Inflammatory pain is a major health care problem that dramatically decreases the quality of life of patients. Inflammatory pain results from peripheral tissue injury/inflammation and is accompanied by sensitization to noxious stimuli (hyperalgesia).1 Tissue injury leads to the release of inflammatory mediators that sensitize and activate peripheral primary nociceptive neurons, resulting in the increased sensitivity of spinal dorsal horn neurons and, eventually, central sensitization.2,3 Central sensitization contributes importantly to the development and maintenance of inflammatory pain.2 Inflammatory pain is generally treated with opioids, nonsteroid anti-inflammatory drugs, paracetamol, and steroids. However, these drugs are limited by their lower efficacy and/or accompanying side effects; thus, there is a significant unmet clinical need for other treatments for inflammatory pain.3,4
Apelin is a bioactive peptide and is highly conserved among different species.5 Apelin exists in at least 3 forms, consisting of 13, 17, or 36 amino acids, all of which are derived from a common 77 amino-acid precursor. Of these forms, apelin-13 is the most effective ligand.6,7 This peptide binds to the apelin receptor (APJ), a G protein-coupled receptor. The apelin/APJ system is expressed widely in various tissues, including the heart, lungs, kidneys, mammary glands, adipose tissue, gastrointestinal tract, and vascular endothelium.8 Accumulating evidence suggests that the apelin/APJ system plays important roles in preventing inflammation, regulating angiotasis, angiogenesis, controlling cell proliferation, energy metabolism, and fluid homeostasis.9,10 Although most studies on the expression and action of the apelin/APJ system have focused on the cardiovascular system,11 recent studies have shown that the apelin/APJ system is also widely expressed in the central nervous system and may contribute to synaptic transmission and neuroprotection.12,13 Furthermore, other recent studies have shown that intracerebroventricular injection or intrathecal injection of apelin-13 not only significantly increases the pain threshold in naïve mice, but also had significant antinociception effects in visceral pain or inflammatory pain models.14–16
Electroacupuncture (EA) is an effective strategy that is widely used to attenuate various types of pain, including inflammatory pain.17 However, the mechanisms underlying the efficacy of EA have not been completely clarified. EA can regulate apelin expression in the rostral ventrolateral medulla.18 In our previous study, the analgesic effect of EA on naïve rats showed marked individual variations.19 Gene expression analyses have revealed that spinal APJ messenger RNA (mRNA) is significantly upregulated by EA stimulation in high-responder rats but is not regulated in the nonresponder rats, suggesting that spinal APJ may be a candidate molecule related to the analgesic effect of EA.19 However, little is known about the involvement of the spinal apelin/APJ system in the inhibition of inflammatory pain by EA. Thus, in the present study, we investigated whether the molecular mechanism underlying EA-induced antihyperalgesic effects was associated with the modulation of the spinal apelin/APJ system in a complete Freund’s adjuvant (CFA)-induced inflammatory pain model.
METHODS
Animals and Drugs
All experiments were performed on male Sprague-Dawley rats, which were provided by the animal experimental center at Shanghai Laboratory Animal Center. The animals weighed 200 ± 20 g at the beginning of the experiment. The animals were housed under a 12-hour light/dark cycle with food and water available ad libitum. The room temperature was maintained at 22° ± 1°C and relative humidity at 45% to 50%. All rats were acclimatized for at least 3 days before the experiments. The animal experiments were approved by the Institutional Animal Care Committee of Shanghai University of Traditional Chinese Medicine and were performed 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. CFA was purchased from Sigma-Aldrich (Catalog Number: F5881, St. Louis, MO). Apelin peptides (apelin-13 and apelin-13A [F13A]) were purchased from Phoenix Pharmaceuticals (Belmont, CA).
Experimental Design
The study consisted of the following 3 experiments. Experiment 1: Effects of EA on apelin and APJ expression—rats were randomly divided into the 4 following groups (n = 10 each): sham injury group (sham), CFA-induced inflammatory pain model group (CFA), EA group (CFA + 2 Hz EA at Zusanli-Yanglingquan acupoints [ST36-GB34]), and sham point electrical stimulation group (NA-EA; CFA + acupuncture without electrical stimulation). Experiment 2: Involvement of the apelin/APJ system in mediating the effects of EA—rats with successful intrathecal catheter installation were randomly divided into the 5 following groups (n = 7–8 each): sham group, CFA group, EA group, apelin/APJ system antagonist group (F13A; CFA + intrathecal injection of F13A), and EA+ F13A group (EA-F13A; CFA + EA + F13A ). Experiment 3: Effects of EA combined with apelin—rats were randomly divided into the 5 following groups (n = 8 each): sham group, CFA group, CFA + apelin-13 group (CFA + intrathecal injection of apelin-13), EA group, and EA + apelin-13 group (CFA + EA + intrathecal injection of apelin-13). Different rats were used in these 3 experiments. A 30-minute EA treatment was administered on days 2 to 4 after CFA. Thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) were determined at baseline and on days 2, 3, and 4 after CFA. In experiment 1, the lumbar L4–5 spinal cord of the rats was removed under deep anesthesia after the last behavior test on day 4, immediately frozen in liquid nitrogen, and stored at –80°C until use. In each group, some animals (n = 4) were used only for RNA, and others (n = 4) were used only for protein isolation. Apelin and APJ mRNA and protein levels were measured by real-time polymerase chain reaction (qRT-PCR) and Western blotting, respectively.
In experiments 2 and 3, F13A or apelin-13 was dissolved in sterile normal saline and administered to the EA-13A group, CFA + apelin-13 group, or EA + apelin-13 group, as appropriate. The other 3 groups received the same volume of sterile normal saline. F13A (1 μg/10 μL, intrathecally), apelin-13 (1 μg/10 μL, intrathecally), or sterile normal saline (10 μL, intrathecally) was administered 10 minutes before EA or sham treatment.
The CFA Model
To induce inflammatory pain, the rats were placed under isoflurane anesthesia (5%), and 100 μL of CFA was injected into the plantar side of the right hindpaws.20 In the sham group, the rats received the same volume of normal saline.
Intrathecal Drug Delivery
As previously described,21 the rats were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally) for the implantation of the intrathecal catheter 5 days before experimentation. Briefly, rats were implanted with a PE-10 polyethylene catheter (AniLab Instruments, Ning Bo, China), which was placed in the lumbar subarachnoid space between the L5 and the L6 vertebrae. The proximal part was tunneled under the skin and led out onto the upper dorsal region. Two days after implanting the intrathecal catheters, 10 μL lidocaine was injected directly into the lumbar spinal cord via the indwelling intrathecal catheters. Transient hind-limb paralysis implied the successful implantation of the intrathecal catheters.
Electroacupuncture
The rats received EA stimulation for 30 minutes each day on days 2 to 4 after CFA. Rats without any anesthetic remained quiet and awake in a well-ventilated Plexiglas restraint barrel during EA treatment. Stainless steel needles 0.3 mm in diameter and 30 mm in length were inserted at a depth of 6 mm into bilateral ST36 (2 mm lateral to the anterior tubercle of the tibia) and GB34 (in the depression anterior and inferior to the fibula capitulum) acupoints. The 2 ipsilateral needles were connected to the output terminals of the HANS Acupuncture Point Nerve Stimulator (LH-200; Beijing Huawei Industrial Developing Company, Beijing, China). The frequency was set at 2 Hz, and the intensity of stimulation was increased stepwise from 0.5 to 1.0 and then 1.5 mA with each step lasting for 10 minutes. The NA-EA group rats received the same subcutaneous needle insertion into ST36 and GB34 (2 mm in depth), and the needles were linked to the electrodes but without electrical stimulation. To eliminate the effects of stress, the rats in the other groups were also immobilized in well-ventilated Plexiglas restraint barrels, similar to the EA group.
Behavioral Tests
Paw withdrawal to thermal and mechanical stimulation was tested on the day before CFA injection and on days 1 to 4 after CFA injection. The behavioral test was performed 20 minutes after EA on days 2 to 4. The rats were placed on an elevated wire grid and acclimatized for 30 minutes before testing. MWT was determined by the 50% withdrawal threshold for the up–down method using a set of von Frey filaments (Stoelting, North Coast, CA).22 TWL was assessed by a method described previously using a 37370 plantar test apparatus (Ugo-Basile, Milan, Italy).23 The rats were acclimatized for 15 minutes before testing. The radiant heat was set at 50°C, and the cutoff time was 40 seconds. The average value of the latency of ipsilateral paw withdrawal of the 3 sessions, which were performed at 15-minute intervals, was used as the TWL.
Quantitative Real-Time Polymerase Chain Reaction
Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) and purified with an RNeasy column (Qiagen, Valencia, CA). RNA quality was assessed with a Lab-on-chip Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). Reverse transcription reactions were primed with oligo(dT). qPCR was conducted in duplicate with glyceraldehyde-3-phosphate dehydrogenase (Gapdh) as the internal control using the Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA) and SYBR Premix Ex TaqTM II (TaKaRa, Dalian, China). The primer sequences of Gapdh are: F-TCCTGCACCACCAACTGCTTAG, R-AGTGGCAGTGATGGCATGGACT. The primer sequences of apelin are: F-CTTCTCTGTCTTTGTTGTTT, R-TCTATCTCTCCTCTTTCTCA. The primer sequences of APJ are: F-GGCTCCTCTTGCTCCTTTGT, R-AGCGGCTTCAGTCACTCTTT. The cycling conditions were 30 seconds at 95°C followed by 40 cycles of 5 seconds at 95°C and 34 seconds at 60°C. After cycling, a melting protocol was performed with 15 seconds at 95°C, 1 minute at 60°C, and 15 seconds at 95°C to control for product specificity. The fold change of the target gene cDNA relative to Gapdh was determined as follows: fold change = 2−ΔΔCt, where ΔΔCt = (CtTarget − CtGapdh) test − (CtTarget − CtGapdh) control. Ct values were defined as the number of the PCR cycles at which the fluorescence signals were detected.
Western Blotting
The tissue was homogenized in cold lysis buffer (Beyotime Biotechnology Co., Haimen, China) and centrifuged at 13,200 g for 15 minutes at 4°C. The supernatant, containing total protein, was quantified by Enhanced BCA Protein Assay Kit (Beyotime Biotechnology Co.). Samples (30 μg total protein per loading) were separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and electrotransferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were blocked with 5% nonfat milk overnight at 4°C and then incubated with primary antibodies recognizing β-actin (mouse monoclonal, 1:5000; Abcam), apelin (rabbit polyclonal, 1:500; GeneTex, Irvine, CA), or APJ (rabbit polyclonal, 1:2000; Millipore, Billerica, CA) for 2 hours at 22°C. Then, the membranes were incubated with horseradish peroxidase-conjugated antimouse (1:5000; Abcam) or goat antirabbit (1:5000; Abcam) secondary antibodies. The signal was visualized with ECL Plus reagent (GE Healthcare, Buckinghamshire, UK) and exposed onto x-ray film (Kodak, Rochester, NY).
Statistical Analysis
All results are expressed as the mean ± standard deviation, and all data were analyzed using SPSS 19.0 software (SPSS Inc, Chicago, IL). Differences in MWT and TWL in the 3 experiments before (1 point) and after (4 points) CFA injection and among the groups were compared by 2-way repeated-measures analysis of variance. When variance homogeneity was not supported, the Kruskal-Wallis H test, which is a rank-based nonparametric test used to determine differences between 2 or more groups, was applied. Meanwhile, if the Mauchly’s test was significant, the Greenhouse-Geisser ε adjustment was used. If there was a significant interaction between the between-groups and within-subjects factors (significant treatment × time interaction), we performed a simple effects analysis of between-groups factors for all time levels, with Bonferroni adjustments, using a multivariate analysis of variance model. Bonferroni correction was performed by dividing the significance level of .01 for each experiment by the number of paired comparisons to be made (for k comparisons, the significance criterion was P < .01/k). Measures of effect size and confidence intervals (CIs) were calculated and reported. Differences in mRNA and protein levels of apelin and APJ were compared using 1-way analysis of variance with the Tukey test. When variance homogeneity was not supported, the Games-Howell test was applied to determine differences between 2 groups. P value <.05 was considered significant. On the basis of our preliminary experiment of 4 rats per group, we performed a power analysis to determine the sample size that was required to obtain significant effects for pain thresholds after CFA injection. Difference in means and estimated standard deviation was planned for primary aims. We calculated that 7 rats per group would be sufficient, the difference detectable with 90% power after making a Bonferroni correction to the type I error, and allocated 10 per group to account for the potential failure of intrathecal drug delivery.
RESULTS
EA Suppressed CFA-Induced Mechanical Allodynia and Thermal Hyperalgesia
;)
Figure 1.: The effects of electroacupuncture (EA) on complete Freund’s adjuvant (CFA)-induced mechanical allodynia (A) and thermal hyperalgesia (B). By 2-way analysis of variance with repeated measurements, mechanical withdrawal threshold and thermal hyperalgesia showed prominent main effects for the groups (P < .0001), time (P < .0001), and significant interaction (P < .0001). Comparisons by Student t test with Bonferroni correction (P < .01/4 = .0025 was defined as significant) displayed significant within-group and between-group differences in mechanical withdrawal threshold and thermal hyperalgesia. *P < .0025 compared with the sham group; # P < .0025 compared with the CFA group. The error bars represent the standard deviations.
On day 0, before the CFA injection, there were no significant differences in the MWT and TWL among the 4 groups (P = .117 and P = .710, respectively). After the rats received the CFA injection, the MWT and TWL of their ipsilateral hindpaws decreased significantly on day 1 compared with the sham group (all P < .001; Figure 1, A and B, respectively), which indicated that CFA induced a prominent mechanical allodynia and thermal hyperalgesia. EA stimulation significantly increased the MWT (overall P < .001, mean difference [99% CI]: 5.86 [4.96–6.77], EA versus CFA; Figure 1A) and TWL (overall P < .001, mean difference [99% CI]: 2.45 [0.91–4.00], EA versus CFA; Figure 1B) from day 2 to day 4. The MWT (mean difference [99% CI]: –0.08 [–0.96 to –0.81], NA-EA versus CFA; Figure 1A) and TWL (mean difference [99% CI]: –0.16 [–1.72 to –1.41], NA-EA versus CFA; Figure 1A) in rats from the NA-EA group did not differ from those in the CFA group (overall P = .10).
EA Increased Apelin/APJ Synthesis at Both the mRNA and Protein Levels in the Spinal Cord of Rats After CFA Injection
Figure 2.: The effects of electroacupuncture (EA) on the gene expression and protein levels of apelin and APJ in the spinal cord of rats with complete Freund’s adjuvant (CFA) injection (n = 4 per group). After the behavior tests (day 4) of experiment 1, the mRNA and protein expression levels of apelin and APJ were assessed in the lumbar L4-5 segments of the spinal cord. A. Apelin mRNA expression levels were analyzed via 1-way analysis of variance (ANOVA) (F (3,12) = 25.592, P < .0001), followed by multiple comparisons using the Games-Howell test (Levene test P = .038 < .05). B. APJ mRNA expression levels were analyzed via a 1-way ANOVA (F (3,12) = 15.026, P < .0001), followed by multiple comparisons using the Tukey test (Levene test P = .483 > 0.05). C. Apelin protein expression levels were analyzed via a 1-way ANOVA (F (3,12) = 55.338, P < .0001), followed by multiple comparisons using the Tukey test (Levene test P = .071 > .05). D. APJ protein expression levels were analyzed via a 1-way ANOVA (F (3,12) = 47.012, P < .0001), followed by multiple comparisons using the Tukey test (Levene test P = .159 > .05). *P < .01 compared with the sham group; # P < .05 compared with the CFA group. The error bars represent the SDs.
The apelin and APJ mRNA levels in the spinal cord are shown in Figure 2, A and B. In the CFA group, CFA injection drastically decreased the mRNA expression levels of both apelin and APJ in the spinal cord compared with the sham group (each P < .01). This decrease was mitigated by EA treatment (each P < .05, EA versus CFA). Similarly, the protein levels of apelin and APJ in the spinal cord were both decreased in the CFA group compared with the sham group (each P < .001) (Figure 2, C and D). EA treatment significantly mitigated the decrease in apelin and APJ protein expression induced by CFA injection (each P < .05). However, in rats in the NA-EA group, the levels of apelin and APJ protein and mRNA did not change significantly compared with the CFA group (each P > .496).
Administration of F13A Inhibited EA-Induced Antihyperalgesia
Figure 3.: Effect of electroacupuncture (EA) on rats after spinal administration of the apelin/APJ system antagonist F13A. A. Mechanical withdrawal threshold (MWT). B. Thermal withdrawal latency (TWL). The 2-way analysis of variance with repeated measurements of mechanical withdrawal threshold and thermal hyperalgesia showed prominent main effects for the groups (P < .0001), time (P < .0001), and significant interaction (P < .0001). Comparisons by Student t test with Bonferroni correction (P < .01/5 = .002 was defined as significant) displayed significant within-group and between-group differences in mechanical withdrawal threshold and thermal hyperalgesia. *P < .002 compared with the sham group; # P < .002 compared with the CFA group; † P < .002 for the EA+F13A group versus the EA group at day 4 after CFA injection. The error bars represent the standard deviations.
Having found that the apelin/APJ system is activated during EA, we next examined whether the apelin/APJ system mediates the antinociceptive effects of EA. The apelin/APJ system antagonist F13A was used. Our results showed that mechanical allodynia and thermal hyperalgesia occurred on day 1 after CFA injection (all P < .0001 compared with sham) (Figure 3, A and B, respectively). Before the intrathecal administration of F13A, EA treatment also significantly increased the MWT (overall P < .0001, mean difference [99% CI]: 6.68 [5.39–7.98], EA versus CFA; Figure 3A) and TWT (overall P < .0001, mean difference [99% CI]: 2.59 [1.08–4.09], EA versus CFA; Figure 3B) of the ipsilateral hindpaws. However, the antinociceptive effect of EA was inhibited after the intrathecal delivery of F13A at day 4 after CFA injection (MWT: P < .0001, mean difference [99% CI]: 8.99 [5.81–12.17]; Figure 3A; TWL: P < .0001, mean difference [99% CI]: 4.22 [1.33–7.12]; Figure 3B; EA versus EA-F13A). This result indicates that the apelin/APJ system antagonist inhibits EA antihyperalgesia. The intrathecal administration of F13A alone had no effect on the MWT (overall P = 1.00, mean difference [99% CI]: 0.20 [–1.09–1.50], F13A versus CFA; Figure 3A) or TWL (overall P = 1.00, mean difference [99% CI]: 1.15 [–0.40–2.70], F13A versus CFA; Figure 3B) of CFA rats.
Administration of Apelin-13 Enhanced EA-Induced Antihyperalgesia
Figure 4.: Effects of electroacupuncture (EA) and intrathecal administration of apelin-13 on mechanical allodynia and thermal hyperalgesia induced by CFA injection. A. Mechanical withdrawal threshold (MWT). B. Thermal withdrawal latency (TWL). The 2-way analysis of variance with repeated measurements of mechanical withdrawal threshold and thermal hyperalgesia showed prominent main effects for the groups (P < .0001), time (P < .0001), and significant interaction (P < .0001). Comparisons by Student t test with Bonferroni correction (P < .01/5 = .002 was defined as significant) displayed significant within-group and between-group differences in the mechanical withdrawal threshold and the thermal hyperalgesia. *P < .002 compared with the sham group; # P < .002 compared with the CFA group; † P < .002 for the EA + apelin-13 group versus the EA group or apelin-13 group at day 3 after CFA injection. The error bars represent the standard deviations.
Because the antihyperalgesia effects of EA are mediated by the apelin/APJ system, we hypothesized that apelin-13 may exert inhibitory effects on inflammatory pain. As predicted, intrathecal injection of apelin-13 at day 3 after CFA injection induced an inhibitory effect on mechanical allodynia and thermal hyperalgesia (MWT: P < .0001, mean difference [99% CI]: 5.44 [1.85–9.03]; Figure 4A; TWL: P < .0001, mean difference [99% CI]: 4.25 [1.20–7.31]; Figure 4B; apelin-13 versus CFA). Meanwhile, in the EA + apelin-13 group, MWT (overall P < .0001, mean difference [99% CI]: 5.05 [3.03–7.07], EA + apelin-13 versus CFA; Figure 4A) and TWL (overall P < .0001, mean difference [99% CI]: 4.03 [2.46–5.60], EA + apelin-13 versus CFA; Figure 4B) of the ipsilateral hindpaws were also significantly increased. Furthermore, a significant enhancement of antihyperalgesia effects was observed when EA was combined with apelin-13 treatment. As shown in Figure 4, EA and the intrathecal administration of apelin-13 elicited stronger analgesic effects by increasing the MWT (P < .0001, mean difference [99% CI]: 5.98 [2.38–9.57], EA + apelin-13 versus apelin-13; P < .001, mean difference [99% CI]: 4.29 [0.72–7.87], EA + apelin-13 versus EA; Figure 4A) and TWL (P < .0001, mean difference [99% CI]: 5.23 [2.19–8.28], EA + apelin-13 versus apelin-13; P < .001, mean difference [99% CI]: 6.43 [3.38–9.48], EA + apelin-13 versus EA; Figure 4A) compared with the single-agent treatment with apelin-13 or EA alone at day 3 after CFA injection.
DISCUSSION
The present study demonstrated that EA treatment attenuates CFA-induced pain and reverses the downregulation of apelin and APJ in the spinal cord. In addition, the combined administration of EA and apelin-13 intrathecal injection remarkably exhibited a synergistic antihyperalgesic effect on inflammatory pain. These data indicate that activation of the apelin/APJ system may be involved in analgesic effects of EA.
EA treatment is an effective method for alleviating various types of pain in preclinical and clinical studies. In the present study, EA significantly increased the mechanical and thermal nociceptive thresholds and reduced the hypersensitivity of CFA rats. This finding indicates that EA treatment had a beneficial effect on the pain thresholds of CFA rats.
Apelin and APJ are expressed in nociception and pain-related structures including the dorsal root ganglia, spinal cord, periaqueductal gray, and dorsal raphe nucleus.24 Previous studies have demonstrated that the apelin/APJ system plays an important role in pain modulation. Apelin-13 results in an increased pain threshold and antinociceptive effects not only in naïve animals, but also in inflammatory and visceral pain models.14–16 However, whether the expression of the endogenous apelin/APJ system changes under pathologic pain conditions is unknown. In this study, we found that the mRNA and protein levels of apelin and APJ decreased markedly in CFA rats in comparison with the normal control group. Meanwhile, EA stimulation could mitigate the reduction in the expression levels of apelin and APJ in the spinal cord in CFA rats. Furthermore, in this study, intrathecal administration of F13A, an apelin/APJ system antagonist, could block EA-induced analgesia. These results indicate the following: (1) in the spinal cord, the apelin/APJ system may be implicated in the initiation and development of inflammatory pain; and (2) the EA treatment is beneficial to the inflammatory pain and acts by restoring the endogenous apelin/APJ system.
Although several experimental studies have demonstrated the analgesic effects of apelin-13, the mechanism underlying the analgesic actions of the apelin/APJ system remains largely unclear. One study showed that μ-opioid receptor signaling is involved in the analgesic response evoked after the intracerebroventricular administration of apelin-13.16 Central injection of apelin-13 activates APJ receptors, which subsequently trigger a Gβγ-dependent activation of phospholipase C-β signaling and inhibit the M-current, eventually enhancing the activity of arcuate nucleus proopiomelanocortin neurons.25 Proopiomelanocortin is a peptide precursor molecule that is broken down to produce β-endorphin, adrenocorticotrophic hormone, and melanocyte-stimulating hormone. Meanwhile, the arcuate nucleus is a crucial specific region that responds to 2 Hz EA to produce an analgesic effect by accelerating the release of β-endorphin.26 There is also functional crosstalk between the apelin/APJ system and the gamma-aminobutyric acid type A (GABAA) receptor because the coadministration (intrathecally) of bicuculline, a GABAA receptor antagonist, significantly antagonizes the hyperalgesic effect of apelin-13.14 Meanwhile, in our previous study, we found that the levels of APJ, GABAA receptor, glycine receptor (GlyR), and 5-hydroxytryptamine receptor 1 [5-HT(1)] mRNA in the spinal horn are related to variations in EA analgesia and are significantly highly upregulated in the high-responder naïve rats.19 Therefore, besides the GABAA receptor, further research is required to determine whether functional crosstalk exists between the apelin/APJ system and the GlyR, or 5-HT(1) receptor. A recent study showed that calcium-mediated signaling in primary sensory neurons is a key pain pathway that regulates nociceptive transmission and pain.27 However, it is not involved in the hyperalgesic effect of the peripheral administration of apelin-13.28 In the future, more physiopathologic and neurochemical experiments should be performed to better understand the mechanisms underlying the pain-modulating actions of the apelin/APJ system.
In this study, we also found that EA and the intrathecal administration of apelin-13 had a synergistic effect on inflammatory pain induced by CFA injection. This effect may be because EA analgesia is essentially a manifestation of integrative biologic processes occurring at the multisystem and multiorgan levels, especially at different levels in the central nervous system.29,30 In addition to the apelin/APJ system, some other substances are also involved in the process of EA analgesia. Furthermore, these different factors, including the apelin/APJ system, may interact with each other to enhance the analgesic effect of EA treatment.
Presently, EA and manual acupuncture are extensively used in clinical practice. For manual acupuncture, the acupuncture needle is inserted into the acupoint and is twisted up and down by hand. For EA, which has evolved from traditional manual acupuncture, the acupuncture needle is inserted into the acupoint and electric pulses are delivered to acupuncture needles. Compared with manual acupuncture, EA stimulation can be accurately adjusted more easily and has good repeatability. Therefore, we choose to use EA in this study. The NA-EA group, which underwent sham acupuncture treatment, did not show any significant change in pain threshold in this study because of the lack of stimulation (either electronically or manually). The intensity of acupuncture stimulation plays a critical role in the therapeutic effect of acupuncture, and optimal higher intensity stimulation is more effective than lower stimulation.31,32
In conclusion, the restoration of apelin and APJ mRNA and protein expression levels in the spine was involved in the antihyperalgesic effect of EA treatment. The present findings also suggest that the spinal apelin/APJ system may be a novel target for inflammatory pain.
DISCLOSURES
Name: Ke Wang, MD.
Contribution: This author helped design the study, conduct the study, analyze the data, and prepare the manuscript.
Name: Ziyong Ju, MD.
Contribution: This author helped perform the experiments, analyze the data, and prepare the manuscript.
Name: Yue Yong, PhD.
Contribution: This author helped perform the experiments and prepare the manuscript.
Name: Tongyu Chen, MM.
Contribution: This author helped analyze the data.
Name: Jiangang Song, MD.
Contribution: This author helped conduct the study and reviewed the analysis of the data.
Name: Jia Zhou, MM.
Contribution: This author helped design the study, conduct the study, analyze the data, and prepare the manuscript.
This manuscript was handled by: Jianren Mao, MD, PhD.
REFERENCES
1. Marchand F, Perretti M, McMahon SBRole of the immune system in chronic pain.Nat Rev Neurosci20056521532
2. Kuner RCentral mechanisms of pathological pain.Nat Med20101612581266
3. Carter GT, Duong V, Ho S, Ngo KC, Greer CL, Weeks DLSide effects of commonly prescribed analgesic medications.Phys Med Rehabil Clin N Am201425457470
4. Dworkin RH, Jensen MP, Gould E, et al.Treatment satisfaction in osteoarthritis and chronic low back pain: the role of pain, physical and emotional functioning, sleep, and adverse events.J Pain201112416424
5. Lee HJ, Tomioka M, Takaki Y, Masumoto H, Saido TCMolecular cloning and expression of aminopeptidase A isoforms from rat hippocampus.Biochim Biophys Acta20001493273278
6. Carpéné C, Dray C, Attané C, et al.Expanding role for the apelin/APJ system in physiopathology.J Physiol Biochem200763359373
7. Tatemoto K, Hosoya M, Habata Y, et al.Isolation and characterization of a novel endogenous peptide ligand for the human APJ receptor.Biochem Biophys Res Commun1998251471476
8. O’Carroll AM, Lolait SJ, Harris LE, Pope GRThe apelin receptor APJ: journey from an orphan to a multifaceted regulator of homeostasis.J Endocrinol2013219R1335
9. Falcão-Pires I, Ladeiras-Lopes R, Leite-Moreira AFThe apelinergic system: a promising therapeutic target.Expert Opin Ther Targets201014633645
10. Chapman NA, Dupré DJ, Rainey JKThe apelin receptor: physiology, pathology, cell signalling, and ligand modulation of a peptide-activated class A GPCR.Biochem Cell Biol201492431440
11. Yu XH, Tang ZB, Liu LJ, et al.Apelin and its receptor APJ in cardiovascular diseases.Clin Chim Acta201442818
12. Cheng B, Chen J, Bai B, Xin QNeuroprotection of apelin and its signaling pathway.Peptides201237171173
13. Telegdy G, Jászberényi MTransmitter mediation of the anxiolytic action of apelin-13 in male mice.Behav Brain Res2014263198202
14. Lv S, Yang YJ, Hong S, et al.Intrathecal apelin-13 produced different actions in formalin test and tail-flick test in mice.Protein Pept Lett201320926931
15. Lv SY, Qin YJ, Wang NB, Yang YJ, Chen QSupraspinal antinociceptive effect of apelin-13 in a mouse visceral pain model.Peptides201237165170
16. Xu N, Wang H, Fan L, Chen QSupraspinal administration of apelin-13 induces antinociception via the opioid receptor in mice.Peptides20093011531157
17. Zhang R, Lao L, Ren K, Berman BMMechanisms of acupuncture–electroacupuncture on persistent pain.Anesthesiology2014120482503
18. Zhang CR, Xia CM, Jiang MY, et al.Repeated electroacupuncture attenuating of apelin expression and function in the rostral ventrolateral medulla in stress-induced hypertensive rats.Brain Res Bull2013975362
19. Wang K, Zhang R, Xiang X, et al.Differences in neural-immune gene expression response in rat spinal dorsal horn correlates with variations in electroacupuncture analgesia.PLoS One20127e42331
20. Peng HY, Chen GD, Hsieh MC, Lai CY, Huang YP, Lin TBSpinal SGK1/GRASP-1/Rab4 is involved in complete Freund’s adjuvant-induced inflammatory pain via regulating dorsal horn GluR1-containing AMPA receptor trafficking in rats.Pain201215323802392
21. Yaksh TL, Rudy TAChronic catheterization of the spinal subarachnoid space.Physiol Behav19761710311036
22. Yu J, Zhao C, Luo XThe effects of electroacupuncture on the extracellular signal-regulated kinase 1/2/P2X3 signal pathway in the spinal cord of rats with chronic constriction injury.Anesth Analg2013116239246
23. Wang WS, Tu WZ, Cheng RD, et al.Electroacupuncture and A-317491 depress the transmission of pain on primary afferent mediated by the P2X3 receptor in rats with chronic neuropathic pain states.J Neurosci Res20149217031713
24. Lee DK, Cheng R, Nguyen T, et al.Characterization of apelin, the ligand for the APJ receptor.J Neurochem2000743441
25. Lee DK, Jeong JH, Oh S, Jo YHApelin-13 enhances arcuate POMC neuron activity via inhibiting M-current.PLoS One201510e0119457
26. Guo ZL, Longhurst JCExpression of c-Fos in arcuate nucleus induced by electroacupuncture: relations to neurons containing opioids and glutamate.Brain Res200711666576
27. Lee Y, Lee CH, Oh UPainful channels in sensory neurons.Mol Cells200520315324
28. Canpolat S, Ozcan M, Saral S, Kalkan OF, Ayar AEffects of apelin-13 in mice model of experimental pain and peripheral nociceptive signaling in rat sensory neurons.J Recept Signal Transduct Res201515
29. Wang K, Xiang XH, Qiao N, et al.Genomewide analysis of rat periaqueductal gray-dorsal horn reveals time-, region- and frequency-specific mRNA expression changes in response to electroacupuncture stimulation.Sci Rep201446713
30. Zhao P, Cheng JEffects of electroacupuncture on extracellular contents of amino acid neurotransmitters in rat striatum following transient focal cerebral ischemia.Acupunct Electrother Res199722119126
31. Ju Z, Guo X, Jiang X, et al.Electroacupuncture with different current intensities to treat knee osteoarthritis: a single-blinded controlled study.Int J Clin Exp Med201581898118989
32. Shao XM, Shen Z, Sun J, et al.Strong manual acupuncture stimulation of ‘Huantiao’ (GB 30) reduces pain-induced anxiety and p-ERK in the anterior cingulate cortex in a rat model of neuropathic pain.Evid Based Complement Alternat Med20152015235491
