Keywords:
Electroacupuncture - Chronic Pain - Adenosine - Substance P
Palavras-chave:
Eletroacupuntura - Dor Crônica - Adenosina - Substância P
Chronic pain (CP) is defined as pain that typically lasts over six months. It can
be initiated with injury or disease and may persist after the triggering injury has
been healed[1]. Chronic inflammatory pain (CIP) is a subtype of CP, which is usually caused by
trauma, bacterial and viral infection, chemical stimulation, and surgery[2]. The prevalence of CIP in adults is estimated to range between 5 and 25%. CIP not
only offers great pain and burden to patients, but it also leads to limited labor
capacity and reduced work efficiency[3]. Currently, several options are available for clinicians and patients to attenuate
CIP. The first choice is using medicines that target the central nervous system to
suppress pain, such as opioid receptor agonist morphine. These drugs lead to strong
analgesia; however, they also produce lots of side effects, such as respiratory depression,
drug resistance, reduction of gastrointestinal motility, and drug addiction[4]. The second option is application of nonsteroidal anti-inflammatory drugs, which
usually induce a weak analgesic effect[4],[5]. Finally, the third choice is the acupuncture therapy for analgesia. It is well
known that acupuncture is a famous analgesic therapy with remarkable curative effect
and a long history in the Asian medicine[6],[7]. Currently, acupuncture has been considered to have a superiority of almost no side
effect compared with drug analgesia. Therefore, it has been widely used around the
world[6],[7]. Nevertheless, the exact analgesia mechanism of acupuncture is still poorly understood.
Therefore, it is urgent to uncover the analgesia mechanism of acupuncture, which not
only provides theoretical basis for clinical application, but also promotes its application.
A previous study reported that adenosine mediates analgesia effect of acupuncture.
Interfering with adenosine metabolism may prolong the clinical benefit of acupuncture[8]. However, little is known about how adenosine contributes to the analgesic effect
of electroacupuncture (EA). Tissue injury causes local releases of inflammatory mediators,
such as substance P (SP), histamine, and prostaglandins E[9]. These mediators activate immune cells, which in turn release many pro-inflammatory
factors, such as interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α). Finally,
pro-inflammatory cytokines induce peripheral nociceptor cells to produce action potentials,
resulting in pain[9],[10]. We have recently noticed adenosine inhibits the release of SP and calcitonin gene-related
peptide from afferent nerve endings[11]. Therefore, we suspect EA improves pain threshold via increasing adenosine levels,
which acts on adenosine receptor and inhibits the release of SP from the dorsal root
ganglion (DRG). In the present study, we used Complete Freund’s Adjuvant (CFA) induced
pain model to investigate the hypothesis that adenosine and its mediated SP release
are involved in EA analgesia.
METHODS
Animals
Male Sprague-Dawley rats (180-220 g) were purchased from the experimental animal center
of Anhui Medical University. All rats were housed in a controlled environment (temperature
- 25±2°C, humidity - 55±5%, and light - 12-hour light/dark cycle) and fed with standard
rodent food and allowed distilled water ad libitum. All procedures were approved by the Animal Care and Use Committee of Anhui Medical
University (Hefei, China, LLSC20183042).
Inflammatory pain induction
Inflammatory pain was induced in rats through intra-plantar injection of 100 μL of
CFA (Sigma-Aldrich Corporation, St. Louis, MO, USA) on the plantar surface of the
left hind paw[12]. Each milliliter of the injection contained 1 mg heat-killed and dried mycobacterium
tuberculosis, 0.85 mL paraffin oil and 0.15 mL mannide monooleate. Control animals
were injected with the same volume of 0.9% saline.
Treatment
All animals were separated randomly into seven groups: regular Control Group, received
0.1 mL of saline injection; CFA Group received 0.1 mL of CFA injection; CFA+EA Group
received 0.1 mL of CFA injection together with daily EA stimulation for seven days;
CFA+EA+Dorsal Nerve Root Transection Group (DNRR) received 0.1 mL of CFA injection,
daily EA stimulation and DNRR surgery performed at 24 hours after immunization; CFA+EA+adenosine
A1 receptor antagonist (ANTAG, rolofylline, Sigma-Aldrich Corporation, St. Louis,
MO, USA) Group received 0.1 mL of CFA injection, daily EA stimulation and injection
of rolofylline at a dose of 3 mg/kg into acupoint (“Zusanli”); CFA+EA+adenosine A1
receptor agonist (AG, N6-cyclopentyladenosine: CPA; Sigma, St. Louis, MO, USA) Group
received 0.1 mL of CFA injection, daily EA stimulation and injection of CPA at a dose
of 0.1 mg/kg into acupoint (“Zusanli”); CFA+SP receptor antagonist (CP96345; Sigma,
St. Louis, MO, USA) received 0.1 mL of CFA injection, daily EA stimulation and subarachnoid
administration of CP96345 at a dose of 2.5 mg/kg.
The EA procedure was conducted based on a previous published method with minor modifications[12]. In brief, the rats received EA treatment with stimulator parameters as follows:
2/100 Hz, 0.5 to 1.5 mA (initial strength of 0.5 mA, increased by 0.5 mA every 10
minutes) for a total of 30 minutes. We applied a total of seven bilateral sessions
of EA therapy (30 minutes per session, one section per day) to CFA rats for seven
days. Electro-stimulation was performed with constant square wave current output (pulse
width: 0.6 ms at 2 Hz, 0.2 ms at 100 Hz). Rats were loosely immobilized. Four stainless
steel acupuncture needles of 0.25 mm in diameter were inserted at a depth of 5 mm
into bilateral “Zusanli” (ST36, 5 mm lateral to anterior tubercle of the tibia) and
“Kunlun” (BL60, at ankle joint level and between the tip of external malleolus and
tendon calcaneus) acupoints. Electrical stimulation was produced by a Trio 300 electrical
stimulator (Trio 300, ITO Corporation, Germany).
Paw withdrawal threshold
The paw withdrawal threshold (PWT) was determined by von Frey behavioral test and
Hargraves’ test according to previous methods[12],[13]. In brief, all stimuli were performed at room temperature and applied when animals
were calm. Mechanical sensitivity was measured by testing the force of responses to
stimulation with three applications of electronic von Frey filaments (North Coast
Medical, Gilroy, CA, USA), while thermal pain was assessed with three applications
using Hargraves’ test IITC analgesiometer (IITC Life Sciences., Model 390 G, CA, USA).
Analysis of adenosine levels
Microdialysis probes were implanted on the left side of the foot in rats of the Control
Group, CFA Group and CFA+EA Group before the study. The microdialysis solution was
collected at different time points during the experiment (one, three, five and seven
days after immunization). Adenosine levels were assessed by a High Performance Liquid
Chromatography (HPLC) method as already described[8].
Real-time quantitative polymerase chain reactions
Total RNA was extracted from tissue homogenization using Trizol (Invitrogen, Carlsbad,
CA, USA). Reverse transcription was performed using 1 μg total RNA by Prime Script
WRT reagent kit (TaKaRa, Japan). The expression of genes was analyzed by real-time
quantitative polymerase chain reactions (qPCR) using CFX96™ real-time PCR detection
system (Bio-Rad, CA, USA). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene
was used as an internal control for target genes, and the relative expression level
was calculated through the 2-ΔΔCT method. The primers described in [Table 1] were designed and synthesized.
Table 1
Primers used in the current study.
|
Gene names
|
Primers
|
|
TNF-α
|
•Forward: 5’-TACTGAACTTCGGG GTGATTGGTCC-3’
•Reverse: 5’-CAGCCTTGTCCCTTGAAGA GAACC-3’
|
|
IL-1β
|
•Forward: 5’-TG AAGCAGCTATGGCAACTG-3’
•Reverse: 5’-CTGCCTTCCTGAAGCTCTTG-3’
|
|
IL-6
|
•Forward: 5′-GCCAGAGTCATTCAGAGCAATA-3′
•Reverse: 5′-TTAGGAGAGCATTGG AAGTTGG-3′
|
|
Neurokinin-1 receptor (NK-1R)
|
•Forward: 5′-CACACT ATGGGCCAGTGAGATC-3′
•Reverse: 5′-GCACACCACGACAATCATCATT-3′
|
|
CD68
|
•Forward: 5’-CTCACAAAAAGGCTGCCACT-3′
•Reverse: 5′-TTCCGGTGGTTGTAGGTGTC-3′
|
|
GAPDH
|
•Forward: 5′-GGTGGTCTCCTCTGACTTCAACA-3′
•Reverse: 5′-GTTGCTGTAGCCA-AATTCGTTGT-3′
|
TNF-α: tumor necrosis factor-alpha; IL-1β: interleukin-1beta; NK-1R: neurokinin-1
receptor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.
Western blotting analysis
The animals were sacrificed after behavioral testing seven days after immunization.
Ipsilateral spinal dorsal horns (L4-L6) were removed and stored at -80°C. The dorsal
horns were homogenized with a mechanical rotary cutter in strong radioimmunoprecipitation
assay (RIPA) buffer (Sigma, St. Louis, MO, USA) containing a cocktail of phosphatase
and proteinase inhibitors. The protein concentration of tissue lysates was detected
with a bicinchoninic acid protein assay kit (Thermo Scientific, Grand Island, NY,
USA). All samples were separated on 10% sodium dodecyl sulfate-polyacrylamide gelelectrophoresis
gels and electrophoretically transferred to polyvinylidene difluoride membranes (Millipore,
MA, USA). The membranes were blocked with 5% low-fat milk in Tris Buffered saline
Tween for one hour and then incubated with NK-1R and CD68 antibodies (Santa Cruz,
CA, USA). Afterwards, the bolts were incubated for one hour at 37°C with HRP-conjugated
secondary antibody and visualized in enhanced chemiluminecence solution (Thermo Scientific,
Grand Island, NY, USA). β-actin was used as an internal control.
Measurement of cytokines
Spinal dorsal horns of rats were homogenized, and a homogeneous section has been collected.
The levels of TNF-α, IL-1β, IL-6 and SP were assessed by using ELISA kits offered
by the manufacturing company (R&D Systems, Minneapolis, MN, USA).
Statistical analysis
All statistical tests were conducted using SPSS 15.0 (SPSS Inc., Chicago, IL, USA)
software. Data were presented as mean±standard deviation. Statistical analysis was
conducted through one-way analysis of variance (ANOVA) followed by individual post hoc multiple comparisons. A statistical difference was considered as significant at p<0.05.
RESULTS
Electroacupuncture stimulation reduced Complete Freund’s Adjuvant-induced pain in
rats
Rats were injected with CFA into the surface of the left hind paw to induce persistent
inflammatory pain. As seen in [Figure 1], both mechanical and thermal hyperalgesia were measured and used to evaluate the
analgesia activity of the animals. No significant difference was observed in mechanical
sensitivity and thermal hyperalgesia among the seven groups at the baseline (before
CFA injection). Moreover, the pain threshold was significantly lower in CFA Groups
in days three, five, and seven after immunization compared with the Control Group
(p<0.01). In contrast, mechanical and thermal hyperalgesias were significantly improved
in the CFA+EA Group (p<0.01) compared with CFA group, suggesting well analgesic effects
induced by EA stimulation. Furthermore, to study whether the analgesic effects of
EA therapy were based on adenosine-regulated SP release, we applied different “tool
reagents” together with EA to treat CFA rats. As shown in [Figure 1], we found that CFA rats who were given DNRR and EA showed increased mechanical and
thermal hyperalgesia compared with those that only received EA (p<0.01). In addition,
CFA rats who were given ANTAG and EA showed an impaired pain tolerance compared with
those that only received EA (p<0.01). As expected, similarly to EA treatment, daily
AG injection could significantly increase pain threshold in CFA rats. Finally, we
noted SP receptor antagonist (CP96345) treatment could undermine the analgesia effects
induced by EA stimulation in rats (p<0.01).
Figure 1 Effects of electroacupuncture stimulation on paw withdrawal threshold in rats. (A)
Mechanical sensitivity of rats was measured by assessing the force of responses to
stimulation with electronic von Frey filaments. (B) Thermal sensitivity of rats was
measured using Hargraves’ test IITC analgesiometer.EA: electroacupuncture; CFA: Complete
Freund’s Adjuvant; AG: antagonist receptor; DNRR: Dorsal Nerve Root Transection Group;
CP: chronic pain; ANTAG: adenosine A1 receptor antagonist. Values represent mean±standard
deviation; n=8 per group for each time point; ##p<0.01 versus Control Group; *p<0.05; **p<0.01 versus Complete Freund’s Adjuvant group; p<0.01 versus CFA+EA Group.
Electroacupuncture stimulation increased adenosine level in the Complete Freund’s
Adjuvant-treated rats
We investigated whether EA therapy could increase adenosine levels in CFA rats. As
shown in [Figure 2], compared with the animals in the Control Group, CFA rats showed decreased level
of adenosine at 24 hours after CFA immunization (p<0.01). However, EA treatment could
significantly increase adenosine levels in CFA rats that began at day three after
stimulation (p<0.01).
Figure 2 Adenosine levels in rats treated by Complete Freund’s Adjuvant together with electroacupuncture.EA:
electroacupuncture; CFA: Complete Freund’s Adjuvant. 1: Control Group; 2: CFA Group;
3: CFA+EA Group. Values represent mean±standard deviation; n=8 per group; ##p<0.01
versus Control Group; **p<0.01 versus CFA Group.
Electroacupuncture stimulation decreased mRNA levels of inflammatory pain-related
genes in Complete Freund’s Adjuvant-treated rats
As shown in [Figure 3], we further investigated mRNA levels of inflammatory pain associated genes in response
to EA stimulation. The results charted in [Figure 3] indicate CFA injection significantly increased SP, NK-1R, CD68, TNF-α, IL-1β and
IL-6 mRNA expression in the DRG of rats (p<0.01). However, EA significantly downregulated
the expression of SP, NK-1R, CD68, TNF-α, IL-1β, and IL-6 mRNA, compared with the
CFA Group (p<0.01). Moreover, we found CFA rats that were given ANTAG and EA showed
decreased levels of SP, NK-1R, CD68, TNF-α, IL-1β and IL-6 mRNA compared with those
that only received EA (p<0.05). AG treatment significantly decreased the mRNA levels
of these genes compared with the CFA Group (p<0.01). In addition, compared with the
EA Group, both blockade of DRG by DNRR and inhibition SP-mediated pathway by SP receptor
antagonist could increase the mRNA levels of SP, NK-1R, CD68, TNF-α, IL-1β and IL-6
(p<0.01).
Figure 3 mRNA levels of inflammatory pain-related genes in dorsal root ganglion of Complete
Freund’s Adjuvant rats that received electroacupuncture stimulation.EA: electroacupuncture;
CFA: Complete Freund’s Adjuvant; AG: antagonist receptor; DNRR: Dorsal Nerve Root
Transection Group; CP: chronic pain; ANTAG: adenosine A1 receptor antagonist. 1: Control
Group; 2: CFA Group; 3: CFA+EA; 4: CFA+EA+DNRR; 5: CFA+EA+ANTAG; 6: CFA+AG; 7: CFA+EA+CP
96345. Values represent mean±standard deviation; n=8 per group; ##p<0.01 versus Control Group; *p<0.05; **p<0.01 versus CFA Group; p<0.01 versus CFA+EA Group.
Electroacupuncture decreased the expression of inflammatory pain-related protein in
Complete Freund’s Adjuvant-treated rats
To investigate changes in the molecule expression of associated inflammatory pain
in DRG following EA stimulation, we used ELISA and western blotting to measure these
protein levels in each group. As shown in [Figure 4], CFA injection significantly increased the levels of SP, NK-1R, CD68, TNF-α, IL-1β,
and IL-6 in DRG (p<0.01). However, after EA stimulation, these molecule levels were
markedly reduced compared with the CFA Group (p<0.01). Moreover, we found that in
rats that were given EA together with DNRR, ATANG or SP receptor antagonist, expression
of SP, NK-1R, CD68, TNF-α, IL-1β, IL-6 were significantly enhanced, compared to those
in rats that only received EA (p<0.05). Finally, we observed AG treatment could significantly
decrease the expression of SP, NK-1R, CD68, TNF-α, IL-1β, IL-6 in DRG of CFA rats
(p<0.01), suggesting the important role of AG in the development of CFA-induced CIP.
Figure 4 Expression of inflammatory pain-related protein in dorsal root ganglion of rats that
received electroacupuncture stimulation. (A) Pro-inflammatory cytokines TNF-α, IL-1β,
IL-6 and SP were detected by ELISA kits; (B) expression of NK-1R and CD68 was assessed
by Western blotting analysis.EA: electroacupuncture; CFA: Complete Freund’s Adjuvant;
AG: antagonist receptor; DNRR: Dorsal Nerve Root Transection Group; CP: chronic pain;
ANTAG: adenosine A1 receptor antagonist. 1: Control Group; 2: CFA Group; 3: CFA+EA;
4: CFA+EA+DNRR; 5: CFA+EA+ANTAG; 6: CFA+AG; 7: CFA+EA+CP 96345. Values represent mean±standard
deviation; n=8 per group. ##p<0.01 versus Control Group; **p<0.01 versus CFA Group; *p<0.05; **p<0.01 versus CFA+EA Group.
DISCUSSION
EA has well analgesia activity and has been proved to be effective in treating various
kinds of pain. In 2004, Huang et al.[14] reported that EA therapy could attenuate mechanical hyperalgesia in a rat model
of CIP. Afterwards, EA was proved to inhibit inflammatory pain elicited by carrageenan
and cold allodynia in a rat model of neuropathic pain[15],[16]. EA, therefore, has been widely used to treat different kinds of pain. However,
it is still not trustworthy due to incomplete decryption of its molecular mechanism.
In the present study, we have observed that EA stimulation provided analgesia in a
rat model of CFA-induced inflammatory pain. In agreement with previous studies, our
data support the conclusion that EA therapy induces well anti-nociceptive effect.
Several clinical and basic studies have been performed to disclose the biological
basis of EA-induced analgesia. At present, several molecules and their mediated pathways
have been found to be involved in EA-induced analgesia. For instance, it has been
previously demonstrated that EA attenuates inflammatory pain by targeting CB2 receptors,
transient receptor potential vanilloid 1 and p38 MAPK pathway[17],[18],[19]. Just like opioids, adenosine plays an important role in CP[8],[20]. In previous studies, direct injection of adenosine A1 receptor agonist could replicate
the analgesic effect of acupuncture, while selective A1 and A2A antagonists completely
prevented antinociception[21],[22].
How does adenosine participate in the analgesic effect induced by EA? Some studies[16],[23] showed EA stimulation that induced analgesia in animal models may target Nav1.8,
COX-2 and pPKCε via regulating adenosine and its mediated pathway. In addition, there
are no other reports focused on the role of adenosine in EA-induced analgesia. SP
is a peptide neurotransmitter from sensory nerve endings and is the main mediator
of neurogenic inflammation[9]. SP plays an important role in the development of pain, tissue damage, and inflammatory
reactions. Interestingly, it has been reported that SP release in rat spinal cord
was mediated by adenosine. Intrathecal adenosine analog administration reduced SP
in cerebrospinal fluid along with antinociception behavioral effects[11],[24]. Therefore, EA treatment may exert analgesia action via inhibiting adenosine mediated
release of SP from DRG neurons.
In the present research, we found EA treatment could increase the level of adenosine,
SP, and NK-1R. It is well known that SP binding with NK-1R could increase the secretion
of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6[25], which directly promote inflammatory pain. Intramuscular injection of TNF-α induces
muscle hyperalgesia in rats[26]. Neutralizing antibodies to IL-1 receptor reduce pain-associated behavior in mice
with experimental neuropathy[27]. Therefore, we have further investigated the genetic and protein levels of TNF-α,
IL-1β and IL-6 in rats that received EA. The results showed that EA treatment reduced
these cytokines at transcription and protein levels. Moreover, we have also observed
the effects of DNRR on the efficacy of EA. Our results showed that DNRR not only blocked
EA-induced analgesia in CFA rats, but also increased the expression of SP, NK-1R and
their downstream inflammatory molecules. These findings strongly suggest that EA-induced
analgesia in CFA rats is dependent on the participation of DRG neurons.
Furthermore, to test whether EA-induced analgesia requires involvement of SP, we used
SP receptor antagonist CP96345 to interfere in the treatment of EA. As expected, similarly
to DNRR, we found that CP96345 almost completely impaired EA-induced analgesia in
CFA rats, implying SP mediated pathway is involved in the analgesia action of EA.
Moreover, we have also treated CFA rats using EA together with ANTAG. Interestingly,
ANTAG weakened EA-induced analgesia and correspondingly increased SP and NK-1R levels,
which suggest that regulation of SP secretion is an important mechanism of adenosine
to exert biological activity in the EA treatment.
In summary, our results suggest that the upregulation of adenosine level and enhancement
of adenosine induced inhibitory effects on SP are both necessary and sufficient for
the clinical benefits of EA. To the best of our knowledge, SP and its relationship
with adenosine have not been previously implicated in the antinociceptive actions
of EA. Therefore, our findings further broaden the current understanding of EA analgesia
mechanism.
