International Immunopharmacology 113 (2022) 109337

Contents lists available at ScienceDirect

International Immunopharmacology
journal homepage: www.elsevier.com/locate/intimp

Systemic administration of dorsomorphin relieves inflammatory

nociception in the mouse formalin test
Yin Xinqiang a, *, 1, Jing Yuanyuan b, 1, You Zhipeng c, Ke Jie c, Tan Xiao c, Hu Yumeng c,
Zhu Chenxi c, Duan Shiyu c, Yi Mingpeng c, Zhu Yanlin c, Chen Sihan c, Yan Hao c
a
School of Pharmacy, North Sichuan Medical College, Nanchong 673000, China
b
Department of Laboratory Medicine, North Sichuan Medical College, Nanchong 673000, China
c
School of Clinical Medicine, North Sichuan Medical College, Nanchong 673000, China

A R T I C L E I N F O A B S T R A C T

Keywords: Inflammatory pain is the most important clinical symptom of inflammatory diseases. Despite intensive research
Dorsomorphin into inflammatory pain mechanisms, the majority of analgesics available are based on mechanistic classes of
Inflammatory pain compounds that have been known for many years, as a result, inflammatory pain control remains a challenge for
Formalin test
drug design in the context of clinically unmet needs in terms of safety and efficacy. A growing literature supports
that pro-inflammatory cytokine signaling plays a crucial role in the development of inflammatory pain. Modu­
lation of the pro-inflammatory cytokine may hold the key to improved pain management. Previous studies have
reported that dorsomorphin played key roles in inflammation. But the role of dorsomorphin in the formalin-
induced inflammatory nociception in mice has never been reported. Here, we report a new function of dorso­
morphin which can inhibit formalin-induced inflammatory nociception in mice. The antinociceptive effect of
dorsomorphin mainly depended on inhibiting the p38 MAPK/c-fos signaling and regulating inflammatory
mediators.

1. Introduction Sestrin2 expression via mitochondria-dependent ROS production [5].

Dorsomorphin prevents the AMPK signaling-independent unfolded
Inflammatory pain, primarily caused by the sensitization of primary protein response during glucose deprivation [6]. What’s more, the broad
sensory neurons (PSN), is the most important clinical symptom of in­ kinase inhibitor alters metabolic fingerprinting of extra cellular matrix
flammatory diseases. Although the presence of several analgesics on the detached cancer cells [7].
market, treatment of inflammatory pain is still an unmet medical need In several studies, the important roles of dorsomorphin related to
because of insufficient efficacy and the occurrence of severe unwanted inflammation have been investigated. Dorsomorphin inhibits ICAM-1
side effects after treatment with these drugs [1]. Thus, it’s urgently and VCAM-1 expression in inflammatory stimulants-activated endo­
needed to develop novel, efficient and safe treatment alternatives for thelial cells independent of AMPK inhibition via inhibition of NK-κB
inflammatory pain. activity along with inhibition of phosphorylation of PI3K and P38 MAPK
Dorsomorphin, also known as Compound c, is a small molecule [8]. In addition, dorsomorphin inhibits macrophage chemotaxis by in­
compound commonly used as an inhibitor of AMPK. However, dorso­ hibition of the focal adhesion kinase, AKT, and NK-κB pathway [9]. In
morphin has been shown to exert various functions in AMPK- LPS plus palmitate-induced THP-1 cells, dorsomorphin attenuates
independent manners in different cell types [2]. Dorsomorphin stimu­ NLRP3 inflammasome through the suppression of mitochondrial ROS
lates ceramide production and promotes Bax redistribution and production despite AMPK knockdown [10]. Dorsomorphin can suppress
apoptosis in MCF7 breast carcinoma cells [3]. In a syngeneic mouse dsDNA-dependent type I interferon induction by inhibiting cGAMP
model, dorsomorphin inhibits B16-F1 tumor growth via the blockage of accumulation [11].
cell cycle progression and angiogenesis [4]. Dorsomorphin increases In this study, we investigated the important role of dorsomorphin in

* Corresponding author at: Preclinical Research Center for Polypeptide Drugs, School of Pharmacy, North Sichuan Medical College, Nanchong 673000, China.
E-mail address: [email protected] (Y. Xinqiang).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.intimp.2022.109337
Received 18 June 2022; Received in revised form 29 September 2022; Accepted 9 October 2022
Available online 20 October 2022
1567-5769/© 2022 Elsevier B.V. All rights reserved.
Y. Xinqiang et al. International Immunopharmacology 113 (2022) 109337

the formalin-induced inflammatory nociception. We found that intra­ electrophoretically transferred onto polyvinylidene fluoride (PVDF)
peritoneal administration of dorsomorphin produced antinociceptive membranes by wet-blotting. Membranes were blocked for 1 h at room
effects through inhibiting the P38 MAP kinases/c-fos pathways and temperature with 5% bovine serum albumin (BSA). Then the blots were
regulating inflammatory mediators. incubated overnight at 4 ◦ C with primary antibodies against P38, p-P38,
NF-κB P65, NF-κB p-P65, IBA-1, GAPDH (Wanleibio) in blocking buffer.
2. Materials and methods The blots were incubated with secondary antibody (Wanleibio) for 60
min after washing 3 times with TBST. The filters were then developed by
2.1. Animals enhanced chemiluminescence reagents (Wanleibio). Data were analyzed
with the Image J2x software.
Male KM mice were obtained from the Animal Center of North
Sichuan Medical College. Food and water were available adlibitum and 2.6. Immunofluorescence
mice were housed in an animal room that was maintained at 22 ± 2 ◦ C
with a 12 h light/dark cycle. All animals were cared for and experiments Mice were deeply anesthetized with chloral hydrate sodium (350
were carried out in accordance with the Provisions and General mg/kg, intraperitonealy) eighty minutes after the formalin injection,
Recommendation of Chinese Experimental Animal Administration then perfused transcardially with 20 ml saline followed by 20 ml 4%
Legislation. All the protocols in this study was approved by the Ethics paraformaldehyde in 0.1 M phosphate buffer. L4 and/or L5 lumbar
Committee of North Sichuan Medical College (SCXK(川)2018-18). The segment was dissected out and post-fixed in 4% paraformaldehyde. The
animals were randomly divided into normal group, control group, and embedded segments were sectioned as 25 μm thick. Sections of each
drug treatment group. The mice in the normal group received intra­ group were incubated with rabbit antibodies for c-fos (1:100) or sub­
peritoneal injection of sterile saline before subcutaneous injection of stance P (1:100). Then the free-floating sections were washed with PBS,
sterile saline (20 µl) into the dorsal surface of the left hind paw. The mice and incubated with the secondary antibody for 2 h. After washing out
in the control group received intraperitoneal injection of sterile saline three times with PBS, the samples were studied under an immunofluo­
before subcutaneous injection of formalin into the dorsal surface of the rescence microscope for mophologic details of the immunofluorescence
left hind paw. The animals in the drug treatment group received intra­ staining. The number of c-fos-positive cells in the ipsilateral dorsal spi­
peritoneal administration of drugs before formalin stimulation. Two nal cord was counted manually by an observer blinded to the treatment
independent observers blinded to the group assignment collected and using ImageJ2x software.
analyzed the experimental data.
2.7. Statistical analysis
2.2. Drugs
The results were expressed as mean ± SEM. Statistical analysis was
Dorsomorphin⋅2HCl was purchased from Selleck, Ibuprofen was performed using IBM SPSS statistics 22 software. In the formalin test, for
obtained from Sigma-Aldrich. Tumor necrosis factor-α (TNF-α), inter­ statistical analysis, data from the phase I and phase IIwere considered
leukine-1β (L-1β), interferon β (IFNβ), and cyclooxygenase-2 (COX-2) indepently. One-way ANOVA with Bonferroni test post hoc test was used
enzyme-linked immunosorbent assays (ELISA) kits were obtained from to evaluate the dose-dependence effect of dorsomorphin on formalin-
Shanghai Enzyme-linked Biotechnology Co., Ltd. (Shanghai, China). induced licking. One-way ANOVA with Bonferroni test post hoc test
Dorsomorphin⋅2HCl and Ibuprofen were dissolved in sterile saline (0.9% was also used to examine the effect of dorsomorphin on the levels of the
NaCl) and PBS respectively. Dorsomorphin and Ibuprofen were intra­ inflammatory cytokines in the mouse serum, feet, and spinal cords. In all
peritoneally injected 20 min prior to formalin injection. statistical comparisons, differences with P < 0.05 were considered
significance.
2.3. Formalin test
3. Results
The formalin test procedure was performed as previously described
[12]. Mice were placed in a plexiglass box (15 cm in diameter and 20 cm 3.1. Dorsomorphin produced antinociceptive effect in a dose-dependent
in height) with a mirror placed under the floor at a 45◦ angle to allow an manner in the mouse formalin test
unobstructed view of the paw and were allowed to habituate for 30 min.
Then, a 5% formaldehyde solution (formalin, 20 μl) was injected sub­ The typical biphasic pain-like behavior with a first licking phase
cutaneously into the dorsal surface of the left hind paw. The time spent from 1 to 10 min and a second phase from 11 to 30 min was found after
licking the formalin-injected paw was recorded at 5-minute intervals up formalin injection in the formalin test (Fig. 1A). Compared to the normal
to 30 min, starting right after formalin injection. group, the licking time was remarkably increased in the both two phases
in the control group (Fig. 1B). The intraperitoneal administration of
2.4. TNF-α, IL-1β, IFN-β, and COX-2 detection in mouse serum, spinal dorsomorphin (1, 2.5, 5, and 10 mg/kg) decreased the time spent on
cord, and left hind paw licking in phase 2 in a dose-dependent manner in the mouse formalin
test (Fig. 1B). The Bonferroni’s test revealed that, in comparison with
The concentrations of TNF-α, IL-1β, IFN-β, and COX-2 in mouse the control group, dorsomorphin (2.5, 5, and 10 mg/kg, i.p.) signifi­
serum, spinal cord homogenate, and inflamed paw homogenate were cantly inhibited the second phase of formalin-induced pain behaviors
measured using ELISA kits according to the manufacturer’s instructions. (Fig. 1B). However, 1 mg/kg dorsomorphin failed to produce a signifi­
The optical density of each well was read at 450 nm. cant antinociceptive effect in comparison with the control group
(Fig. 1B). What’s more, 10 mg/kg dorsomorphin also significantly
2.5. Western blot decreased the licking time in the first phase (Fig. 1B). Under the same
condition, 10 mg/kg ibuprofern significantly attenuated the nociceptive
For Western blot analysis, mice were injected with formalin into the response induced by formalin in both phases (Fig. 1C). Compared to the
left hind paw. Lumbar spinal cords were dissected out 2 h after formalin ibuprofen group, the antinociceptive effect produced by dorsomorphin
injection. Total proteins in tissues were extracted immediately after (10 mg/kg) in both phase was more effective (Fig. 1C). In addition,
preparation using Whole Cell Lysis Assay kit (Wanleibio). The protein compared to the saline + dorsomorphin (i.p. 10 mg/kg) group, intra­
concentrations were quantified using a BCA protein assay kit (Wanlei­ peritoneally administrated AICAR (100 mg/kg), naloxone (10 mg/kg),
bio). The whole sample lysates were separated by SDS-PAGE and propranolol (5 mg/kg), phentolamine (5 mg/kg), and L-NMMA (10 mg/

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kg) 15 min prior to dorsomorphin have no significant effects on the

antinociceptive effect of dorsomorphin (Fig. 1D).

3.2. Dorsomorphin regulated TNF-α, IL-1β, IFN-β, and COX-2 in the

mouse serum

To assess the effects of dorsomorphin treatment on formalin-induced

inflammatory cytokines and COX-2 production in mouse serum, we
measured the contents of TNF-α, IL-1β, IFN-β, and COX-2 in the mouse
serum using ELISA kits according to the manufacturer’s instructions.
Compared to the normal group, the levels of TNF-α, IL-1β, IFN-β, and
COX-2 in the mouse serum were significantly increased in the control
group (Fig. 2A-D). Meanwhile, in comparison with the control group,
dorsomorphin treatment significantly reduced the contents of TNF-α, IL-
1β, IFN-β, and COX-2 in the dorsomorphin treatment group (Fig. 2A-D).

3.3. Dorsomorphin regulated TNF-α, IL-1β, IFN-β, and COX-2 in the

mouse spinal cord

We performed ELISA to investigate the expression of TNF-α, IL-1β,

IFN-β, and COX-2 in the mouse spinal cord homogenate from all groups.
Formalin induced significant increase of TNF-α, IL-1β, IFN-β, and COX-2
levels in control group in comparison with the normal group (Fig. 3A-D).
Pretreatment mice with dorsomorphin significantly decreased formalin-
induced the increase of TNF-α, IL-1β, IFN-β, and COX-2 (Fig. 3A-D) in the
mouse spinal cord in comparison with the control group.

3.4. Dorsomorphin regulated TNF-α, IL-1β, IFN-β, and COX-2 in the

mouse inflamed paw

The concentrations of TNF-α, IL-1β, IFN-β, and COX-2 in the mouse

inflamed paw homogenate were measured by ELISA. Productions of
TNF-α, IL-1β, IFN-β, and COX-2 in the control group were significantly
increased in comparison with the normal group (Fig. 4A-D). Moreover,
dorsomorphin (10 mg/kg) treatment significantly reduced the levels of
TNF-α, IL-1β, IFN-β, and COX-2 (Fig. 4A-D) in the mouse inflamed paw
in comparison with the control group.

3.5. Dorsomorphin suppressed formalin-induced phosphorylation of P38

and NF-κB P65, activation of microglia in the spinal cord

We measured changes of several protein expressions in the mouse

spinal cord using western blotting to explore the mechanisms by which
dorsomorphin inhibits formalin-induced inflammatory nociception in
mice. Formalin induced a significantly activation of p38 MAP-kinase
(Fig. 5A), NF-κB P65 (Fig. 5B), and microglia cells (Fig. 5C) in com­
parison with the normal group. Compared to the control group, dorso­
morphin (10 mg/kg, i.p.) treatment remarkably inhibited
Fig. 1. Dorsomorphin attenuated inflammatory nociception in the formalin phosphorylation of P38 (Fig. 5A) and NF-κB P65 (Fig. 5B), activation of
test. (A) Time course of the licking behavior in mice after injection of formalin
microglia (Fig. 5C) in the spinal cord, which indicated that these effects
(5%, 20 μl) into the left hind paw pretreatment with saline or dorsomorphin.
may contribute to the antinociceptive effects observed after dorsomor­
(B) The effects of intraperitoneally administrated saline, dorsomorphin (1.0,
2.5, 5.0, and 10.0 mg/kg) on the licking time in phase 1 (0–10 min) and phase 2 phin (10 mg/kg, i.p.) treatment.
(11–30 min) in the mouse formalin test. (C) The effects of intraperitoneally
administrated saline, dorsomorphin (10 mg/kg), and ibuprofen (10 mg/kg) on 3.6. Dorsomorphin inhibited formalin-induced c-fos protein expression in
the licking time in phase 1 (0–10 min) and phase 2 (11–30 min) in the mouse the spinal cord
formalin test. (D) The effects of intraperitoneally administrated saline, AICAR
(100 mg/kg), naloxone (10 mg/kg), propranolol (5 mg/kg), phentolamine (5 c-fos is a downstream effector of MAP-kinases and a well-accepted
mg/kg), and L-NMMA (10 mg/kg) on the antinociceptive effects of dorsomor­ marker for inflammatory and noxious responses. Substance P can
phin (10 mg/kg) in the mouse formalin test. Formalin was injected at time 0. directly or indirectly participate in pain transmission by promoting the
Saline, dorsomorphin, or ibuprofen was intraperitoneally administrated 20 min
release of glutamate and its analgesic effect is caused by the release of
prior to formalin. Saline, naloxone, propranolol, phentolamine, L-NMMA was
encephalin. We detected the effect of dorsomorphin (10 mg/kg, i.p.) on
intraperitoneally administrated 15 min prior to dorsomorphin. The time spent
licking the injected paw was measured in 5 min intervals for 30 min. Data are
c-fos and substance P protein expression in the mouse spinal cord.
presented as the mean number of licking time ± S.E.M. (n = 6 for each group). Compared to the normal group, formalin stimulation induced up-
*P < 0.05, **P < 0.01, ***P < 0.001 versus saline according to one-way regulated protein expression of c-fos in the ipsilateral dorsal horn of
ANOVA followed by Bonferroni test. the spinal cord (Fig. 6A). Dorsomorphin treatment remarkably
decreased formalin-induced c-fos expression (Fig. 6B). However,

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Fig. 2. Dorsomorphin regulated the levels of

TNF-α, IL-1β, IFN-β, and COX-2 in the mouse
serum in the formalin test. 2 h after formalin in­
jection, mice were killed and blood samples were
collected and the concentrations of TNF-α (A), IL-
1β (B), IFN-β (C), and COX-2 (D) were measured
by ELISA in the normal, control (10 μl/g, i.p.),
and dorsomorphin (10 mg/kg, i.p.) group. Results
are expressed as mean ± S.E.M. (n = 6 for each
group). ***p < 0.001, ****p < 0.0001, and
*****p < 0.00001according to one-way ANOVA
followed by Bonferroni test.

Fig. 3. Dorsomorphin regulated the levels of

TNF-α, IL-1β, IFN-β, and COX-2 in the mouse
spinal cord homogenate in the formalin test. 2 h
after formalin injection, mice were killed and the
spinal cords were harvested and the concentra­
tions of TNF-α (A), IL-1β (B), IFN-β (C), and COX-
2 (D) in mouse spinal cord homogenate were
measured by ELISA in the normal, control (10 μl/
g, i.p.), and dorsomorphin (10 mg/kg, i.p.) group.
Results are expressed as mean ± S.E.M. (n = 6 for
each group).**p < 0.01, ***p < 0.001, ****p <
0.0001, and ******p < 0.000001 according to
one-way ANOVA followed by Bonferroni test.

dorsomorphin had no significant effect on the expression of substance P painful pathologies [13]. Dorsomorphin is a small molecule compound
in the ipsilateral dorsal horn of the spinal cord of mouse in the mouse widely used as an inhibitor of AMPKs. The important roles of dorso­
formalin test (Fig. 6B). morphin related to inflammation have been reported [8–11]. However,
the role of dorsomorphin in the formalin-induced inflammatory noci­
4. Discussion ception remains unclear. In the present study, the role of dorsomorphin
in the formalin-induced inflammatory nociception was explored. Our
Pain is a fundamental feature of inflammation. The immune system data demonstrated that dorsomorphin (1–10 mg/kg) produced anti­
plays a critical role in the activation of sensory neurons and there’s nociceptive effect in a dose-dependent manner in the second phase of
growing evidence of neuro-inflammatory mechanisms contributing to the mouse formalin test. Moreover, 10 mg/kg dorsomorphin also

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Fig. 4. Dorsomorphin regulated the levels of

TNF-α, IL-1β, IFN-β, and COX-2 in the mouse
inflamed paw homogenate in the formalin test. 2
h after formalin injection, mice were killed and
the inflamed paw were harvested and the con­
centrations of TNF-α (A), IL-1β (B), IFN-β (C), and
COX-2 (D) in the inflamed paw homogenate were
measured by ELISA in the normal, control (10 μl/
g, i.p.), and dorsomorphin (10 mg/kg, i.p.) group.
Results are expressed as mean ± S.E.M. (n = 6 for
each group). **p < 0.01, ***p < 0.001, ****p <
0.0001, *****p < 0.00001, and ******p <
0.000001according to one-way ANOVA followed
by Bonferroni test.

reduced the first phase nociception in the mouse formalin test. dorsomorphin significantly suppressed the producing of TNF-α in the
Since dorsomorphin is a widely used inhibitor of AMPK and our and mouse serum, spinal cord, and inflamed paw and then produced anti­
other teams’ studies have shown that activation of AMPK produced nociceptive effect in the mouse formalin test. These results suggest that
antinociceptive effects in several types of pain models, including formalin-induced production of TNF-α may be one of the causes of the
formalin-induced inflammatory pain, thus we think that the anti­ inflammatory nociception and the inhibition of TNF-α contributes to the
nociceptive effect of dorsomorphin is independent of AMPK path way. anti-nociceptive effect.
We tried to explore the possible acting mode of dorsomorphin using IL-1β is released primarily by monocytes and macrophages as well as
AICAR (the activator of AMPK), naloxone (the classical opioid receptor by nonimmune cells, such as fibroblasts and endothelial cells, during cell
antagonist), propranolol (the β-adrenoceptor antagonist), phentolamine injury, infection, invasion, and inflammation. A study found that IL-1β is
(the α-adrenceptor antagonist), and L-NMMA (the total NOS inhibitor). expressed in nociceptive DRG neurons [20]. The expression of IL-1β is
Unfortunately, all of them failed to reverse the antinociceptive effects of enhanced following crush injury to peripheral nerve and after trauma in
dorsomorphin in this study. Since dorsomorphin inhibits several other microglia and astrocytes in the CNS [21]. IL-1β can produce hyper­
kinases much more potently than AMPK, it need to further study the algesia following either intraperitoneal, intracerebroventricular or
acting mechanism of dorsomorphin in the mouse formalin test. intraplantar injection [16,22]. What’s more, IL-1β was found to increase
Pro-inflammatory cytokines are produced predominantly by acti­ the production of substance P and PEG2 in a number of neuronal and
vated macrophages and are involved in the up-regulation of inflamma­ glial cells [23,24]. Administrations of IL-1ra, a specific IL-1 receptor
tory reactions. Abundant evidence suggests that certain pro- antagonist, and other anti-inflammatory cytokines have been demon­
inflammatory cytokines and inflammatory mediators are involved in strated to prevent or attenuate cytokine-mediated inflammatory
the process of inflammatory pain. hyperalgesia [25] and nerve-injury induced mechanical allodynia [26].
TNF-α has been shown to play important roles in inflammatory What we have found in the current is in good agreement with the pre­
hyperalgesia. Intraplantar injection of complete Freund’s adjuvant in vious studies described above. An increased expression of IL-1β has been
adult rats resulted in significant elevation in the levels of TNF-α, IL-1β, detected in the mouse serum, spinal cord, and inflamed paw after
and NGF in the inflamed paw. A single injection of anti-TNF-α antiserum formalin injection in the mouse formalin test. Dorsomorphin treatment
before the CFA significantly delayed the onset of the resultant inflam­ reduced the contents of IL-1β in the mouse serum, spinal cord, and
matory hyperalgesia and reduced IL-1β but not NGF levels [14]. Intra­ inflamed paw, contributing to the antinociceptive effect.
plantar injection of TNF-α also produces mechanical [15] and thermal We detected the contents of IFN-β in the mouse serum, spinal cord,
hyperalgesia [16]. It has been found that TNF-α injection into nerves and inflamed paw in the present study. The results demonstrated that
induces Wallerian degeneration [14,17] and generates the transient formalin induced significant increase of IFN-β levels in control group in
display of behaviors and endoneurial pathologies found in experimen­ comparison with the normal groupand pretreatment mice with dorso­
tally painful nerve injury [18]. When TNF-BP, an inhibitor of TNF, is morphin significantly decreased formalin-induced the increase of IFN-β
administered systemically, the hyperalgesia normally observed after in the mouse serum, spinal cord, and inflamed paw. We assumed that
lipopolysaccharide (LPS) administration is completely eliminated. formalin-induced IFN-β promotes nociception in the mouse formalin
Intrathecal administration of a combination of TNF-BP and IL-1 antag­ test. However, the role of IFN-β in pain is still controversial, as both
onist attenuated mechanical allodynia in rats with L5 spinal nerve pronociceptive actions and antinociceptive actions of IFN-β have been
transection [19]. In the present study, we found that formalin induced reported, depending on the locations, doses, conditions (physiological
paw edema and increased level of TNF-α in the mouse serum, spinal vs. pathological), and phases (acute vs. chronic) [27].
cord, and inflamed paw. Intraperitoneal administration of PGE2 is produced by COX-2 during inflammation. COX-2 is critical to

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Fig. 5. Dorsomorphin suppressed formalin-induced phosphorylation of P38 (A) and P65 (B), activation of microglia (C) in the spinal cord. The spinal cord was
dissected out 2 h after formalin injection and the levels of phosphorylated P38 and P65 were evaluated by densitometric analysis of the blots. The blots showed
representative samples. All samples derived from the same experiment and that blots were processed in parallel. Each phosphoprotein was normalized to the
expression of the corresponding total protein of the same sample in Fig. 5A-B. IBA-1 was normalized to the expression of GAPDH ON the same membrane. Results are
expressed as mean ± S.E.M. (n = 4 for each group). ***P < 0.001, ****P < 0.0001, *****P < 0.00001 according to one-way ANOVA followed by Bonferroni test.

the inflammatory state, and PGE2 is an important marker of inflamma­ Activation of p38 MAPK in microglia increases the synthesis and release
tion [28]. Prostanoids contribute to the development of peripheral of many pro-inflammatory mediators such as TNF-α, IL-1β, IFN-β, and
sensitization in nociceptor terminals, increasing excitability and COX-2 in the spinal cord in the different pain states [30]. Therefore, we
reducing pain thresholds [29]. Inhibiting production of COX-2 produce further detected the regulation of P38 after dorsomorphin treatment in
specific analgesia for inflammatory pain both in central and peripheral vivo in the mouse spinal cord. Formalin-induced activation of P38 was
sites [29]. In the current study, formalin induced significant elevation of remarkably suppressed by treating mice with dosomorphin, possibly
COX-2 and dorsomorphin treatment suppressed the increase of COX-2 in contributing to the antinociceptive effects in the inflammatory models.
the mouse serum, spinal cord, and inflamed paw. The inhibition of COX- However, it must be point out that distinct dorsomorphin target proteins
2 favored the anti-inflammatory nociception effect. may also contribute to the effects on nociception and that an interplay of
Accumulating evidence suggests that p38 mitogen-activated protein different downstream effectors may lead to the drastic improvement of
kinase, one member of the MAPK family, activated in neuronsand glia the nociceptive behavior.
and contributes importantly to inflammatory pain [30]. Inflammatory NF-κB is an important regulator of the production of inflammatory
cytokines such as TNF-α, IL-1β, IFN-β and inflammatory mediator (COX- cytokines and is involved in a variety of responses that play a pivotal role
2) cause phosphorylation of p38 MAPK in both neurons and glial cells in regulating the immune response to inflammation, infection, and
and favor the development of neuropathic and inflammatory pain [30]. nociception [31]. The results of this study showed that formalin

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Fig. 6. The number of c-fos (A) and substance P (B) positive cells in the normal group, the control group and the dorsomorphin (i.p. 10 mg/kg) treatment group.
Results are expressed as mean ± S.E.M. (n = 6 for each group). ****p < 0.0001 according to one-way ANOVA followed by Bonferroni test.

significantly increased NF-κB P65 phosphorylation in the mouse spinal neurons. Previous studies and our current results indicate that inhibition
cord, whereas dorsomorphin treatment remarkably inhibited the acti­ of the p38 MAPK/c-fos signaling pathway by dorsomorphin plays a key
vation of NF-κB P65 signaling. It can thus be suggested that the inhibi­ role in mediating anti-inflammatory nociception effect.
tion of the NF-κB pathway also plays a role in the antinociceptive effect Pro-inflammatory cytokines were mainly produced by glia cells and
of dorsomorphin. c-fos was normally expressed in neurons in the spinal cord. Our results
In the present study, we also found that dorsomorphin treatment suggest that dorsomorphin may act on both microglia cells and neurons.
significantly reduced formalin-induced c-fos positive cell number in the In addition, although our data indicate that dorsomorphin likely inhibits
mouse spinal cord. The transcription factor c-fos is a well-known NF-κB in microglia and acts on p38 pathway in c-fos-positive neurons,
downstream effector of MAP-kinases and a well-accepted marker for respectively, experiments are still needed to determine the changed
inflammatory and nociceptive response in the spinal cord neurons expression of p-p38 and p- NF-κB were occurred in glia cells or in
following peripheral stimulation [32,33]. Thus, the strength of noci­ neurons.
ceptive signaling can be correlated with the number of c-fos positive Taken together, the present study suggested that systemic

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