints significantly alleviates the mechanical allodynia and thermal hyperalgesia of the ipsilateral paws of rats with CCI, at least partly, by suppressing P2X3 receptors in the spinal cord. LEA has a better analgesic effect than HEA on CCI-induced pain.
Administration of U0126 Intrathecally and Treatment of EA at Acupoint: Each Attenuated CCI-Induced Mechanical Allodynia and Thermal Hyperalgesia
In experiment 2, to investigate whether ERK1/2 activation would regulate P2X3 receptor expression in the spinal cord, a group of rats (n = 10) was given U0126 (5 µg/10 µL IT) twice a day on post-CCI days 4 to 9 to mimic the effect of EA. Five percent DMSO (10 µL IT) was given as vehicle control in the other 4 groups. Our results showed that mechanical allodynia and thermal hyperalgesia occurred on day 3 after CCI (P < 0.001, when compared with sham-CCI; Fig. 3, 3A and 3B). Both administration of U0126 and EA at treatment acupoints significantly increased TWL and MWT of ipsilateral hindpaws from day 5 (P < 0.001, U0126 versus CCI; P = 0.007 for MWT and P = 0.002 for TWL, EA versus CCI) to day 9 (P < 0.001 when compared with CCI). Compared with the CCI group, the NA-EA group did not show a statistically significant analgesic effect.
Inhibition of ERK1/2 Activation May Be Involved in the Effect of EA at Acupoint Treatments on Downregulating the P2X3 Receptor Expression in the Spinal Cord of Rats with CCI
The activation of ERK1/2 and P2X3 receptor protein level was significantly higher in the spinal cord in the CCI group than in the sham-CCI group (P < 0.001; Fig. 4, 4A and 4B). Compared with the CCI group, ERK1/2 activation and P2X3 receptor protein level in the EA group and U0126 group were significantly decreased (P < 0.001 EA versus CCI for P2X3 protein expression; P = 0.004 EA versus CCI for p-ERK1/2 expression; P < 0.001 U0126 versus CCI). The mRNA expression of P2X3 receptor in the spinal cord was significantly increased in the CCI group compared with the sham-CCI (P < 0.001; Fig. 4C). Compared with the CCI group, the mRNA expression of P2X3 receptor in the EA group and in the U0126 group was significantly decreased (P < 0.001). These data demonstrated that inhibition of ERK1/2 activation may be partly involved in the effect of EA at acupoint treatment on downregulating P2X3 receptor expression in the spinal cord of rats with CCI.
Our findings showed that 2 Hz or 15 Hz EA at acupoints significantly attenuates CCI-induced hyperalgesia and allodynia. Moreover, we demonstrated that EA at SB36-GB34 acupoints significantly attenuated CCI-induced upregulation of protein and mRNA of P2X3 receptor in the spinal cord. In addition, EA administration and U0126 IT injection exhibited potency in decreasing ERK1/2 activation as well as protein and mRNA of P2X3 receptor in the spinal cord. The data indicated that the effect of EA on NP behavior may be mediated by the attenuation of ERK1/2 activation and P2X3 receptor synthesis in the spinal cord.
ATP released from injured tissue or the spinal cord is implicated in the initiation of pain by activating P2X3 receptors on sensory nerve terminals.30,31 In the present study, the protein and mRNA level of P2X3 receptor significantly increased in the spinal cord of rats with CCI injury, which is consistent with previous studies.32 Persistent nerve injury causes long-lasting excitement to nociceptive neurons, which relates to ATP-induced P2X3 receptors activation and could cause activation of the neurokinin 1 receptor in the spinal cord and glutamate release from presynaptic nerve endings.33 These mechanisms participate in changing neuron plasticity and the maintenance of NP. Our results showed that CCI-induced hyperalgesia and allodynia and an increase in protein and mRNA expression of P2X3 receptor in the spinal cord were significantly attenuated by EA stimulation. These results suggest that EA treatment enhances the analgesic effect at least partly by decreasing the expression of P2X3 receptor in the spinal cord of CCI rats. Notably, it was recently reported that EA can relieve NP by increasing P2X3 receptor expression in the lateral periaqueductal gray, the upregulation of which inhibits the progression of NP.34 All in all, these studies delineated the crucial role of P2X3 receptor in EA’s analgesic effect.
Several studies have shown various responses to different frequencies of EA stimulation. Two hertz had a better analgesic effect on NP than 100 Hz.35 Fifteen hertz EA had a significant analgesic effect on hyperalgesia induced by arthritis in rats compared with 100 Hz EA treatment.36 In a rat model of complete Freund adjuvant-induced hyperalgesia, HEA-induced potent and shorter-lasting analgesic effects were observed, whereas LEA induced moderate and longer-lasting effects.37 In addition, EA at different frequencies induces the release of different types of endogenous opioids.38 In the present study, we observed 2 Hz EA had a better analgesic effect by increasing TWL and MWT significantly on day 9 after CCI compared with 15 Hz EA treatment. The expression of P2X3 receptor in rats treated with 2 Hz EA was significantly lower than that in rats with 15 Hz EA treatment. These findings may have implications for the use of EA in various clinical practice settings.
Furthermore, in the spinal cord, intense peripheral noxious stimulation induces ERK activation, which contributes to central sensitization.39 The inhibition of ERK activation reduces the behavioral measures of activity-dependent central sensitization.40 Consistent with behavioral pain hypersensitivities, partial sciatic nerve ligation, spinal nerve ligation, and CCI of the sciatic nerve–induced persistent ERK activation in the spinal cord.41–43 Studies have shown that activation of ERK, not p38 MAPK in the spinal cord, contributes to the induction phase and early maintenance phase of long-lasting allodynia induced by IT administration of ATP.44 It was also shown that the depression of ERK activation in the spinal cord may contribute to EA’s analgesic effect.21 In our results, ERK1/2 activation and P2X3 receptor expression were increased in the spinal cord of rats after CCI, and EA administration decreased these effects. Moreover, the IT administration of U0126 increased TWL and MWT and decreased the ERK1/2 activation as well as P2X3 receptor protein and mRNA expression in the spinal cord of rats after CCI, which confirmed the effect of ERK1/2 on regulating P2X3 receptor expression.
In addition, several studies have demonstrated that EA exhibited therapeutic effects on specific acupoints.45,46 Modulation of the limbic system may differentiate between specific and nonspecific components of acupuncture.47 Acupuncture at acupoints induces specific patterns of brain activity.48 In the present study, however, there were no statistically significant changes in nociceptive behaviors and P2X3 protein and mRNA expression in EA between the nonacupoint and the CCI groups.
In conclusion, the current study demonstrated that EA produces a significant analgesic effect on nerve injury–induced NP, and this effect may be mediated through regulation of spinal ERK1/2/P2X3 signaling pathway.
Name: Jianbo Yu, PhD.
Contribution: This author helped design and conduct the study, analyze the data, and write the manuscript.
Attestation: Jianbo Yu has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Cong Zhao, MD.
Contribution: This author helped design and conduct the study, analyze the data, and write the manuscript.
Attestation: Cong Zhao has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Xiaoqin Luo, MD.
Contribution: This author helped analyze the data and write the manuscript.
Attestation: Xiaoqin Luo has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
This manuscript was handled by: Jianren Mao, MD, PhD.
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