European Journal of Pharmacology 786 (2016) 128–136

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European Journal of Pharmacology

journal homepage: www.elsevier.com/locate/ejphar

Molecular and cellular pharmacology

Pharmacological activation of cannabinoid 2 receptor attenuates

inflammation, fibrogenesis, and promotes re-epithelialization during
skin wound healing
Lin-Lin Wang a,1, Rui Zhao a,1, Jiao-Yong Li a, Shan-Shan Li a, Min Liu a, Meng Wang a,
Meng-Zhou Zhang a, Wen-Wen Dong a, Shu-Kun Jiang a, Miao Zhang a, Zhi-Ling Tian a,
Chang-Sheng Liu b, Da-Wei Guan a,n
a
Department of Forensic Pathology, China Medical University School of Forensic Medicine, Shenyang, China
b
Institute of Forensic Science, Anshan Municipal People's Procuratorate, Anshan, China

art ic l e i nf o a b s t r a c t

Article history: Previous studies showed that cannabinoid 2 (CB2) receptor is expressed in multiple effector cells during
Received 11 January 2016 skin wound healing. Meanwhile, its functional involvement in inflammation, fibrosis, and cell pro-
Received in revised form liferation in other organs and skin diseases implied CB2 receptor might also regulate skin wound healing.
2 June 2016
To verify this hypothesis, mice excisional wounds were created and treated with highly selective CB2
Accepted 2 June 2016
receptor agonist GP1a (1-(2,4-dichlorophenyl)-6-methyl- N-piperidin-1-yl-4H-indeno[1,2-c]pyrazole-3-
Available online 3 June 2016
carboxamide) and antagonist AM630 ([6-iodo-2- methyl-1-(2-morpholin-4-ylethyl)indol-3-yl]-(4-
Keywords: methoxyphenyl)methanone) respectively. The inflammatory infiltration, cytokine expression, fibrogen-
Cannabinoid 2 receptor esis, and wound re-epithelialization were analyzed. After CB2 receptor activation, neutrophil and mac-
Skin wound healing
rophage infiltrations were reduced, and expressions of monocyte chemotactic protein (MCP)-1, stromal
Inflammation
cell-derived factor (SDF)-1, Interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF)-α, transforming growth
Fibrogenesis
Re-epithelialization factor (TGF)-β1 and vascular endothelial growth factor (VEGF)-A were decreased. Keratinocyte pro-
liferation and migration were enhanced. Wound re-epithelialization was accelerated. Fibroblast accu-
Chemical compounds studied in this article: mulation and fibroblast-to-myofibroblast transformation were attenuated, and expression of pro-col-
GP1a (PubChem CID: 10252734) lagen I was decreased. Furthermore, HaCaT cells in vitro were treated with GP1a or AM630, which re-
AM630 (PubChem CID: 4302963) vealed that CB2 receptor activation promoted keratinocyte migration by inducing the epithelial to me-
senchymal transition. These results, taken together, indicate that activating CB2 receptor could ameliorate
wound healing by reducing inflammation, accelerating re-epithelialization, and attenuating scar for-
mation. Thus, CB2 receptor agonist might be a novel perspective for skin wound therapy.
& 2016 Elsevier B.V. All rights reserved.

1. Introduction The endocannabinoid system (ECS; comprising the en-

Skin wound healing is a complicated process proceeding by
three stages that overlap in time and space: inflammation, new
tissue formation, and remodeling. During this process, a series of
cytokines, growth factors, extracellular matrix molecules, and
various cells including leukocytes, fibroblasts, and keratinocytes
coordinately interacted with each other to reform the epidermal
barrier and dermal structure (Gurtner et al., 2008; Martin, 1997;
Martin and Nunan, 2015).
n
Corresponding author.
E-mail addresses: dwguan@mail.cmu.edu.cn,
dwguan@aliyun.com (D.-W. Guan).
1
These authors have contributed equally to the work.
docannabinoids, G-protein-coupled cannabinoid receptors, bio-
synthetic and metabolism enzymes) in the mammals has attracted
great attention in the last decades for its multiple functions both in
health and diseases, and the ECS of the skin is recognized as a
novel therapeutic target for cutaneous pathologies (Biro et al.,
2009; Maccarrone et al., 2015). Cannabinoid 2 (CB2) receptor is an
important constituent of the ECS, which has been increasingly
emphasized in recent years because it doesn’t arouse psychotropic
and cardiovascular side-effects (Yrjölä et al., 2015). CB2 receptor is
mainly distributed in peripheral and immune cells, including
lymphocytes, NK cells, monocytes, macrophages, fibroblasts, ker-
atinocytes, etc., and functionally involved in a series of pathologies
referring to these cells (Battista et al., 2012; Kupczyk et al., 2009;
Munro et al., 1993; Witkamp and Meijerink, 2014). Previous

http://dx.doi.org/10.1016/j.ejphar.2016.06.006
0014-2999/& 2016 Elsevier B.V. All rights reserved.
 L.-L. Wang et al. / European Journal of Pharmacology 786 (2016) 128–136
studies have revealed that CB2 receptor interruption results in
enhanced allergic inflammation in cutaneous contact hypersensi-
tivity (Karsak et al., 2007), while activating CB2 receptor reduces
inflammation in murine pancreatitis (Michler et al., 2013), cystitis
(Wang et al., 2014), traumatic brain injury (Amenta et al., 2014),
and sepsis (Tschop et al., 2009). CB2 receptor activation also at-
tenuates pathological liver fibrosis in mice (Guillot et al., 2014;
Mahmoud et al., 2014; Munoz-Luque et al., 2008), and exerts an-
tifibrotic effect in bleomycin-induced dermal fibrosis (Akhmet-
shina et al., 2009). In addition, absence of CB2 receptor improves
keratinocyte differentiation and decreases proliferation in a tape-
stripping-induced epidermal barrier abrogation model (Roelandt
et al., 2012).
As aforementioned, CB2 receptor is involved in the regulation of
inflammation, fibrogenesis, and epidermal growth, which are the
key biological processes during skin wound healing. Taking into
account our previous finding that expression of CB2 receptor was
up-regulated during skin wound healing in mice (Zheng et al.,
2012), we presumed CB2 receptor might regulate skin wound
healing, and pharmacomodulation of its activity might be a novel
perspective for wound therapy. In the present study, we verified
this hypothesis using Gp1a (highly selective CB2 receptor agonist)
and AM630 (highly selective CB2 receptor antagonist) in mice skin
excisional wounds in vivo and HaCaT keratinocytes in vitro. Our
results indicate that activating CB2 receptor could ameliorate skin
wound healing by attenuating inflammation and fibrogenesis, as
well as promoting re-epithelialization.
2. Materials and methods
2.1. Reagents and Abs
See details in Supplementary materials and methods.
2.2. Wound model and experimental grouping
Eight-weeks-old male BALB/c mice, each weighing 20–25 g,
were used in this study. Wound model was established according
to the instruction of Birch et al. (2005) with slight modification
(see details in Supplementary materials and methods). Two 6 mm-
diameter full-thickness dermal excisional wounds were created
symmetrically on the dorsal skin of each mouse.
After surgery, mice were randomly divided into three groups
and daily intraperitoneal injected with vehicle (5% DMSO/2%
tween-80/physiological saline, 2.5 μl/g), Gp1a (3 mg/kg, dissolved
in vehicle), or AM630 (3 mg/kg, dissolved in vehicle) respectively.
Mice were euthanized at 12 h, 1 d, 3 d, 5 d, 7 d, 10 d, 13 d, 17 d and
21 d post-injury (6 mice at each time point). The original wound
sizes (measured by tracing the dermal border of the wound) were
analyzed. Six mice without surgery were used as control. Two
1 cm
1 cm skin specimens centered on the wound were col-
lected from each mouse. One specimen was used for morpho-
metric, and another for Western blotting and real-time quantita-
tive PCR (qRT-PCR) analyses.
Experiments were conformed to the Guide for the Care and Use
of Laboratory Animals (National Institutes of Health published
no.86-23, revised 1985), and the Guidelines for the Care and Use of
Laboratory Animals of China Medical University.
2.3. Morphological analyses of wound re-epithelialization, in-
flammatory infiltration, fibrogenesis, and qRT-PCR and Western
blotting assays
For morphometrical analyses, wound sections were per
formed with H&E-staining; Masson's trichrome-staining;
129
immunohistochemical staining of myeloperoxidase (MPO), F4/80,
von Willebrand factor (vWF), proliferating cell nuclear antigen
(PCNA), E-cadherin, and vimentin; as well as triple immuno-
fluorescent staining of CD45/pro-collagen I/α-smooth muscle actin
(SMA). mRNA expressions of monocyte chemotactic protein
(MCP)-1, stromal cell-derived factor (SDF)-1, tumor necrosis factor
(TNF)-α, transforming growth factor (TGF)-β1, epidermal growth
factor (EGF), and keratinocyte growth factor (KGF) were quantified
by qRT-PCR. Protein expressions of interleukin (IL)-6, IL-1β, in-
terferon (IFN)-γ, vascular endothelial growth factor (VEGF)-A, and
pro-collagen I were assayed by Western blotting. See details in
Supplementary materials and methods.
2.4. Cell culture and in vitro wound healing study
HaCaT cells were cultured as described previously (Zhao et al.,
2013). Cells were treated with vehicle (0.1% DMSO), Gp1a (40 nM
to 25 μM, dissolved in vehicle), and AM630 (40 nM to 25 μM,
dissolved in vehicle) respectively. Wound scratch assay was per-
formed. Cell viability was measured by cell counting kit (CCK)-8
assay. Protein expressions of involucrin, E-cadherin, vimentin,
PCNA, and cleaved caspase 3 were measured by Western blotting.
Co-localization of E-cadherin and vimentin was performed by
immunofluorescent staining. See details in Supplementary mate-
rials and methods.
2.5. Statistical analysis
Data were expressed as means 7 standard deviation and ana-
lyzed using SPSS for Windows 13.0. The one-way ANOVA with a
Bonferroni post hoc test was used for data analysis. Difference
associated with Po 0.05 was considered statistically significant.
3. Results
3.1. The general morphology changes after CB2 receptor modulation
To investigate the regulatory function of CB2 receptor during
skin wound healing, we firstly detected the general morphology
changes after the agonist/antagonist treatment. Macroscopically,
CB2 receptor activation by GP1a inhibited wound contraction after
decrustation (Fig. 1A, B). The original wound sizes (measured by
tracing the dermal border of the wound) in GP1a group were
larger than vehicle group at 10–21 d post-injury. No significant
difference was observed between AM630 and vehicle group. Mi-
croscopically, wound re-epithelialization was accelerated in GP1a
group (Fig. 1C, D). The epithelial sheets were longer at 3–10 d, with
larger cross-sectional areas at 5 d and 7 d post-injury. The in-
flammatory infiltration and granulation formation in the wound
cavity were apparently decreased as compared with vehicle group.
In AM630 group, wound re-epithelialization was delayed. The
epithelial sheet length and cross-sectional area were both declined
at 5 d and 7 d post-injury.
Keratinocyte proliferation continues for many days after com-
plete re-epithelialization, resulting in hypertrophic epidermis that
is about four times as thick as unwounded skin (Gallant-Behm
et al., 2011). Similar appearances were observed in the present
study. At 21 d post-injury, the epidermal thicknesses in three
groups were all elevated as compared with the normal skin.
However, the epidermal hypertrophy in GP1a group was greatly
ameliorated when compared with vehicle group (Fig. 2A, C). In
addition, the dermal scar in GP1a group was thinner and the
collagen fibers were much slenderer (Fig. 2B, D). AM630 group
showed similar epidermal and dermal morphologies with vehicle
group.
130 L.-L. Wang et al. / European Journal of Pharmacology 786 (2016) 128–136

Fig. 1. General morphology changes after GP1a/AM630 treatment. (A) Representative macrophotographs of the wounds at different posttraumatic intervals. Bar ¼5 mm.
(B) Percentage of original wound size, N ¼6. *Po 0.05, GP1a versus vehicle; #Po 0.05, AM630 versus vehicle. (C) Microphotographs of H&E-stained wound sections at 3 d, 7 d
and 10 d post-injury. Bar¼ 1 mm. Arrows represent the dermal border; arrowheads represent the epidermal margin. (D) Rate of wound re-epithelialization, and mea-
surement of epithelial sheet length, cross section area and average thickness. N ¼ 6. *Po 0.05, GP1a versus vehicle; #Po 0.05, AM630 versus vehicle.

These morphology changes, taken together, highly indicated
that CB2 receptor was functionally involved in re-epithelialization,
inflammation, and fibrogenesis. Thus, further experiments were
carried out in these aspects.
3.2. CB2 receptor activation attenuates inflammation
Inflammation was evaluated by neutrophil and macrophage
infiltrations, as well as cytokine expressions. Neutrophils and
macrophages were identified by MPO and F4/80 immunostaining
(Fig. S1). In GP1a group, neutrophil content was decreased at 3–
7 d, and macrophage content was decreased at 1–13 d (Fig. 3A). In
AM630 group, neutrophil and macrophage contents were in-
creased at 3–5 d and 3–10 d respectively.
mRNA levels of MCP-1, SDF-1, TNF-α, TGF-β1, EGF, and KGF
were measured by qRT-PCR (Fig. 3B), and protein expressions of IL-
6, IL-1β, IFN-γ, and VEGF-A were measured by Western blotting
(Fig. S2, Fig. 3C). In general, mRNA expressions of MCP-1, SDF-1,
TNF-α and TGF-β1 were decreased in GP1a group, whereas in-
creased in AM630 group. mRNA expression of EGF was not sig-
nificantly changed, and that of KGF was decreased after GP1a
treatment at 10–21 d. Protein expressions of IL-6, IL-1β, and VEGF-
A were decreased in GP1a group and increased (VEGF-A un-
changed) in AM630 group. Protein expression of IFN-γ showed a
different pattern that was decreased after injury. This result was
similar to the finding of Maeda et al. (2011) in a mouse excisional
wound study. IFN-γ is predominantly secreted by NK cells and T
lymphocytes (Farrar and Schreiber, 1993). We presumed that a
large amount of other cytokines secreted by the massively in-
filtrated neutrophils and macrophages resulted in relative de-
crease in quantity of IFN-γ at the early inflammation stage. The
relatively increased IFN-γ expression in GP1a group and decreased
expression in AM630 group as compared with vehicle group
(Fig. 3C) were consistent with their dynamics in neutrophil and
macrophage infiltration. The aforementioned results, taken to-
gether, highly indicated that wound inflammation was attenuated
after CB2 receptor activation.
3.3. CB2 receptor activation attenuates fibrogenesis
Fibroblasts (pro-col I þ ), myofibroblasts (pro-col I þ /α-SMA þ ),
and fibrocytes (CD45 þ /pro-col I þ ) were simultaneously detected
by triple immunofluorescent staining (Fig. 4A). In GP1a group, fi-
broblast content was significantly reduced at 5–21 d (Fig. 4B). The
 L.-L. Wang et al. / European Journal of Pharmacology 786 (2016) 128–136 131

Fig. 2. Wound morphology at 21 d post-injury. (A) Representative microphotographs of epidermis in H&E-stained wound sections. Bar ¼50 mm. (B) Representative mi-
crophotographs of Masson's-stained wound sections. Bar ¼1 mm. (C) Analysis of the epidermal thickness at 21 d post-injury & Po 0.05, wound epithelium versus normal
skin epithelium. *Po 0.05, GP1a versus vehicle; #P o0.05, AM630 versus vehicle. (D) Analysis of the dermal thickness. *Po 0.05, GP1a versus vehicle; #P o0.05, AM630
versus vehicle.

Fig. 3. Inflammatory infiltration and cytokine expression changes after GP1a/AM630 treatment. (A) Cell counting of neutrophils and macrophages in the wounds. NS
represents the results obtained from normal skin as control. (B) mRNA levels of MCP-1, SDF-1, TNF-α, TGF-β1, EGF, and KGF. N ¼6. *P o 0.05, GP1a versus vehicle; #P o0.05,
AM630 versus Vehicle. (C) Protein expressions of IL-6, IL-1β, IFN-γ, and VEGF-A. N ¼6. *P o0.05, GP1a versus Vehicle; #Po 0.05, AM630 versus Vehicle.
132 L.-L. Wang et al. / European Journal of Pharmacology 786 (2016) 128–136

Fig. 4. Fibrogenesis alteration after GP1a/AM630 treatment. (A) Representative immunofluorescence stains of CD45/pro-col I/α-SMA in the wound sections at 7 d and 10 d
post-injury. Enlarged areas show the fibrocytes (arrows). Bar ¼ 100 mm. (B) Cell counting of fibroblasts, myofibroblasts, and fibrocytes in the wounds. N ¼ 6. *Po 0.05, GP1a
versus Vehicle; #Po 0.05, AM630 versus Vehicle. (C and D) Representative immunoblotting results and quantitative analysis of the expression of pro-collagen I at different
posttraumatic intervals. N¼ 6. *Po 0.05, GP1a versus Vehicle; #P o0.05, AM630 versus Vehicle.

ratios of fibroblast-to-myofibroblast differentiation (generally de-
fined by the ratios of myofibroblasts to fibroblasts) were decreased
(23.5 75.3% at 5 d; 38.9 712.2% at 7 d; 47.67 5.3% at 10 d) as
compared with vehicle (31.675.1% at 5 d; 63.4 77.2% at 7 d;
62.7 78.6% at 10 d). Correspondingly, the myofibroblast content
was much more reduced in Gp1a group (Fig. 4B). In AM630 group,
the fibroblast and myofibroblast contents were not significantly
changed as compared with vehicle group.
Fibrocytes are bone marrow-derived progenitor cells that co-
express hematopoietic and mesenchymal markers, possess in-
flammatory properties, and have potential to differentiate into
myofibroblasts (Bucala et al., 1994; Reilkoff et al., 2011). There is a
controversy on whether or not fibrocytes participate in skin
wound repair. A few experiments revealed that no fibrocyte was
detected during skin wound healing (Barisic-Dujmovic et al., 2010;
Higashiyama et al., 2011). However, many others certified the re-
cruitment of fibrocytes after skin injury (Ishii et al., 2005; Mori
et al., 2005; Ou et al., 2015; Suga et al., 2014). The present study
was in support of the latter opinion. In vehicle group, fibrocytes
were detected in the wound cavity at 3–17 d post-injury (Fig. 4A,
B). After GP1a treatment, fibrocyte recruitment was greatly in-
hibited at 5–10 d. No significant difference was observed in AM630
group as compared with vehicle. In accordance with the reduced
contents of fibroblasts, myofibroblasts and fibrocytes, protein ex-
pression of pro-collagen I was decreased after GP1a treatment
(Fig. 4C, D).
3.4. CB2 receptor activation promotes re-epithelialization in vivo
In the present study, the cross-sectional area of epithelial sheet
in GP1a group was larger than vehicle at 5–7 d post-injury. Fur-
thermore, the epithelial sheet was extended to a higher degree
and even resulted in decreased epidermal thickness (Fig. 1C, D).
These morphologies implied that both the proliferation and mi-
gration of keratinocyte were enhanced. During skin wound heal-
ing, keratinocytes at the wound margin undergo partial epithelial
to mesenchymal transition (EMT), losing intercellular adhesions
with each other and acquiring mesenchymal property that facil-
itate their migration (Barriere et al., 2015; Hudson et al., 2009;
Lamouille et al., 2014). Therefore, besides proliferation marker
PCNA, the classic EMT markers E-cadherin and vimentin were
immunostained to further reveal the alteration of keratinocyte
biology.
In GP1a group, keratinocytes adjacent to the wound margin
were less-stratified with PCNA expressed throughout the epithe-
lium. The PCNA þ cell number per unit area was increased at 5–7 d
post-injury (Fig. 5A, B). Expression of E-cadherin was down-
regulated. Keratinocytes over the basal layer were stretched and
orientated to the wound center, and their expression of vimentin
were up-regulated (Fig. 5A, B). In AM630 group, keratinocytes
adjacent to wound margin were more-stratified with PCNA ex-
pression restricted to the basal layer. The PCNA þ cell number per
unit area was decreased at 5–7 d post-injury (Fig. 5B). The ex-
pression of E-cadherin was up-regulated, and vimentin down-
regulated. These morphologies further certified that GP1a
 L.-L. Wang et al. / European Journal of Pharmacology 786 (2016) 128–136 133

Fig. 5. Morphology changes of the epithelial sheet after GP1a/AM630 treatment. (A) Representative microphotographs of H&E stained, as well as PCNA, E-cadherin and
vimentin immunostained epithelium adjacent to wound margin at 7 d post-injury. Red dotted lines indicate roughly the boundary between epidermis and dermis.
Bar ¼ 100 mm. (B) Analyses of PCNA þ cell number per unit area, and the expressions of E-cadherin and vimentin at 3 d, 5 d, and 7 d post-injury. N ¼6. *Po 0.05, GP1a versus
Vehicle; #P o0.05, AM630 versus Vehicle. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

promoted, whereas AM630 inhibited keratinocyte proliferation
and migration.
3.5. CB2 receptor activation promotes re-epithelialization in vitro
To testify the direct effect of CB2 receptor on keratinocyte
biology, further experiments were carried out in HaCaT cells in
vitro. Cell proliferation was assessed by CCK-8 assay (Fig. 6A). As
compared with vehicle group, cell viability was not significantly
elevated after GP1a treatment at concentrations of 40 nM to 5 μM,
while was slightly decreased at the concentration of 25 μM
(95.0 72.5%). AM630 significantly decreased cell viability at con-
centrations of 5 μM (94.0 73.0%) and 25 μM (86.7 73.6%). Sub-
sequently, HaCaT cells were treated respectively with GP1a and
AM630 at concentrations of 0.2 μM, 1 μM, 5 μM, and 25 μM. Cell
morphology alteration and expressions of relevant proteins were
detected.
GP1a dose-dependently down-regulated the expressions of
involucrin and E-cadherin, while up-regulated the expression of
vimentin (Fig. 6B, C). Expression of PCNA was not significantly
changed except for a slight decrease after 25 μM GP1a treatment.
Morphologically, after 5 μM GP1a treatment for 24 h, lots of HaCaT
cells changed from a typical cobblestone-like to a spindle-like
shape, and stretched out pseudopodia. Decreased expression of
E-cadherin and increased expression of vimentin were re-
confirmed by immunofluorescence staining (Fig. 6D). 25 μM GP1a
treatment caused more obvious cell morphology alteration. Lots of
cells detached from the cell colony and migrated out with pseu-
dopodia (Fig. S3A). However, epithelial cell growth and survival
critically rely on cell-matrix and cell-cell adhesion, and severe
disruption of these interactions could induce cell programmed
death by anoikis (Bretland et al., 2001; Frisch and Francis, 1994). It
is likely in this manner some detached cells were induced to
death. This effect was supported by the slight increase of cleaved
caspase 3 expression. Meanwhile, it also explained the decrease of
PCNA expression (Fig. 6B, C). AM630 dose-dependently increased
the expressions of involucrin, E-cadherin, and reduced the ex-
pression of PCNA (Fig. 6B, C). The cell morphology was not ob-
viously changed after 5 μM AM630 treatment (Fig. 6D). However,
25 μM AM630 treatment increased the expression of cleaved
caspase 3 (Fig. 6B, C), and induced cell apoptosis (Fig. S3A). Cor-
responding results were observed in wound scratch assay. GP1a
accelerated wound closure at concentrations of 1 μM and 5 μM,
while retarded it at the concentration of 25 μM (Fig. 6E).
134 L.-L. Wang et al. / European Journal of Pharmacology 786 (2016) 128–136

Fig. 6. HaCaT cell biology alteration after GP1a/AM630 treatment. (A) The cell viability alteration determined by CCK-8 assay. (B and C) Representative immunoblotting
results and quantitative analyses of involucrin, E-cadherin, vimentin, PCNA, and cleaved caspase 3 expressions in HaCaT cells. N ¼ 5. *P o0.05, GP1a versus vehicle; #Po 0.05,
AM630 versus vehicle. (D) Representative phase-contrast microphotographs of HaCaT cells, and immunofluorescent stains of E-cadherin and vimentin. (E) Representative
wound-scratch assay and quantitative analyses of migration ratios at 24 h. Bar ¼ 0.5 mm. N ¼ 5. *Po 0.05, GP1a versus vehicle; #P o0.05, AM630 versus vehicle.

Microscopically, 25 μM GP1a treatment induced cells at the
wound margin to migrate out of the cell colony quickly. However,
the separated HaCaT lost interactions with each other, and were
induced to anoikis subsequently (Fig. S3B). AM630 at concentra-
tions of 5 μM and 25 μM inhibited wound closure (Fig. 6E).
These in vitro results, taken together, indicated that activating
CB2 receptor could inhibit keratinocyte differentiation (involucrin
was decreased) and induce EMT (E-cadherin was decreased and
vimentin was increased), but not promote cell proliferation (PCNA
was not increased). Inhibiting CB2 receptor could enhance kerati-
nocyte adhesion (E-cadherin was increased), promote differentia-
tion (involucrin was increased), and inhibit proliferation (PCNA
was decreased).
4. Discussion
In the present study, we demonstrated that pharmacological
activation of CB2 receptor could ameliorate skin wound healing by
attenuating inflammation and fibrogenesis, and promoting re-
epithelialization.
CB2 receptor has been recognized as a novel therapeutic per-
spective for various inflammatory diseases (Biro et al., 2009; Klein,
2005; Maccarrone et al., 2015). In this study, we further confirmed
its anti-inflammation property during skin wound healing. After
CB2 receptor agonist treatment, neutrophil content was decreased
only to a small extent. However, macrophage content was about
half declined. This change was accompanied with decreased ex-
pression of MCP-1, which is mainly secreted by macrophages and
acts as their powerful chemokine in turn (Deshmane et al., 2009).
Macrophages are key-regulators of wound repair that phagocytize
pathogens and necrotic tissue, and serve as important sources of
numerous cytokines and growth factors (Brancato and Albina,
2011; Kondo and Ishida, 2010). In accordance with their reduction,
 L.-L. Wang et al. / European Journal of Pharmacology 786 (2016) 128–136
expressions of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α
were decreased, thus further attenuated inflammation. Mean-
while, expression of VEGF-A, an essential factor for angiogenesis,
was decreased. This down-regulation did not alter the neovascular
density, but reduced granulation tissue content in the wound
cavity (Fig. S4). Expression of IFN-γ was relatively increased after
CB2 receptor activation. Since IFN-γ could active macrophages,
promoting phagocytosis and cytokines production (Farrar and
Schreiber, 1993; Gordon and Taylor, 2005), this effect might act as
a feedback mechanism to enhance macrophage activity in the
circumstance of decreased leukocyte infiltration.
Fibrogenesis is a dynamic process predominantly conducted by
fibroblasts, and to some extent regulated by inflammatory med-
iators (Stramer et al., 2007; Wynn and Ramalingam, 2012). TGF-β1
is a key driver of fibrosis which stimulates fibroblasts to synthesize
collagen, and induces fibroblast-to-myofibroblast differentiation.
Myofibroblasts possess stronger collagen synthesizing ability and
contribute to wound contraction (Duffield et al., 2013; Wynn and
Ramalingam, 2012). IL-1β, IL-6, and TNF-α could also promote fi-
brogenesis by increasing fibroblast proliferation and collagen
synthesis (Barnes et al., 2011; Duffield et al., 2013; Zhang et al.,
1993). IFN-γ could inhibit fibroblast proliferation, and suppresses
the production and functional activity of TGF-β1 (Gurujeyalakshmi
and Giri, 1995; Ishida et al., 2004). Since CB2 receptor doesn’t di-
rectly modulate collagen synthesis by fibroblasts in vitro (Akh-
metshina et al., 2009), we presumed the anti-fibrogenesis effect of
CB2 receptor in the present study was achieved indirectly by
down-regulating IL-1β, IL-6, TNF-α, TGF-β1 expressions and up-
regulating IFN-γ expression. The reduced wound contraction could
be attributed to decreased fibroblast-to-myofibroblast differ-
entiation. Activating CB2 receptor also decreased the expression of
SDF-1, an important chemokine ligand for fibrocyte recruitment
(Phillips et al., 2004), which might account for the fibrocyte con-
tent reduction, and to a small extent for the fibrosis alleviation.
In the present study we found that CB2 receptor activation
exerted obvious anti-inflammation and anti-fibrogenesis effects
during skin wound healing, while inhibition with antagonist
moderately exaggerated inflammation, and did not exhibit sig-
nificant effect on fibrogenesis. Presumably, CB2 receptor might be
activated by the endocannabinoids as endocannabinoids are pro-
duced ‘on demand’ from their membrane lipid precursors when
and where needed on pathological or physiological stimuli (Mac-
carrone et al., 2015). The suppressive action on CB2 receptors by
AM630 might be partially reversed by the endocannabinoids.
Correspondingly, the expressions of profibrotic cytokines were
slightly increased, which did not give rise to a significant
fibrogenesis.
Previous knowledge about the effect of CB2 receptor on non-
tumorigenic keratinocytes is limited and controversial. It was re-
ported that mixed CB1/CB2 receptor agonist WIN-55,212-2 at a low
concentration (25 nM) (Casanova et al., 2003) and CB2 receptor
antagonist AM630 at a low concentration (1 μM) (Toth et al., 2011)
were ineffective in modulating keratinocyte growth in vitro.
However, CB2 receptor knockout mice showed improved epi-
dermal differentiation and decreased proliferation (Roelandt et al.,
2012). The present study provided an interpretation to this con-
troversy, and further expanded our cognition about CB2 receptor.
CB2 receptor inhibition by low concentrations of AM630 (40 nM to
1 μM) did not affect HaCaT cell proliferation or differentiation.
However, high concentrations of AM630 treatment attenuated cell
proliferation and induced differentiation. These results were co-
incident with the findings in CB2 receptor knockout mice. CB2
receptor agonist GP1a did not promote HaCaT cell proliferation,
but dose-dependently inhibited its differentiation. Furthermore, it
induced keratinocytes to take place EMT and promoted cell mi-
gration. These results were coinciding with the morphological
135
appearances in vivo, that the wound epithelium was less-stratified
and more extended after CB2 receptor activation.
During skin wound healing, EGF and KGF are potent promoters
of keratinocyte proliferation and migration, while TGF-β1 could
reverse activated keratinocytes to the basal cell phenotype and
inhibit their proliferation (Gurtner et al., 2008; Pastar et al., 2014).
In the present study, the expressions of EGF and KGF were not
significantly changed in the early stage. Thus, it might be pre-
sumed that activating CB2 receptor accelerated re-epithelialization
indirectly by decreasing TGF-β1 expression and directly by pro-
moting keratinocyte migration, while inhibiting CB2 receptor at-
tenuated re-epithelialization by increasing TGF-β1 expression and
promoting keratinocyte differentiation. We also observed the
epidermal hypertrophy at the late stage was ameliorated after CB2
receptor agonist treatment. Most probably it was associated with
decreased KGF expression which reduced keratinocyte prolifera-
tion. Since fibroblasts are the major effector cells at the late stage
and are also important sources of KGF (Martin, 1997), this phe-
nomenon might be attributed to reduced fibroblast accumulation
after CB2 receptor activation.
There is a notion that in mammals, wound repair is evolutio-
narily optimized for speed of healing under dirty conditions,
where a multiply redundant, compensating and rapid in-
flammatory response allows the wound to heal quickly without
infection. Scar formation is the price mammalians have to pay for
their survival after wounding (Bayat et al., 2003). Nowadays, it is
increasingly accepted that reducing inflammation might speed
wound healing and improve the final scar appearance (Rosique
et al., 2015). Our present study revealed that activating CB2 re-
ceptor could curb inflammation and attenuate scar formation.
Furthermore, CB2 receptor activation could promote keratinocyte
migration, rendering it more beneficial for wound re-epitheliali-
zation. From the perspective of wound therapy, it should be no-
ticed that inflammation per se is important in orchestrating
wound healing, and an excessive blockade of inflammation would
not be a useful strategy. Appropriate collagen deposition is also
necessary for the skin to maintain its mechanical property. Thus,
we suggest that a moderate CB2 receptor agonist treatment in the
early stage, and gradually ceased it at the late stage (for example
after decrustation) might be more favorable for skin wound
healing.
In conclusion, pharmacological activation of CB2 receptor could
ameliorate wound healing by attenuating inflammation and fi-
brogenesis, as well as promoting re-epithelialization, suggesting
that CB2 receptor agonist might be a novel perspective for skin
wound therapy.
Acknowledgments
The study was financially supported by Grants from projects
funded by National Natural Science Foundation of China (Grant No.
81273342, 81372943, 81102156), Shenyang Scientific and Techno-
logical Plan (F12-277-1-03), and Anshan Scientific and Technolo-
gical Plan (2060499).
Appendix A. Supplementary material
Supplementary data associated with this article can be found
in the online version at http://dx.doi.org/10.1016/j.ejphar.2016.06.
006.
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