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https://doi.org/10.1590/0004-282X-ANP-2021-0149
ARTICLE
1Wen Zhou Medical University, The First Affiliated Hospital, Department of Anesthesiology, Wenzhou, China.
QD https://orcid.org/0000-0002-2516-5798; SL https://orcid.org/0000-0002-6737-5873; MR https://orcid.org/0000-0002-0025-8955;
XW https://orcid.org/0000-0001-8309-5380; XY https://orcid.org/0000-0002-9805-5485; FL https://orcid.org/0000-0001-5914-295X;
XN https://orcid.org/0000-0002-2118-1918; YM https://orcid.org/0000-0001-8671-6322; JW https://orcid.org/0000-0002-1525-3938
Correspondence: Yun-chang Mo; Email: myc1104@163.com.
Conflict of interest: There is no conflict of interest to declare.
Authors’ contributions: QD: writing-original draft and writing-review & editing; SL: formal analysis and methodology; MR, XW: software; XY: validation and
visualization; LF: investigation and validation; XN: data curation and formal analysis; YM: data curation and investigation; JW: resources and writing-review &
editing.
Support: This work was supported by the Natural Science Foundation of Zhejiang Province of China, No. LY19H290008.
Received on April 16, 2021; Received in its final form on May 30, 2021; Accepted on June 03, 2021.
Analgesia with 5’ extracellular nucleotidase-
mediated electroacupuncture for neuropathic
pain
Analgesia com eletroacupuntura mediada por nucleotidase extracellular 5’ para dor
neuropática
Qin-xue DAI1, Shan LI1, Miao REN1, Xinlu WU1, Xin-yu YAO1, Fei-hong LIN1, Xu-qing NI1, Yun-chang MO1,
Jun-lu WANG1
ABSTRACT
Background: Acupuncture is a treatment for neuropathic pain, but its mechanism remains unclear. Previous studies showed that analgesia
was induced in rats with neuropathic pain when their spinal cord adenosine content increased after electroacupuncture (EA); however, the
mechanism behind this electroacupuncture-induced increase has not been clarified. Objective: This study aimed to determine the role
that ecto-5’-nucleotidase plays in EA-induced analgesia for neuropathic pain. Methods: We performed electroacupuncture at the Zusanli
acupoint on the seventh day after establishing a rat model of neuropathic pain induced through chronic constriction injuries. We observed the
mechanical withdrawal threshold and thermal pain threshold and detected the expression of ecto-5’-nucleotidase in the spinal cord using
Western blot. Chronic constriction injury rat models were intraperitoneally injected with α,β-methyleneadenosine 5’-diphosphate, an ecto-
5’-nucleotidase inhibitor, 30 min before electroacupuncture. The adenosine content of the spinal cord was detected using high-performance
liquid chromatography. Lastly, the adenosine A1 receptor agonist N6-cyclopentyladenosine was intrathecally injected into the lumbar
swelling of the rats, and the mechanical withdrawal and thermal pain thresholds were reevaluated. Results: Analgesia and increased ecto-
5’-nucleotidase expression and adenosine content in the spinal cord were observed 1 h after electroacupuncture. α,β-methyleneadenosine
5’-diphosphate was able to inhibit upregulation of adenosine content and electroacupuncture-induced analgesia. After administration of
N6-cyclopentyladenosine, electroacupuncture-induced analgesia was restored. Conclusions: Our results suggest that electroacupuncture at
Zusanli can produce analgesia in chronic constriction injury rat models, possibly via the increased ecto-5’-nucleotidase expression induced
through electroacupuncture, thus leading to increased adenosine expression in the spinal cord.
Keywords: Electroacupuncture; Analgesia; 5’-nucleotidase; Neuralgia.
RESUMO
Antecedentes: A acupuntura é um tratamento para a dor neuropática, mas seu mecanismo permanece obscuro. Estudos anteriores
mostraram que a analgesia foi induzida em ratos com dor neuropática quando o conteúdo de adenosina da medula espinhal aumentou após
a eletroacupuntura (EA); no entanto, o mecanismo por trás desse aumento induzido por eletroacupuntura não foi esclarecido. Objetivo: Este
estudo teve como objetivo determinar o papel que a ecto-5’-nucleotidase desempenha na analgesia induzida por EA para dor neuropática.
Métodos: Realizamos eletroacupuntura no ponto de acupuntura de Zusanli no sétimo dia após estabelecer um modelo de rato de dor
neuropática induzida por lesão de constrição crônica. Observamos o limiar de retirada mecânica e o limiar de dor térmica, e detectamos
a expressão de ecto-5’-nucleotidase na medula espinhal usando Western blot. Modelos de ratos com lesão de constrição crônica foram
injetados intraperitonealmente com α, β-metilenadenosina 5’-difosfato, um inibidor de ecto-5’-nucleotidase, 30 min antes da eletroacupuntura.
O conteúdo de adenosina da medula espinhal foi detectado por meio de cromatografia líquida de alta eficiência. Por último, o agonista do
receptor de adenosina A1 N6-ciclopentiladenosina foi injetado por via intratecal no edema lombar dos ratos, e a retirada mecânica e os
limiares de dor térmica foram reavaliados. Resultados: Analgesia e aumento da expressão de ecto-5’-nucleotidase e conteúdo de adenosina
na medula espinhal foram observados 1 h após a eletroacupuntura. α, β-metilenadenosina 5’-difosfato foi capaz de inibir a sobrerregulação
do conteúdo de adenosina e analgesia induzida por eletroacupuntura. Após administração de N6-ciclopentiladenosina, a analgesia induzida
por eletroacupuntura foi restaurada. Conclusões: Nossos resultados sugerem que a eletroacupuntura em Zusanli pode produzir analgesia
290 Arq Neuropsiquiatr 2022;80(3):289-295
em modelos de ratos com lesão de constrição crônica, possivelmente por meio do aumento da expressão de ecto-5’-nucleotidase induzida
por eletroacupuntura, levando ao aumento da expressão de adenosina na medula espinhal.
Palavras-chave: Eletroacupuntura; Analgesia; 5’-Nucleotidase; Neuralgia.
INTRODUCTION
Neuropathic pain has a high incidence rate and its treat-
ment is dicult. is seriously aects the work and lives of
patients and poses challenges to clinicians
1
. erefore, the
search for an eective, nontoxic treatment with minimal side
eects has received close attention from the scientic com-
munity. In the late 1990s, the U.S. National Institutes of Health
claimed that acupuncture is an eective alternative treatment
for lumbago and leg pain2, but the mechanism of acupuncture
analgesia remains unclear. Our previous studies showed that
the adenosine content in the spinal cord of rats with chronic
constriction injuries (CCIs) can signicantly rise 60 min after
electroacupuncture (EA), thus producing analgesic eects in
these rats2. However, we could not determine how EA caused
the adenosine content in the spinal cord of CCI rats to increase.
Previous studies have shown that adenosine triphosphate
(ATP), adenosine diphosphate (ADP), adenosine monophos-
phate (AMP) and adenosine are all categorized as purine sub-
stances3,4. ATP is stripped of a phosphate group by a dephos-
phatase to become ADP, and ADP is then stripped of another
phosphate group by a dephosphatase to form AMP. Loss of a
phosphate group from AMP under the action of ecto-5’-nu-
cleotidase (5’-NT) results in formation of adenosine. Via the
action of adenosine deaminase, adenosine is then deaminated
to become creatinine, which is eventually metabolized by the
kidneys and excreted in urine4. Recent studies have suggested
that 5’-NT is the key enzyme for adenosine production5, and
it is worth exploring whether 5’-NT is the target of EA when
promoting adenosine production.
erefore, this study investigated whether EA produces
analgesic eects on CCI rats via promotion of increased spinal
adenosine, through upregulating 5’-NT expression.
METHODS
Animals
e study protocol was approved by the Institutional Animal
Experimental Ethics Committee at the First Aliated Hospital
of Wenzhou Medical University, China (approval no. 12045). e
experimental animal center of Wenzhou Medical University
provided 42 healthy adult male Sprague-Dawley rats (specic-
pathogen-free grade) weighing 220 to 250 g for this study (animal
No: 2007000517448). e rats were purchased three days before
surgery and were made to fast for one day but were allowed
to drink freely before surgery to prevent reux and aspiration.
CCI model
In accordance with the method described by Bennett6, the
rats were anesthetized with intraperitoneal chloral hydrate
(10%; 350 mg/kg) and routinely disinfected. e femoral bicep
muscles in the right lower extremities were separated, and the
sciatic nerve trunks were exposed and tied at four sites at 1-mm
intervals using 4-0 chromium-containing catgut. e tying force
was sucient to induce a slight leg muscle or toe jerk. e inci-
sions were then sutured, and the rats were returned to their
cages. In the sham group, the sciatic nerve was exposed, but no
knots were tied, while the other procedures remained identical.
After surgery, the right lower limb showed lameness, and wan-
dering and was held in a defensive position when the rats had
fully awakened from anesthesia; they would also occasionally
lick the limb. ese cases conrmed successful induction of
chronic sciatic nerve compression. Seven rats with autophagy
or wound infections were excluded and replaced.
Experimental grouping
e 42 rats were randomly divided into seven groups with
six rats in each: sham group; model group; EA group; EA +
α,β-methyleneadenosine 5’-diphosphate (AOPCP) group; EA +
normal saline (NS) group; EA + AOPCP + adenosine A1 recep-
tor agonist N6-cyclopentyladenosine (CCPA) group; and EA +
AOPCP + dimethylsulfoxide (DMSO) group.
Administration method
In accordance with the method proposed by Bennett6, a
sterile PE-10 catheter was inserted slowly into the subarachnoid
space to prevent lumbar enlargement (L4-6) under anesthesia,
three days before establishment of the CCI model. Subsequently,
lidocaine (2%; 10 μL) was injected. If the rats developed tempo-
rary paralysis of both lower extremities, the catheterization was
considered successful. e rats were then observed for signs
of neurobehavioral defects, such as paralysis or limping. Two
rats were abandoned because they showed these symptoms,
and the rejected rats were replaced.
AOPCP powder was dissolved in 0.9% sodium chloride
solution, and CCPA powder was dissolved in 10% DMSO. e
rats in the EA + AOPCP and EA + NS groups were intraperito-
neally injected with AOPCP (20 mg/kg)7 and an equal amount
of 0.9% sodium chloride solution, respectively, 30 min before
EA. Following intraperitoneal AOPCP injection, the rats in the
EA + AOPCP + CCPA and EA + AOPCP + DMSO groups were
intrathecally injected with CCPA (1 mmol/L; 10 μL) and DMSO
through a pre-prepared PE catheter, respectively.
291
Dai Q, et al. 5’ NT mediated analgesic effect of electroacupuncture.
EA treatment
e Zusanli acupoint is located approximately 5 mm below
the bula head in the knee joint8. An acupuncture needle was
used to puncture the Zusanli acupoints in the legs, followed
by electrical stimulation at a current intensity, frequency and
duration of 1 mA, 2/100 Hz, and 30 min, respectively.
Mechanical pain threshold measurement
To determine the baseline value of the mechanical with-
drawal threshold before experimental treatment, the rats were
placed in a special transparent glass box with a grid bottom.
After 30 min of silence, the rats were stimulated with laments
of dierent weights (0.008, 0.02, 0.04, 0.07, 0.16, 0.4, 0.6, 1, 1.4,
2 and 4) in ascending order, and their foot shrinking reaction
was observed. e right hind paws were stimulated 10 times
with each lament for a duration of 1 to 2 s each time, and
the interval between each instance was 1 min. e number of
instances of foot shrinkage was recorded, and the correspond-
ing foot shrinkage rate (foot shrinkage rate = ( foot shrinkage
instance number / 10 times) × 100%) with dierent gram weights
of laments for all of the rats was estimated, in order to nd
the laments that induced the foot shrinkage rate closest to
50%8. In this experiment, the shrinkage rate induced by a 0.4
g ber was 45%. ereafter, a 0.16 g ber was used to measure
the number of instances of foot shrinkage among the rats in
each group and to calculate the foot shrinkage rate of the rats,
as follows: mechanical withdrawal threshold (MWT) = 100%
– foot shrinkage rate.
Thermal pain threshold measurement
e rats were placed in a glass box with a glass plate of thick-
ness 3 mm at the bottom. After the rats had been allowed to
settle in the glass box for 20 min, the skin on the bottom of the
right feet of the rats was irradiated with a claw thermal sense
tester, with a cuto time of 20 s, to prevent scalding of the rats’
feet, which is often caused by a superuously long test dura-
tion. When the right hind limb was raised or retracted to cut
o contact with the tester, the heat source was automatically
disconnected, and the exposure time was recorded as the ther-
mal pain threshold (TPT; s). e measurement was repeated
three times for each rat and was repeated every 5 min, and the
average value was used as the TPT of the rat9.
Detection of adenosine levels through high-
performance liquid chromatography
e rats’ brains were removed and stored at -80 °C. After
adding perchloric acid (0.4 mol/L; 10 mL/g) by weight, the
sample was homogenized at a high speed (-4 °C) and centri-
fuged (4000 r/min, 15 min). e pH value of the sample was
adjusted to 6.0–7.0 with potassium hydroxide (4 mol/L), and
the liquid was centrifuged again (4000 r/min; 15 min). e rest
of the high-performance liquid chromatography process was
the same as published in previous papers10.
Detection of 5’-NT expression through western blot
analysis
After the rats had been euthanized, the L
4-6
spinal cord seg-
ments were removed, and the total protein content of the spinal
cord tissue was extracted using a cellular protein extraction kit.
Equal amounts of samples were transferred to a nitrocellulose
(NC) membrane by means of polyvinylidene uoride mem-
brane protein isolates. e NC membrane was incubated with
bovine serum albumin for 90 min, and then sealed with bovine
serum. Rabbit anti-rat 5’-NT protein monoclonal antibodies
were added, and the NC membrane was then incubated at 4 °C
overnight. e secondary antibody was rinsed by shaking
and incubating at room temperature, followed by chemilumi-
nescence, development and imaging. β-actin was used as an
internal reference. e target protein was analyzed using an
AlphaImager 2200 gel image processing system (Protein Simple,
CA, USA). e ratio of the 5’-NT band optical density to that of
the β-actin band indicated the protein expression level.
Statistical analysis
e SPSS 25.0 software (SPSS Inc., Chicago, IL, USA) was
used for statistical analyses, and the data were expressed as
mean ± standard error of the mean (SEM). One-way analysis
of variance (ANOVA) was used to assess dierences in 5’-NT
expression and adenosine content between the groups. If the
variance was homogeneous, a least signicant dierence test
was used for pairwise comparison, and if the variance was not
homogeneous, a Tamhane test was used for pairwise com-
parison. e MWT and TPT data of the rats were analyzed by
means of repeated-measurement ANOVA, and P < 0.05 indi-
cated a statistically signicant dierence.
RESULTS
Effect of EA on the expression of 5’-NT in CCI rats
e 5’-NT content of the spinal cord of the rats in the EA
group was signicantly greater than that of the rats in the
model group (P = 0.026) (Figure 1).
AOPCP inhibited EA from upregulating adenosine
content in the spinal cord of CCI rats
e adenosine content of the spinal cord of the rats in the
EA group was signicantly higher (P = 0.018) than that of the
model group. Also. the adenosine content of the spinal cord of
the rats in the model group was higher than that of the rats in
the sham group. Compared with the EA and EA + NS groups,
the EA + AOPCP group had signicantly lower adenosine con-
tent in the spinal cord (P = 0.002 and P = 0.016, respectively).
ere was no signicant dierence in spinal adenosine content
between the EA and EA + NS groups (P = 0.9630), thus indicat-
ing that the solvent of AOPCP (normal saline) did not aect
the experimental results (Figure 2).
292 Arq Neuropsiquiatr 2022;80(3):289-295
CCI: chronic constriction injury; EA: electroacupuncture; SEM: standard error
of the mean; 5-NT: ecto-5’-nucleotidase.
Figure 1. EA increased the expression of 5’-NT in CCI rats. (A):
Representative western blots of 5’-NT in the L4-L6 spinal
dorsal horn on day seven after CCI induction (B): Quantification
analysis on the optical density of these bands. The values are
presented as means ± SEM. EA group vs. model group (*P =
0.026)
AOPCP: α,β-methyleneadenosine 5’-diphosphate; CCI: chronic constriction
injury; EA: electroacupuncture; SEM: standard error of the mean.
Figure 2. AOPCP can inhibit EA from upregulating the
adenosine content in the spinal cord of CCI rats.The values
are presented as means ± SEM. EA group vs. model group (*P
= 0.018); EA + AOPCP group vs. EA group (#P = 0.002); EA +
AOPCP group vs. EA + NS group (&P = 0.016).
AOPCP reversed the analgesic effect of EA on CCI
rats
On the seventh day after the model had been established,
the MWT and TPT of the rats in all the groups, except the
sham group, had decreased signicantly, compared with those
before modeling (P < 0.0001 for MWT and TPT), thus indicat-
ing that the models were successfully established in all groups.
Compared with the MWT and TPT of the model group, those
of the EA group were signicantly higher at 60 min after EA
(P = 0.012 for MWT; P < 0.0001 for TPT). In the EA group, com-
pared with the MWT and TPT of the rats on the seventh day
after modeling, they were signicantly higher 60 min after EA
(P = 0.003 for MWT; P < 0.0001 for TPT), thus indicating that
EA had an analgesic eect on CCI rats. Compared with the
EA and EA + NS groups, the EA + AOPCP group had signi-
cantly decreased MWT and TPT at 60 min after EA (P = 0.003
for MWT; P < 0.0001 for TPT). ere was no signicant dif-
ference in MWT or TPT between the EA and EA + NS groups
(P = 0.9995 for MWT; P = 0.7489 for TPT), thus indicating that
the solvent of AOPCP (normal saline) did not aect the experi-
mental results (Figure 3).
CCPA restored the analgesic effect of EA on CCI rats
On the seventh day after the model had been established,
the MWT and TPT of the rats in each group had decreased sig-
nicantly, compared with the values obtained before modeling
(P < 0.0001 for both MWT and TPT), thus indicating that the
models were established successfully in each group. Compared
with the EA + AOPCP and EA + AOPCP + DMSO groups, the
EA + AOPCP + CCPA group had signicantly increased MWT
and TPT levels at 60 min after EA (P = 0.0003 for MWT; P <
0.0001 for TPT). ere was no statistical dierence in MWT
or TPT between the EA + AOPCP and EA + AOPCP + DMSO
groups (P = 0.6867 for A; P = 0.7886 for B), which indicated that
the solvent of CCPA (DMSO) did not aect the experimental
results (Figure 4).
293
Dai Q, et al. 5’ NT mediated analgesic effect of electroacupuncture.
AOPCP: α,β-methyleneadenosine 5’-diphosphate; CCI: chronic constriction injury; CCPA: N6-cyclopentyladenosine; EA: electroacupuncture; MWT: mechanical
withdrawal threshold; TPT: thermal pain threshold; SD: standard deviation.
Figure 4. CCPA can restore the analgesic effect of EA in CCI rats. The MWT (A) and TPT (B) of the right hind paw at pre-surgery, 7 d
post-surgery and 1 h post-EA are shown. The values are presented as means ± SEM. On the seventh day after modeling, the rats
in each group, except for the sham group, were compared with those before modeling (%P < 0.0001 for A and B). At 60 min after EA,
the following comparisons were conducted: EA + AOPCP group vs. EA + AOPCP + CCPA group (*P = 0.0003 for A; *P < 0.0001 for B);
and EA + AOPCP + DMSO group vs. EA + AOPCP + CCPA group (#P = 0.0003 for A; #P < 0.0001 for B).
AOPCP: α,β-methyleneadenosine 5’-diphosphate; CCI: chronic constriction injury; EA: electroacupuncture; MWT: mechanical withdrawal threshold; TPT: thermal
pain threshold; SD: standard deviation.
Figure 3. AOPCP can reverse the analgesic effect of EA in CCI rats. The MWT (A) and TPT (B) of the right hind paw at pre-surgery, 7
days post-surgery and 1 h post-EA are shown. The values are presented as means ± SEM. On the seventh day after modeling, the
rats in each group, except for the sham group, were compared with those before modeling (%P < 0.0001 for A and B). At 60 min after
EA, the following comparisons were conducted: EA group vs. model group ($P = 0.012 for A; $P < 0.0001 for B); EA + AOPCP group vs.
EA group (#P = 0.003 for A; #P < 0.0001 for B); and EA + AOPCP group vs. EA + NS group (&P = 0.003 for A; &P < 0.0001 for B).
294 Arq Neuropsiquiatr 2022;80(3):289-295
DISCUSSION
At present, acupuncture is widely used for clinical treat-
ment of pain. e purpose of central regulation by acupunc-
ture is primarily to induce neurons to release neurotransmit-
ters, neuromodulators or other chemical substances, such as
endogenous opioids and adenosine, which have an impact
on the body, into the brain tissue9-11. Among these, adenosine
(which is a neurotransmitter) has received extensive atten-
tion in studies that focused on the mechanism of acupuncture
analgesia. Adenosine mainly acts on adenosine receptors on
the cell membrane, thus playing a biological function12. ere
are four adenosine receptor subtypes: A1, A2a, A2b and A3.
Among these, adenosine A1 receptors are widely distributed
in the spinal dorsal horn neurons, and activation of adenosine
A1 receptors can inhibit the excitability of spinal neurons and
achieve analgesic eects13.
e most revered study on adenosine and acupuncture
analgesia was published in Nature Neuroscience in 201014. It
claimed that the increase in adenosine content around the
Zusanli acupoint in mice after EA was the main mechanism of
acupuncture analgesia and that the analgesic eect of acupunc-
ture was eliminated in adenosine A1 receptor knockout mice
14
.
In addition, during human experiments, some researchers found
that adenosine content could increase around the Zusanli acu-
points, further corroborating the evidence that adenosine is
involved in the mechanism for acupuncture analgesia15. It has
also been shown that electrical stimulation increases the con-
tent of ATP and its metabolite, adenosine, in the thalamus16.
Moreover, our own previous studies showed that adenosine
content in the spinal cord of CCI rats can increase 60 min after
EA, while silencing the adenosine A1 receptors can reverse the
analgesic eect of EA11. However, how EA regulates adenosine
production remains to be claried.
According to the existing research, 5’-NT is the most criti-
cal enzyme in the adenosine generation pathway5. In earlier
studies, 5’-NT was found to be an enzyme with innate pro-
tective eects against lung injury17. Subsequent studies used
5’-NT knockout mice to establish a model for inammatory
pain and found that, compared with wildtype mice, knockout
mice were more sensitive to pain and had signicantly lower
levels of adenosine18.
In this experimental study, we rst found that EA could
upregulate 5’-NT expression in the spinal cord of CCI rats.
Second, when AOPCP was used to inhibit the activity of 5’-NT,
we found that the adenosine content in the spine in the EA +
AOPCP group was signicantly lower than that of the EA and
EA + NS groups, thus indicating that AOPCP can reverse the
upregulation eect of EA on adenosine content in the spinal
cord of CCI rats. At the same time, the MWT and TPT levels in
the EA + AOPCP group were signicantly lower than those in
the EA and EA + NS groups, which provided strong evidence
that AOPCP could reverse the analgesic eect of EA on CCI
rats. Lastly, we found that the MWT and TPT levels were sig-
nicantly higher than those of rats treated with AOPCP alone,
after administration of adenosine A1 agonist and AOPCP in
CCI rats. Compared with the EA group, there was no signi-
cant dierence in the MWT or TPT of rats in the EA + AOPCP
+ CCPA group, thus indicating that EA analgesia could still
appeared againafter supplementation with exogenous adenos-
ine. erefore, we hypothesized that EA may increase the level
of adenosine and produce analgesic eects through upregu-
lating the expression of 5’-NT in the spinal cord of CCI rats.
However, our study had some shortcomings: we only used
AOPCP to inhibit the activity of 5’-NT; this could only demon-
strate the involvement of 5’-NT in the analgesic eect of EA
at the pharmacological level. If an adenovirus vector can be
designed to knock down the expression of 5’-NT in rat spinal
cords, we may be able to demonstrate the relationship between
5’-NT and EA analgesia at the gene level, which is considered
more conclusive than pharmacological evidence.
In conclusion, we believe that EA can upregulate the expres-
sion of 5’-NT in the spinal cord of CCI rats and that AOPCP can
signicantly reverse the upregulation of adenosine content in
the spinal cord of CCI rats and the analgesic eect of EA on CCI
rats, while exogenous adenosine supplementation can restore
the analgesic eect of EA. erefore, we hypothesize that 5’-NT
mediates the analgesic eect of EA in CCI rats.
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