Resveratrol suppresses glial activation and alleviates trigeminal neuralgia via activation of AMPK
Yang et al. Journal of Neuroinflammation
Resveratrol suppresses glial activation and alleviates trigeminal neuralgia via activation of AMPK
Yan-jing Yang 0 3
Liang Hu 2
Ye-peng Xia 0 3
Chun-yi Jiang 2
Chen Miao 1
Chun-qing Yang 1
Miao Yuan 0 3
Lin Wang 0 3
0 Jiangsu Key Laboratory of Oral Disease, Nanjing Medical University , 140 Hanzhong Road, Nanjing, Jiangsu 210029 , People's Republic of China
1 Department of Pathology, First Affiliated Hospital of Nanjing Medical University , 300 Guangzhou Road, Nanjing, Jiangsu 210029 , People's Republic of China
2 Jiangsu Key Laboratory of Neurodegeneration, Department of Pharmacology, Nanjing Medical University , 140 Hanzhong Road, Nanjing, Jiangsu 210029 , People's Republic of China
3 Jiangsu Key Laboratory of Oral Disease, Nanjing Medical University , 140 Hanzhong Road, Nanjing, Jiangsu 210029 , People's Republic of China
Background: Glial activation and neuroinflammation in the spinal trigeminal nucleus (STN) play a pivotal role in the genesis and maintenance of trigeminal neuralgia (TN). Resveratrol, a natural compound from grape and red wine, has a potential anti-inflammatory effect. We hypothesized that resveratrol could significantly suppress neuroinflammation in the STN mediated by glial activation and further relieve TN. In this study, we evaluated whether resveratrol could alleviate trigeminal allodynia and explore the mechanism underlying the antinociceptive effect of resveratrol. Methods: Animals were orally injected with resveratrol after chronic constriction injury (CCI) of the infraorbital nerve. Mechanical thresholds of the affected whisker pad were measured to assess nociceptive behaviors. The STN was harvested to quantify the changing levels of p-NR1, p-PKC, TNF-α, and IL1-β by western blotting and detect the expression of calcitonin gene-related peptide (CGRP) and c-Fos by immunofluorescence. Glial activation was observed by immunofluorescence and western blotting. Mitogen-activated protein kinase (MAPK) phosphorylation in vivo and in vitro was examined by western blotting. Results: We found that resveratrol significantly attenuated trigeminal allodynia dose-dependently and decreased the increased expression of CGRP and c-Fos in the STN. Additionally, resveratrol showed an inhibitory effect on CCI-evoked astrocyte and microglia activation and reduced production of pro-inflammatory cytokines in the STN. Furthermore, the antinociceptive effect of resveratrol was partially mediated by reduced phosphorylation of MAP kinases via adenosine monophosphate-activated protein kinase (AMPK) activation. Conclusions: AMPK activation in the STN glia via resveratrol has utility in the treatment of CCI-induced neuroinflammation and further implicates AMPK as a novel target for the attenuation of trigeminal neuralgia.
TN; Resveratrol; AMPK; Glia activation; Cytokines; MAPK
Trigeminal neuralgia (TN) is the most common type of
neuropathic pain which is distributed at the branches of
the trigeminal nerve. It is characterized by unilateral
pain attacks which are sharp, shooting, lancinating,
electric shock-like, burning, and excruciating. Therefore,
TN is known as one of the most painful complaints of
humans. However, the underlying mechanisms of TN
have not been completely elucidated, and the outcomes
of pharmacological or surgical treatments are often
disappointing . Thus, it is urgent to develop new
approaches and discover new agents to treat TN.
Previous studies related to the therapy of neuropathic
pain focus on the primary sensory neurons and their
influence on the activity of spinal dorsal horn neurons
[2, 3]. However, emerging evidence implicates that both
microglia and astrocytes which are robustly activated
and persist in the spinal trigeminal nucleus play an
important role in TN [4, 5]. Activation of glial cells in
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the central nervous system (CNS) continues to release
pro-inflammatory cytokines, such as interleukin-1β
(IL1β), interleukin-6 (IL-6), and tumor necrosis factor-α
(TNF-α), which increase pain hypersensitivity [6–9] and
participate in the pathogenesis of neuropathic pain .
These upregulated cytokines construct a cytokine network
and elicit chronic neuroinflammation leading to
neuropathic pain [11, 12]. Therefore, targeting inhibition of glial
activation is a potentially novel treatment target for TN.
Resveratrol is a natural plant antibiotic found in various
plants and fruits, especially abundant in grapes and red
wine [13, 14]. Not only is resveratrol widely known for its
anti-oxidant and anti-inflammation properties, but it also
presents the neuroprotective benefits according to
ameliorating kainate-induced excitotoxicity  and improves
pathological and behavioral outcomes in various types of
CNS injuries including stroke , traumatic brain injury
, and spinal cord injury . The mechanism of
resveratrol-induced neuroprotection is not clear, but many
of its benefits are thought to activate adenosine
monophosphate (AMP)-activated protein kinase (AMPK) .
Multiple lines of evidence have indicated that AMPK is
emerging as a vital target to promote chronic pain
through the sensitization of peripheral nociceptors .
However, few relevant reports assess the inhibition of
resveratrol on glial activation via upregulating AMPK to
alleviate mechanical allodynia in TN.
Herein, we tested the hypothesis that AMPK could
represent a novel and efficacious opportunity for the
treatment of TN. In the present study, we firstly determined
whether oral administration of resveratrol, as an AMPK
activator, could relieve mechanical allodynia of the
trigeminal nerve. Further, we investigated the effect of resveratrol
on glial activation and consequently examined its
underlying mechanism. The results showed that resveratrol
could significantly attenuate trigeminal neuralgia
behaviors in the chronic constriction injury (CCI) model.
Moreover, activated microglia and astrocytes markedly
increased in the CCI model, whereas resveratrol decreased
the activation of microglia and astrocytes and decreased
the release of inflammatory cytokines in the CCI rat.
Finally, we found that mitogen-activated protein kinase
(MAPK) signal pathway was involved in the analgesic
effect of resveratrol, and the involvement of MAPK signal
pathway was confirmed in BV-2 cell line and primary
astrocytes. Based on these findings, our postulation was
that AMPK plays an analgesic action in TN by inhibiting
the activation of microglia and astrocytes.
for the Care and Use of Laboratory Animals (The
Ministry of Science and Technology of China, 2006).
All animal experiments were approved by Nanjing
Medical University Animal Care and Use Committee and
were designed to minimize suffering and the number of
Sprague-Dawley male rats (200–220 g at the start of the
experiment) were provided by the Experimental Animal
Center at Nanjing Medical University, Nanjing, China.
Five to six animals were housed per cage under
pathogen-free conditions with soft bedding under
controlled temperature (22 ± 2 °C) and photoperiods
(12:12h light-dark cycle). They were allowed to acclimate to
these conditions for at least 2 days before inclusion in
experiments. Animals were randomly divided into
groups (n = 8). The sample size was designed based on
the previous experience and to be limited to the minimal
as scientifically justified. For each group of experiments,
the animals were matched by age and body weight. All
surgeries were performed under anesthesia with
pentobarbital (Sigma, USA, 50 mg/kg, intraperitoneally (i.p.)).
Drugs and reagents
Fetal bovine serum (FBS) and other cell culture media and
supplements were purchased from Hyclone (USA).
Compound C and resveratrol were purchased from Sigma.
Primary antibody p-NR1 (Ser897) was purchased from
Millipore; TNF-α, p-ERK1/2 (Thr202/Tyr204), p-JNK
(Thr183/Tyr185), p-p38 (Thr180/Tyr182), and p-PKCγ
(Thr514) were purchased from Cell Signaling Technology;
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was
purchased from Sigma; glial fibrillary acidic protein (GFAP)
and IL-1β were purchased from Santa Cruz Biotechnology;
and IBA-1 was purchased from Abcam. Secondary
antibodies were purchased from Cell Signaling Technology. All
other chemicals were purchased from Sigma Chemical Co.
Rats were anesthetized with sodium pentobarbital
(50 mg/kg, i.p.), and all surgeries were conducted in
sterile conditions under a surgical microscope. The hair on
the top of the head was shaved, and the rat was placed
in a stereotaxic frame. Ophthalmic cream was applied to
the corner of both eyes to prevent drying damage. An
anterior-posterior 15-mm skin incision was made at
midline of the head. The infraorbital muscle was gently
dissected from the bone until the orbit could be gently
retracted. A piece of gelfoam or a tiny cotton ball was
packed into the orbital cavity to minimize bleeding. The
infraorbital nerve can be seen deep within the orbital
cavity, lying in the infraorbital bony fissure. The
infraorbital nerve was dissected free from the bone at its most
rostral extent in the orbital cavity, and a single 2-mm
length of chromic gut suture (4-0) was inserted between
the infraorbital nerve and the maxillary bone. In the
sham operation control group, only skin incision and
muscle dissection were performed. The nerve was not
touched, and no chromic gut suture was inserted. All
skin incisions were sutured with 4-0 nylon
Assessment of mechanical allodynia on the whisker pad
Mechanical sensitivity of the whisker pad, the
infraorbital nerve receptive field, was measured with a series of
von Frey fiber filament (Stoelting, Wood Dale, IL) by
modified up-down method. Rats were handled several
times before experiments. One experimenter held the
rat with two hands in insulating cotton gloves until the
animal was calm. The animal moved freely in the
holder’s hands with its head exposed. During testing,
one experimenter slightly restrained the rat in their
hands so that another experimenter could accurately
apply the von Frey filament onto the center of the rat
whisker pad, both ipsilateral and contralateral to the
surgery site. For consistency of results, each filament was
applied five times at intervals of a few seconds. If head
withdrawal was observed at least three times after
probing with a filament, the rat was considered responsive to
that filament according to the up-down method. For this
approach, whenever a positive response to the
mechanical stimulus occurred, the next weaker von Frey
filament was applied. If no positive response was evoked,
the next stronger filament was applied. Testing
proceeded in this manner until four fibers were applied after
the first one successfully caused positive responses. This
allowed estimation of the 50 % mechanical withdrawal
threshold (in gram) using a curve-fitting algorithm.
Microglial BV-2 cells were incubated under humidified
5 % CO2 and 95 % O2 at 37 °C in Dulbecco’s modified
Eagle’s medium (DMEM, Invitrogen, USA) containing
10 % FBS and 1 % streptomycin and penicillin
(Invitrogen). Twenty-four hours before experimentation, the
culture medium was replaced by 0.5 % FBS high-glucose
DMEM. Then, the cells were stimulated with IL-1β
(1 ng/ml) and TNF-α (50 ng/ml) for 30 min with or
without resveratrol (5, 25, and 125 μM). Glia were
isolated from the brains of postnatal day 2 pups as
described earlier. In brief, cerebral cortices were
removed from the rat brains for glial cultures. The tissue
was dissociated in 0.0025 % trypsin/EDTA and passed
through a 70-mm pore nylon mesh. After centrifugation,
the cell pellet was resuspended in DMEM containing 10 %
FBS, 50 U/ml penicillin, and 50 mg/ml streptomycin. The
cell culture dishes were coated with 10 μg/ml type I
collagen (BD Biosciences, USA) for 12 h at 37 °C. Excess
collagen was removed by PBS solution at 37 °C. Then, the
glial cells in DMEM containing 10 % FBS were loaded into
the dishes. After 7 to 10 days, glial cells typically reached
80 confluence and were ready for the experiment. In
general, the cultures consisted of more than 95 % astrocytes
(as indicated by GFAP-positive staining). The cells were
activated with IL-1β (1 ng/ml) and TNF-α (50 ng/ml) for
24 h with resveratrol (5, 25, and 125 μM). As for the
measurement of proteins in astrocytes, cells were
collected 30 min after stimulation.
To identify temporal expression of p-AMPK, GFAP,
IBA-1, IL-1β, TNF-α, p-NR1, p-PKCγ, p-p38, p-ERK,
p-JNK, and GAPDH, whole-cell protein extract lysates
were used. Under anesthesia and immediately after
perfusion with PBS, the rat spinal cord tissue was rapidly
removed and homogenized. The filters were blocked with
5 % bovine serum albumin (BSA) and then incubated
overnight at 4 °C with the primary antibodies (p-NR1
(Ser897), 1:800; p-AMPK (Thr172), 1:1000; TNF-α,
1:1000; p-ERK1/2 (Thr202/Tyr204), 1:1000; p-JNK
(Thr183/Tyr185), 1:1000; p-p38 (Thr180/Tyr182),
1:1000; p-PKCγ (Thr514), 1:1000; GAPDH, 1:1000;
GFAP, 1:500; IL-1β, 1:500; and IBA-1, 1:1000). The
filters were developed using ECL reagents
(PerkinElmer) with secondary antibodies from Millipore
Bioscience Research Reagents. Data were analyzed with a
Molecular Imager (Gel Doc TMXR, 170-8170) and
the associated software Quantity One-4.6.5
Under deep anesthesia, rats were transcardially perfused
with PBS followed by 4 % paraformaldehyde with 1.5 %
picric acid in 0.16 M PB (pH 7.2–7.4, 4 °C), and then,
the rat spinal cord tissue was dissected out and postfixed
in the same fixative overnight. The embedded blocks
were sectioned (30 μm thick) and processed for
immunofluorescence. Sections from each group (five rats
in each group) were incubated with rabbit anti-c-Fos
polyclonal antibody (1:100, sc-52, Santa Cruz
Biotechnology), rabbit anti-calcitonin gene-related peptide
(CGRP) polyclonal antibody (1:1000, Millipore), rabbit
polyclonal anti-GFAP (1:500, ab7260, Abcam), and
rabbit polyclonal anti-IBA (1:100, 019-19741, Wako Pure
Chemical Industries), respectively. Rabbit IgG (1:200,
Vector Laboratories) was used as an isotype control. For
double immunofluorescence staining, the free-floating
sections were incubated in PBS containing 10 % donkey
serum and 1 % BSA for 2 h, incubated at 4 °C in primary
antibody, then washed three times in 50 mM Tris-HCl
(pH 7.4) PBS, and incubated in the secondary antibody
either for 2 h at room temperature or overnight at 4 °C.
After washing three times in PBS, sections were
reincubated in blocking serum for 1 h. Morphologic details
were examined with a confocal microscope (Leica TCS
SP2). Images were randomly coded and transferred to a
computer for further analysis. Fos-immunoreactive
neurons were counted in a blind fashion. The number of
Fos-like-immunoreactive neurons in the spinal
trigeminal nucleus was determined by averaging the counts
made in 20 spinal cord sections for each group. To
obtain quantitative measurements of CGRP
immunofluorescence, 15–20 fields covering the spinal trigeminal
nucleus in each group were evaluated and photographed
at the same exposure time to generate the raw data.
Fluorescence intensities of the different groups were
analyzed using MicroSuite image analysis software
(Olympus America). The average green fluorescence
intensity of each pixel was normalized to the background
intensity in the same image.
SPSS Rel 15 (SPSS Inc., Chicago, IL) was used to conduct
all the statistical analyses. Alteration of the expression of
the proteins was detected, the behavioral responses were
tested with one-way ANOVA, and the differences in
latency over time among groups were tested with two-way
ANOVA. Bonferroni post hoc tests were conducted for all
ANOVA models. Results are expressed as mean ± SEM of
three independent experiments. Results described as
significant are based on a criterion of P < 0.05.
Resveratrol ameliorates mechanical allodynia in a
trigeminal neuralgia rat model
In this study, rats that received CCI of the trigeminal
nerve exhibited mechanical allodynia (Fig. 1). There
were no significant differences in pain-related behaviors
between the control group and the other groups before
surgery. A marked decrease in mechanical withdrawal
was observed 7 days after CCI and followed by a peak
on day 14. Thus, resveratrol was administered to the
CCI rats on day 14. These pain-related behaviors were
greatly ameliorated by resveratrol (100, 200, and
400 μg/ml) orally single administrated at
postoperative 14 days in a dose-dependent manner, and the effect
lasted for more than 24 h (Fig. 1a). However, the effects of
resveratrol were abolished by the AMPK inhibitor
compound C (Fig. 1c). In addition, consecutive administration
of resveratrol at postoperative days 14, 15, 16, 17, and 18
reversed the mechanical allodynia in the CCI rats (Fig. 1b).
Fig. 1 Resveratrol ameliorates mechanical allodynia in a trigeminal neuralgia rat model. Rats administered Res only on day 14 (a) or on days 14,
15, 16, 17, and 18 (b) post-CCI showed a significant increase in mechanical withdrawal compared with that of the control group. a Resveratrol
(100, 200, and 400 μg/ml) orally single administrated at day 14 ameliorated pain-related behaviors in a dose-dependent manner, and this effect
lasted for more than 24 h. b Consecutive administration of resveratrol at postoperative days 14, 15, 16, 17, and 18 reversed the mechanical allodynia in
the CCI rats. c The AMPK inhibitor compound C reversed the effects of Res on CCI-induced mechanical allodynia. Drug administration is indicated by
the arrows (n = 8 each group). Two-way ANOVA revealed a significant difference at *P < 0.05 and **P < 0.01 versus control; #P < 0.05 and ##P < 0.01
versus the CCI group; and &P < 0.05 and &&P < 0.01 versus the CCI + Res 400 mg/kg group
These results suggest that AMPK activation via resveratrol
had a clearly positive effect on mechanic analgesia in the
Resveratrol inhibits phosphorylation of c-Fos and CGRP in
the spinal trigeminal nucleus
CGRP has an essential role in trigeminal nociceptive
processing. As shown in Fig. 2a, significantly higher
CGRP level was induced by CCI than that in the
control group. Treatment with resveratrol suppressed the
increase of the CGRP level. Resveratrol alone had no
effect on the expression of CGRP. Furthermore, CCI
remarkably increased the number of
c-Fosimmunoreactive neurons in the spinal trigeminal
nucleus (STN), and the increase of positive neurons was
significantly attenuated by administration of
resveratrol. Similarly, resveratrol alone did not influence the
number of c-Fos-immunoreactive neurons in the STN
in normal rats (Fig. 2b).
Resveratrol inhibits the activation of microglia and
astrocytes in rat STN
Astrocytes and microglia play a key role in the central
sensitization process that occurs in neuropathic pain. The
level of GFAP and IBA-1 was determined by western
blotting and used as an indicator of astrocyte and microglia
activation. To investigate whether resveratrol affects the
activation of glia, we examined the levels of GFAP and
IBA-1 in the STN. As shown in Fig. 3a and b, the
expression of both GFAP and IBA-1 was enhanced in the STN
of the CCI rats when compared with the control group,
whereas resveratrol (400 mg/kg, p.o.) downregulated the
expression of both markers (P < 0.05). These findings
suggested that AMPK activation downregulated the activation
of astrocytes and microglia in the CCI rat model.
In addition to immunoblotting, immunofluorescence
staining was performed to confirm increased GFAP and
IBA-1 immunoreactivity in the STN. As shown in Fig. 4a,
b, compared with the control group, increased green
Fig. 2 Resveratrol inhibits phosphorylation of c-Fos and CGRP in the STN. Immunofluorescence analysis data show CGRP expression and
c-Fos-immunoreactive neuron number. The CGRP and c-Fos level was significantly increased by CCI, and resveratrol suppressed the
increase of CGRP expression (a) and attenuated the increase of c-Fos-immunoreactive neuron number (b). Resveratrol alone had no effect
on CGRP expression and c-Fos-immunoreactive neuron number. n = 5, five images per animal. *P < 0.05 and **P < 0.01 versus control; and
#P < 0.05 and ##P < 0.01 versus the CCI group
Fig. 3 Resveratrol inhibits the activation of microglia and astrocytes in rat STN. a, b Representative western blot bands and a data summary
(n = 4 each group) of the expression of GFAP and IBA-1, which are markers of astrocytes and microglia, respectively. GFAP and IBA-1 expression
were enhanced in the STN of the CCI rats, whereas resveratrol (400 mg/kg, p.o.) downregulated the expression of both markers. *P < 0.05 and
**P < 0.01 versus control; and #P < 0.05 and ##P < 0.01 versus the CCI group
fluorescence was observed in the STN ipsilateral to CCI,
suggesting the activation of both astrocytes and
microglia. Similar to the results from immunoblotting analysis,
this activation was alleviated by resveratrol. These
results supported the notion that AMPK was involved in
regulating glial activation in TN.
Resveratrol reduces CCI-induced production of
pro-inflammatory factors and phosphorylation of NR1
and PKCγ in the STN
AMPK activation has been reported to inhibit
neuroinflammation . IL-1β and TNF-α are characterized
pro-inflammatory cytokines that play an important
Fig. 4 Resveratrol inhibits the activation of microglia and astrocytes in rat STN. a, b Immunofluorescence analysis data show GFAP and IBA-1
expression (n = 5, five images per animal). CCI increased GFAP and IBA-1 immunoreactivity in the STN, whereas resveratrol (400 mg/kg, p.o.)
downregulated the expression of both markers. *P < 0.05 and **P < 0.01 versus control; and #P < 0.05 and ##P < 0.01 versus the CCI group
role in inflammatory response by stimulating glial cells.
The expression levels of IL-1β and TNF-α in the
STN were examined by western blotting, showing
that the levels of IL-1β and TNF-α were increased in
the CCI rats compared to the control group (P <
0.01). Treatment with resveratrol (400 mg/kg, p.o.)
inhibited the increased production of IL-1β and
TNFα in the STN (P < 0.01) (Fig. 5a). There were no
significant differences between control and resveratrol
The NMDA receptor, which regulates neuronal
activity and synaptic efficacy, has a well-established
role in various pain states. NMDA receptor
phosphorylation can be activated by pro-inflammatory factors.
The NMDA receptor 1 (NR1) subunit also can be
phosphorylated by protein kinase C (PKC). Thus, we
examined whether trigeminal nerve injury was able to
elicit phosphorylated NR1 and PKCγ. Using CCI of a
trigeminal nerve rat model, we found that CCI
significantly increased the levels of phosphorylated NR1 and
PKCγ, whereas repetitive treatment with resveratrol
(400 mg/kg, p.o.) reversed the increase of their
expression (Fig. 5b). Neither the production of
proinflammatory factors nor the phosphorylation of NR1
and PKCγ was altered by resveratrol treatment alone
in naïve rats.
Resveratrol regulates phosphorylation of MAPK in vivo
and in vitro via AMPK activation
Previous studies indicated that the production of both
IL-1β and TNF-α might be mediated by MAPK signaling
pathways in glia [22, 23]. As shown in Fig. 6a,
administration of resveratrol (400 mg/kg, p.o.) significantly
reduced the levels of phosphorylated MAPKs in the
STN, including p38, extracellular signal-regulated
protein kinase (ERK), and c-Jun N-terminal kinase (JNK),
induced by CCI. These effects were reversed by the
coadministration of the AMPK inhibitor compound C
(30 μg, i.t.). These results suggested that
resveratrolmediated suppression of MAPKs may be secondary to
AMPK signaling activation.
Our findings suggested that resveratrol may act
through MAPK signaling pathways to attenuate CCI via
the inhibition of spinal cord glial activation in vivo. We
further sought to expose cultured primary astrocytes
from newborn rats to IL-1β (1 ng/ml)/TNF-α (50 ng/ml)
to mimic the effect of neuroinflammation in rats to
investigate the role of resveratrol. IL-1β/TNF-α treatment
induced activation in astrocytes and was characterized
by increased phosphorylation of JNK and ERK/MAPKs.
Treatment with resveratrol (5, 25, and 125 μM) before
IL-1β/TNF-α administration significantly reduced these
effects (Fig. 6b).
Fig. 5 Resveratrol reduces CCI-induced production of pro-inflammatory factors and phosphorylation of NR1 and PKCγ in the STN. a Resveratrol
reduces the production of pro-inflammatory factors IL-1β and TNF-α. b Resveratrol inhibits the phosphorylation of NR1 and PKCγ. Representative
western blot bands and a data summary (n = 4 each group) are shown. *P < 0.05 and **P < 0.01 versus control; and #P < 0.05 and ##P < 0.01 versus
the CCI group
Fig. 6 Resveratrol regulates phosphorylation of MAPK via AMPK activation in vivo and in vitro. a Resveratrol downregulates the phosphorylation of
MAPKs in vivo, including p38, ERK, and JNK; these effects were reversed by compound C. Compound C (30 μg/20 μl, i.t.) was coadministered. b
Resveratrol increased p-JNK and p-ERK expression in primary astrocytes in a dose-dependent manner. c Resveratrol increased p-p38 and p-ERK
expression in BV2 cells in a dose-dependent manner. Western blot bands and a data summary (n = 4 each group) are shown. *P < 0.05 and **P < 0.01
versus control; and #P < 0.05 and ##P < 0.01 versus the CCI group
To investigate the in vitro effects of resveratrol on
pro-inflammatory factor-induced microglia activation,
we used the immortalized murine microglial cell line
BV-2 to examine the effect of resveratrol on primary
mouse microglia cells [24, 25]. BV-2 cells have a similar
reaction pattern to that of primary microglia after
stimulation with lipopolysaccharide  and have been used
in vitro to replace primary microglia, which pose
experimental difficulties when being separated from the STN
in vitro. In the present study, IL-1β (1 ng/ml)/TNF-α
(50 ng/ml) treatment induced BV-2 cell activation,
which was characterized by increased phosphorylation of
p38 and ERK/MAPKs (Fig. 6c). Treatment with
resveratrol (5, 25, and 125 μM) before IL-1β/TNF-α
administration significantly reduced these effects.
demonstrated that resveratrol suppresses mechanical
allodynia of the trigeminal nerve and increases the
expression of CGRP and c-Fos induced by CCI in
rats. Resveratrol had a significant impact on the
CCIinduced activation of astrocytes and microglia and
decreased the expression level of pro-inflammatory
cytokine in TN. Resveratrol reduced the upregulated
phosphorylation of MAPKs both in vivo and in vitro
in an AMPK-dependent manner. These studies
provide an assessment of resveratrol for TN and for
the future development of more efficacious AMPK
activators for the treatment of chronic trigeminal pain.
Infraorbital nerve (the second branch of the trigeminal
nerve) ligation is a validated model for producing
allodynia in the ipsilateral vibrissae area in rats . In the
present study, we have revealed that resveratrol, an AMPK
activator, produced a marked increase in pressure
threshold to evoke a nocifensive response in trigeminal
nerveligated rats (Fig. 1). Furthermore, we noted that CCI of
the trigeminal nerve induced upregulation of CGRP in the
STN (Fig. 2). The neurotransmitter CGRP plays a crucial
role in the pathophysiology of neuropathic pain-related
behavior in the CCI rats . It is well known that
activation of the trigeminal nerve system induces the release of
CGRP . Our group has demonstrated that the increase
of CGRP release induced by CCI of the trigeminal nerve
could be almost completely abolished by reducing the
central sensitization of primary trigeminal sensory
neurons with resveratrol treatment. We observed a similar
pattern in the CCI of the trigeminal nerve, with the
number of the immediate early gene c-Fos IR cells increased
remarkably, indicating the activation of the trigeminal
system. Our findings pond well with a previous report that
cFos in the STN was elevated after stimulation of the
infraorbital nerve . Treatment with resveratrol was shown
to effectively block c-Fos upregulation. Previous data
demonstrated that resveratrol significantly alleviated the
behavior and pathophysiology of trigeminal neuropathic
pain. However, the mechanism by which resveratrol exerts
an analgesic effect in TN has not been illuminated.
Trigeminal neuropathic pain following trigeminal
nerve injury is often difficult to diagnose and treat due
to complexity of TN mechanisms [30–33].
Accumulating evidence suggests that translation regulation at
the level of the primary afferent neuron is vital for
the establishment and maintenance of enhanced pain
states [34, 35]. Neuropathic pain treatment is
currently aimed only at reducing symptoms, generally by
suppressing neuronal activity. In contrast, targeting
neuroglia may provide more opportunities for disease
management by aborting neurobiological alterations
that support the development of persistent pain.
Multiple lines of evidence show that glia cells, notably
microglia and astrocytes, contribute to the central
sensitization process that occurs in the peripheral nerve
injury. The activated microglia and astrocytes release
numerous substances, including pro-inflammatory cytokines
such as IL-1β, TNF-α, and COX-2 [36–38]. IL-1β is one of
the most vital cytokines, and directly sensitizes the
nociceptors, such as the transient receptor potential cation
channel subfamily V member 1 (TRPV1), a heat and
chemical-sensitive cation channel, in primary sensory
neurons . It has been reported that the upregulation of
TNF-α in the DRG and spinal dorsal horn is induced by
injury of the periphery nerve [40–42]. It is possible that
TNF-α also participates in neuropathic pain. Our study
demonstrated that peripheral injury of the trigeminal
nerve also activated glial activation and enhanced the
secretion of IL-1β and TNF-α (Figs. 3 and 4a). The influence
of resveratrol on other pro-inflammatory cytokines in TN
will be the focus in our future work.
AMPK has been regarded as the energy sensor for
more than one decade [43, 44]. AMPK is a ubiquitous
kinase endogenously activated by AMP and ADP and
exogenously regulated by a variety of pharmacological
entities including the widely prescribed anti-diabetes
drug metformin and natural products such as resveratrol
. AMPK is regarded as a regulator of neuronal
function, plasticity, and neurodegeneration [46, 47], but the
potential mechanisms of AMPK activation on neuronal
excitability, as an important component of neuropathic
pain conditions , is not known completely. Previous
studies mostly focus on discovering the mechanism of
effect of activating AMPK on sensory neuronal
excitability, with few reports on the regulation of AMPK
activation on the function of glia in neuropathic pain.
It was reported that AMPK activation in the spinal
glia by resveratrol participates in the treatment of
tumor cell implantation-induced neuroinflammation
. Moreover, pharmacological activation of AMPK
enhanced glial glutamate transporter activity by
attenuating glial glutamate transporter-1 internalization
in neuropathic pain mice . Our study revealed
that resveratrol could activate AMPK to inhibit
CCIevoked astrocyte and microglial activation and
reversed the production of IL-1β and TNF-α (Figs. 3
and 4a). These results have well documented the role
of AMPK in neuroglial cells. We validated the hypothesis
that AMPK activation could inhibit glia activation and
relieve glia-mediated neuroinflammation, and thus alleviate
trigeminal neuralgia (Figs. 1, 2, 3, and 4a), which is in
agreement with previous related studies [51, 52].
The NMDA receptor has an ionotropic property which
regulates Ca2+ influx and Ca2+-dependent physiological
effects, further neuronal activity, and synaptic efficacy.
Resveratrol administration reversed the production of
pro-inflammatory cytokines IL-1β and TNF-α which
regulate synaptic plasticity. It is widely accepted that the
phosphorylation of NMDA receptors (NMDARs) also
has a momentous role in neural plasticity and various
pain states . The activation of PKCγ plays a
welldeveloped role in central sensitization in neuropathic
pain, which may contribute to increasing the excitability
of nociceptive neurons . Our results showed that
resveratrol inhibited in the phosphorylation of NMDAR
NR1 and the activation of PKCγ in the STN in the CCI
rats (Fig. 4b). Thus, we provided the evidence for the
first time that resveratrol administration in the
peripheral nerve may inhibit glial activation and relieve
gliamediated neuroinflammation via AMPK activation, and
further suppress central sensitization present in the
STN, which may explain why resveratrol has a sustaining
MAPK plays a key role in the induction and
maintenance of neuropathic pain . MAPK signal
transduction pathways are evolutionarily conserved in eukaryotic
cells and transducer signals in response to a variety of
extracellular stimuli . Among the well-characterized
MAPK subfamilies, the p38 kinases are first defined for
drugs inhibiting TNF-α-mediated inflammatory response
. Because the MAPKs are vital regulators of the
expression of many cytokines, they appear to be involved in
neuroinflammation of TN. Our studies demonstrated that
the significantly increased phosphorylative level of
MAPKs in the STN was induced by CCI. Oral
administration of resveratrol downregulated the expression levels of
MAPKs (Fig. 5a). We also verified the change trend of the
phosphorylation of MAPKs in primary astrocytes and
microglia cell line BV-2 cell and obtained consistent
results (Fig. 5b, c). These results indicated that MAPKs in
astrocytes and microglia might be involved in the
mechanism of resveratrol managing the glial activation and release
of inflammatory cytokines. Our results were consistent
with other relevant reports that resveratrol affords
antineuroinflammatory effects by inhibiting microglial
activation after suppressing the activation of MAPK signaling
pathways [33, 40, 58].
In this study, we have found a novel pathway for the
potential treatment of TN, activating glial AMPK.
Pharmacological AMPK activation resolves trigeminal
neuropathic allodynia and decreases sensory neuron
excitability by inhibiting glial activation and release of
cytokines, which is regulated by MAPK pathway. Due
to natural production and safety of resveratrol, these
preclinical results have the potential to be rapidly
translated into the clinic.
YY, LH, and LW designed and performed the experiments. CJ, YX, and MY
performed the immunoassays and behavioral measure. LH and CJ performed
the western blotting analysis. CM and CY carried out the cell cultures. LH
analyzed the results. YY and LW drafted the manuscript. YY, LH, and CJ
secured funding for the project. All authors read and approved the final
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