European Journal of Pharmacology 559 (2007) 227 – 235

www.elsevier.com/locate/ejphar

Mechanisms underlying the inhibitory actions of the pentacyclic

triterpene α-amyrin in the mouse skin inflammation induced by
phorbol ester 12-O-tetradecanoylphorbol-13-acetate
Rodrigo Medeiros a,1 , Michel F. Otuki a,1,2 , Maria Christina W. Avellar b , João B. Calixto a,⁎
a
Departamento de Farmacologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil
b
Departamento de Farmacologia, Universidade Federal de São Paulo, São Paulo, SP, Brazil
Received 4 May 2006; received in revised form 5 December 2006; accepted 8 December 2006
Available online 29 December 2006

Abstract

The present study evaluated some of the mechanisms through which α-amyrin, a pentacyclic triterpene isolated from Protium Kleinii and other
plants, exerts its effects against 12-O-tetradecanoylphorbol-acetate (TPA)-induced skin inflammation in mice. Topical application of α-amyrin
(0.1–1 mg/ear) dose-dependently inhibited TPA-induced increase of prostaglandin E2 (PGE2) levels. In contrast with the selective cyclooxygenase
(COX)-1 SC560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole] or COX-2 rofecoxib inhibitors, α-amyrin failed to alter
either COX-1 or COX-2 activities in vitro. Western blot analysis revealed that α-amyrin dose-dependently inhibited TPA-induced COX-2
expression in the mouse skin. The evaluation of nuclear factor-κB (NF-κB) pathway revealed that topical treatment with α-amyrin is able to
prevent IκBα degradation, p65/RelA phosphorylation and NF-κB activation. Moreover, α-amyrin given topically dose-dependently inhibited the
activation of upstream protein kinases, namely extracellular signal-regulated protein kinase (ERK), p38 mitogen-activated protein kinase (MAPK)
and protein kinase C (PKC)α, following topical TPA treatment. Collectively, present results suggest that topical skin application of α-amyrin
exerts a strong and rapid onset inhibition of TPA-induced inflammation. These effects seem to be associated with the suppression of skin PGE2
levels by mechanisms involving the suppression of COX-2 expression, via inhibition of upstream protein kinases – namely ERK, p38 MAPK and
PKCα – and blocking of NF-κB activation. These results indicate that α-amyrin-derivative could be potentially relevant for the development of a
topical agent for the management of inflammatory diseases.
© 2006 Elsevier B.V. All rights reserved.

Keywords: α-amyrin; Skin inflammation; PGE2; NF-κB; TPA; COX-2

1. Introduction and inflammatory systems play a pivotal role. Thus, a large

The skin is the largest organ in the body and one of its main
functions is to protect the body from environmental and
endogenous noxious conditions, such as injury, infection and
inflammation. In the pathogenesis of skin diseases, the immune
⁎ Corresponding author. Departamento de Farmacologia, Centro de Ciências
Biológicas, Universidade Federal de Santa Catarina, Campus Universitário
Trindade, 88049-900, Florianópolis, SC, Brazil. Tel.: +55 48 33319491; fax:
+55 48 32224164.
E-mail addresses: calixto@farmaco.ufsc.br, calixto3@terra.com.br
(J.B. Calixto).
1
The first two authors contributed equally to this work.
2
Current address: Departamento de Farmacologia, Setor de Ciências
Biológicas, Universidade Federal do Paraná, PR, Brazil.
0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2006.12.005
production of inflammatory mediators, such as cytokines
(tumor necrosis factor-α, TNF-α; interleukin-(IL)1β) chemo-
kines (monocyte chemotactic protein-1, MCP-1; IL-8), cell
adhesion molecules (intercellular adhesion molecule-1, ICAM-
1; vascular cell adhesion molecule-1, VCAM-1), growth factors
and proteases, are produced and released at the site of skin
injury (Homey, 2005; Okayama, 2005; Pivarcsi and Homey,
2005; Numerof et al., 2005).
There is a substantial amount of experimental evidence
indicating that the mitogen activated protein kinases (MAPKs)
and nuclear factor-κB (NF-κB) have a critical role in both acute
and chronic inflammatory processes (Karin, 2006; Kyriakis and
Avruch, 2001). The stimulation of these cascades occurs in
response to several stimuli, such as mechanical stress, inflam-
228 R. Medeiros et al. / European Journal of Pharmacology 559 (2007) 227–235
matory cytokines, heat and chemical shock, or bacterial
products, all of them also capable of stimulating cyclooxygen-
ase (COX)-2 inductions. It has been shown that binding of NF-
κB to its specific cognate κB sites is functionally important for
COX-2 induction by the prototype tumor promoter 12-O-
tetradecanoylphorbol-13-acetate (TPA) (Chen et al., 2000). In
addition, the participation of activator protein-1 (AP-1) in cox-2
gene expression was also reported (Chun et al., 2004).
Accumulating evidence indicates that IκB kinase (IKK)-
dependent IκB degradation is a pre-requisite for NF-κB
activation, and p38 mitogen-activated protein kinase (MAPK),
extracellular signal-regulated kinase (ERK) and protein kinase
C (PKC) are also involved in NF-κB activation and COX-2
expression in response to TPA (Schulze-Osthoff et al., 1997;
Janssen-Heininger et al., 2001).
α-Amyrin (Fig. 1) is a naturally-occurring pentacyclic
triterpene found in various plants that are widespread in tropical
regions (eg., Protium kleinii, Protium heptaphyllum and Mol-
denhawera nutans) (do Vale et al., 2005; Oliveira et al., 2005;
Otuki et al., 2005b). Some experimental evidence shows that α-
amyrin exhibits systemic antinociceptive, antiinflamatory,
antipruritic, hepatoprotective and gastroprotective properties
when assessed in vivo (Kweifio-Okai et al., 1994; Recio et al.,
1995; Oliveira et al., 2004a,b). We have shown previously that
the mixture of triterpene α-amyrin and β-amyrin produces
consistent peripheral, spinal, and supraspinal antinociception in
rodents, especially when assessed in inflammatory models of
pain. The mechanisms involved in the actions of this mixture
seem to involve the inhibition of both protein kinase A- and
PKC-dependent pathways (Otuki et al., 2005a). Furthermore,
our previous study has also demonstrated that the topical
application of pentacyclic triterpene α-amyrin is capable of
reducing, in a dose-dependent manner, three important events
related to the TPA-induced skin inflammatory response:
oedema formation, migration of polymorphonuclear leuko-
cytes, and increase in tissue IL-1β levels (Otuki et al., 2005b).
However, the mechanisms underlying these topical anti-
inflammatory properties of triterpene pentacyclic α-amyrin
are so far, only partially understood.
The aim of the present study was therefore to investigate
further, using functional, biochemical and molecular proce-
dures, some of the mechanisms through which α-amyrin exerts
its topical anti-inflammatory action on TPA-induced skin
inflammation in mice.
Fig. 1. Structure of α-amyrin.
2. Materials and methods
2.1. Reagents
The following substances were used: TPA, α-amyrin,
dexamethasone acetate, phosphate-buffered saline (PBS), 4-
(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),
indomethacin, Tween-20, Triton-X 100, glycerol, phenyl-
methylsulphonyl fluoride, leupeptin, pepstatin A, aprotinin,
soybean trypsin inhibitor, benzamidine, sodium orthovanadate,
β-glycerophosphate, sodium fluoride, dithiothreitol, β-mercap-
toethanol, dimethyl sulfoxide (DMSO), bromophenol blue
(Sigma Aldrich Co., São Paulo, Brazil), ethylenediamine
tetraacetic acid (EDTA), Tris–HCl, MgCl2, NaCl, KCl, sodium
acetate, methanol, acetone, absolute ethanol, Rofecoxib (Merck
and Co., Inc., Whitehouse Station, USA), NF-κB double-
stranded consensus oligonucleotide probe, T4 polynucleotide
kinase (Promega, Madison, USA), poly(dI–dC), [γ32P]ATP,
protein A-sepharose (GE Healthcare, São Paulo, Brazil), 5-(4-
chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole
(SC560) (Boehringer-Ingelheim, Ingelheim, Germany), anti-
COX-2, anti-α-actin, anti-IκBα, anti-PKCα, anti-ERK, anti-
phosphorylated-ERK, anti-p38 MAPK, anti-phosphorylated-
p38 MAPK, anti-p65 NF-κB antibodies (Santa Cruz Biotech.
Inc., Santa Cruz, USA) and anti-phosphoserine antibody
(Calbiochem–Novabiochem Co., San Diego, USA).
2.2. Animals
Experiments were conducted using male Swiss mice (20–30 g)
kept in a controlled room temperature (22 ± 1 °C) and humidity
(60 to 80%) under a 12 h:12 h light–dark cycle (lights on 06:00 h).
All the procedures used in the present study complied with the
“Principles of laboratory animal care” from NIH publication No.
85-23 and were approved by the Institutional Ethics Committee of
the Federal University of Santa Catarina (protocol numbers 262/
CEUA and 23080.035334/2003-16/UFSC).
2.3. TPA induced skin inflammation
The skin inflammation was induced in the right ear by the
topical application of 2.5 μg/ear of TPA dissolved in 20 μl of
acetone. Control animals received the same volume of acetone
in the right ear. Both α-amyrin (0.1–1.0 mg/ear) and
dexamethasone (0.05 mg/ear, used as a positive control) were
dissolved in 20 μl of acetone, and were applied topically in
conjunction with TPA. Control animals received the same
volume of vehicle. Animals were sacrificed at 1 or 6 h after
TPA-treatment. Tissues were frozen in liquid N2, and stored at
− 70 °C until use.
2.4. Cyclooxygenase-1/cyclooxygenase-2 ovine enzyme assay
The activity COX-1/COX-2 in vitro was carried out by using
a colorimetric assay for detecting COX inhibitors (Cayman
Chemical Company, Ann Arbor, USA), according to the
manufacturer's protocol. The reaction was performed in the
 R. Medeiros et al. / European Journal of Pharmacology 559 (2007) 227–235
presence of vehicle (DMSO, 0.5%) or α-amyrin (1–30 μM).
SC560 and rofecoxib were used as positive controls for the
inhibition COX-1 and COX-2 activity, respectively. Data is
expressed as the percentage of the activity in relation to the
control group.
2.5. Measurement of prostaglandin E2 levels
The tissue PGE2 levels were determined 6 h after TPA
application in 6 mm punch-biopsies obtained from the mouse
ears. The tissues were homogenized in 100 mM phosphate
buffer (pH 7.4) containing 1 mM EDTA and 10 μM
indomethacin. After addition of ethanol, the sample was
centrifuged at 1500 ×g for 10 min. Then 50 mM citrate buffer
(pH 3.5) was added to the supernatant, after which it was
centrifuged again at 2500 ×g for 10 min. The resulting
supernatant was applied to a 6 ml Sep-Pak C18 cartridge
(Waters Associate, Milford, USA) pre-activated with 5 ml
methanol and then with 5 ml distilled water. The cartridge
was rinsed with 5 ml distilled water, followed by 5 ml
hexane. The final eluate was obtained with 5 ml ethyl acetate
containing 1% methanol and dried under N2 stream. After
dissolving the residue in a small amount of assay buffer, the
prostaglandin E2 concentration was measured with an ELISA
kit (Cayman Chemical Company) according to the manufac-
turer's instructions.
2.6. Preparation of cytosolic and membrane fractions
Protein extraction was performed as previously described
(Medeiros et al., 2004; Ferreira et al., 2005). In brief, tissues
were homogenized in ice-cold buffer A (10 mM HEPES, pH
7.4; containing 1.5 mM MgCl2, 10 mM KCl, 1 mM
phenylmethylsulphonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml
pepstatin A, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 mM
sodium orthovanadate, 10 mM β-glycerophosphate, 50 mM
sodium fluoride and 0.5 mM dithiothreitol). The homogenates
were chilled on ice under vigorously shaken for 15 min. The
membrane fraction was precipitated by centrifugation at
14,000 ×g for 60 min. The supernatant containing the cytosolic
fraction was stored at − 70 °C until use. The membrane pellet
was resuspended in 50 μl of buffer A containing 1% Triton X-
100 and incubated under continuous shaking at 4 °C for 30 min.
The membrane fraction was then centrifuged for 60 min at
14,000 ×g and the supernatant was aliquoted and stored at
− 70 °C. The protein concentration was determined using the
Bio-Rad protein assay kit (Bio-Rad, São Paulo, Brazil).
2.7. Western blot analysis
Cell lysates (30 μg protein) were boiled in sodium dodecyl
sulfate (SDS) sample buffer for 5 min before electrophoresis on
12% SDS-polyacrylamide gel. After transferring to a polyvinyli-
dene fluoride (PVDF) membrane (GE Healthcare), the blots were
blocked with 5% fat-free dry milk-TBST buffer (tris-buffered
saline containing 0.5% Tween-20) for 1 h at room temperature and
then washed with TBST buffer. The membranes were incubated
229
for 2 h at room temperature with 1:1000 dilutions of primary
antibodies for COX-2, β-actin, IκBα, PKCα, ERK or phosphory-
lated-ERK and for 16 h at 4 °C with 1:1000 dilutions of primary
antibodies for p38 MAPK and phosphorylated-p38. Blots were
washed three times with TBST at 5 min intervals followed by
incubation with 1:5000 dilution of appropriate horseradish
peroxidase conjugated secondary antibodies for 1 h and again
washed in TBST for three times. The transferred proteins were
visualized with an enhanced chemiluminescence (ECL) detection
kit according to the manufacturer's instructions (GE Healthcare).
2.8. Immunoprecipitation of p65 NF-κB
Equivalent amounts of cytosolic extracts (200 μg for each
sample) were mixed with 40 μl of protein A-sepharose and 2 μl of
anti-p65 NF-κB antibody, and left overnight at 4 °C with
continuous shaking. Immunocomplexes were washed three times
with 500 μl of ice-cold buffer (10 mM Tris–HCl pH 7.5, 1 M
NaCl, 0.2% Triton-X 100 and 2 mM EDTA), mixed with 40 μl of
SDS gel loading buffer and then boiled for 3 min. Samples were
then subjected to western blot analysis as described above. Proteins
were incubated overnight with 0.1 μg/ml of anti-phosphoserine
antibody. Following the detection of the phosphoserine, the
membranes were washed, stripped and then re-probed with anti-
p65 NF-κB antibody (1:1000).
2.9. Electrophoretic Mobility Shift Assay (EMSA)
Nuclear protein extraction was performed as previously
described (Medeiros et al., 2004). In brief, tissues were suspended
in 1 ml of ice-cold hypotonic lysis (10 mM HEPES, 1.50 mM
MgCl2, 10 mM KCl, 0.5 mM PMSF, 1.5 μg/ml soybean trypsin
inhibitor, 7 μg/ml pepstatin A, 5 μg/ml leupeptin, 0.1 mM
benzamidine, and 0.5 mM DTT) and were then homogenized in a
Polytron (Brinkmann Instruments, Westbury, USA) for 1 min. The
homogenate was chilled on ice for 15 min, and then vigorously
shaken for 15 min in the presence of 10 μl of Nonidet P-40. The
nuclear fraction was precipitated by centrifugation at 1500 ×g for
5 min. The nuclear pellet was resuspended in 100 μl of high salt
extraction buffer (20 mM HEPES (pH 7.9), 420 mM NaCl,
1.5 mM MgCl2, 0.2 mM EDTA, 25% (v/v) glycerol, 0.5 mM
PMSF, 1.5 μg/ml soybean trypsin inhibitor, 7 μg/ml pepstatin A,
5 μg/ml leupeptin, 0.1 mM benzamidine, and 0.5 mM DTT),
incubated under continuous shaking at 4 °C for 30 min, and then
centrifuged for 15 min at 14,000 ×g. The supernatant was
aliquoted and stored at −70 °C. The protein concentration was
determined using a protein assay kit (Bio-Rad).
For EMSAs, an NF-κB double-stranded consensus oligonu-
cleotide probe (5′-AGTTGAGGGGACTTTCCCAGGC-3′) was
end-labeled with [γ-32P]ATP in the presence of T4 polynucleotide
kinase (10 U) for 10 min at 37 °C. Unincorporated nucleotides
were removed by passing the reaction mixture over a Sephadex G-
25 spin column (GE Healthcare). In a total volume of 20 μl,
nuclear extracts (10 μg) were incubated with gel shift binding
buffer [10 mM Tris–HCl (pH 7.5), 1 mM MgCl2, 50 mM NaCl,
0.5 mM DTT, 0.5 mM EDTA, 4% glycerol, and 1 μg of poly(dI–
dC)] for 20 min at room temperature. Each sample was further
230 R. Medeiros et al. / European Journal of Pharmacology 559 (2007) 227–235

incubated for 30 min at room temperature with 25,000 cpm of

32
P-labeled NF-κB consensus oligonucleotide. Protein–DNA
complexes were resolved by nondenaturing 6% acrylamide/bis-
acrylamide (37.5/1) in 0.25 X Tris–borate/EDTA buffer at
150 V for 2 h. Finally, the gel was dried and exposed to an X-
ray film.

2.10. Software

All western blot and EMSA experiments were scanned to

acquire digital images using a Genius ColorPage Scanner (KYE
Systems Corp., Taipei, Taiwan). Digital images were processed
with the Photoshop software package (Adobe Systems, San
Jose, USA), complying with strict standards (Rossner and
Yamada, 2004). Band density measurements were made using
Scion Image software package (Scion Corporation, Maryland,
USA).

2.11. Statistical analysis

The results are presented as mean ± S.E.M. The statistical
significance between the groups was assessed by means of one-
way analysis of variance (ANOVA) followed by post-hoc
Newman–Keuls test. The accepted level of significance for the
tests was P < 0.05. All tests were carried out using the Statistica
software package (StatSoft Inc., Tulsa, USA).
Fig. 3. Effect of α-amyrin on COX activity in vitro. The (A) COX-1 and (B)
COX-2 in vitro assay was carried out using a colorimetric assay for detection of
COX inhibitors according to the manufacturer's protocol. α-Amyrin was tested
at 10 or 30 μM concentration. Selective COX-1 SC560 (0.03 μM) and COX-2
rofecoxib (Rofe, 10 μM) inhibitors were used as a control. Figures are
representative of 3 similar experiments and each column represents the mean ±
S.E.M. The asterisks denote the significance levels: ⁎⁎P < 0.01, compared with
vehicle (control) (one-way ANOVA followed by post-hoc Newman–Keuls test).

3. Results

3.1. α-Amyrin reduced TPA-induced PGE2 production in mouse

skin through inhibition of COX-2 expression, but not COX-1 or
COX-2 activity

High levels of prostaglandins are produced by COX enzymes

during the inflammatory response. As shown in Fig. 2, topical
application of TPA in the mouse ear induced a significant (about

Fig. 4. Effect of α-amyrin on TPA-induced COX-2 expression in mouse ear in

Fig. 2. Effect of α-amyrin on TPA-induced PGE2 production in mouse skin in
vivo. The PGE2 levels were measured 6 h after topical application of acetone
(control, C) or TPA (2.5 μg/ear) as described in the Material and methods
section. Vehicle (V), α-amyrin (0.1–1.0 mg/ear) or dexamethasone (0.05 mg/
ear, Dexa) were administered topically in combination with TPA. Each column
represents the mean ± S.E.M. of 3 independent experiments. The asterisks denote
the significance levels: ⁎⁎P < 0.01, compared with control-treated mice, and
#P < 0.05 and ##P < 0.01, compared with vehicle-treated mice (one-way
ANOVA followed by post-hoc Newman–Keuls test).
vivo. (A) Western blot analysis of COX-2 expression was performed on
cytosolic extract 6 h after acetone (control, C) or TPA (2.5 μg/ear) treatment.
Animals were topically treated, at same time of TPA administration, with vehicle
(V), α-amyrin (0.1–1.0 mg/ear) or dexamethasone (0.05 mg/ear, Dexa).
Immunoblot for β-actin was used as a loading control. (B) Quantification of
COX-2 protein expression normalized by β-actin. Each column represents the
mean ± S.E.M. of 3 independent experiments. The asterisks denote the
significance levels: ⁎⁎P < 0.01, compared with control-treated mice, and
#P < 0.05 and ##P < 0.01, compared with vehicle-treated mice (one-way
ANOVA followed by post-hoc Newman–Keuls test).
 R. Medeiros et al. / European Journal of Pharmacology 559 (2007) 227–235 231

3.6-fold) increase of PGE2 levels. Local administration of α-
amyrin (0.1–1.0 mg/ear), applied in conjunction with TPA,
dose-dependently inhibited the increase of PGE2 formation,
when evaluated 6 h after treatment (Fig. 2). The percentage of
inhibition obtained for α-amyrin at 1.0 mg/ear was 68 ± 4%
(P < 0.01; n = 3). Additionally, when the animals were treated
with the positive control drug dexamethasone (0.05 mg/ear), a
complete inhibition of TPA-induced PGE2 increase was
observed (Fig. 2). To gain further insights into the mechanisms
involved in the inhibitory effects of α-amyrin on PGE2
production following TPA application in the mouse ear, we
firstly evaluated the possible influence of this compound on
COX-1 and COX-2 activity in vitro. In contrast to that reported
for the selective inhibitors of COX-1 (SC 560) or COX-2
(rofecoxib), the α-amyrin triterpene (10–30 μM) failed to
significantly affect either COX-1 (Fig. 3A) or COX-2 (Fig. 3B)
activities in vitro. We next verified whether α-amyrin could
inhibit PGE2 in the ear by interfering with COX-2 expression. It
has been previously shown that topical application of TPA
induces expression of COX-2 in mouse skin in vivo (Chun et al.,
2003). Supporting this data, western blot analysis revealed that
topical application of TPA in the ears of mice resulted in a
dramatic (4.5-fold) up-regulation of COX-2 protein expression
(Fig. 4). Topical application of the same doses of α-amyrin
which were effective in inhibiting the increase of PGE2 levels
after application of TPA (0.1–1.0 mg/ear) caused a dose-
dependent suppression of the COX-2 expression (Fig. 4). The
maximal inhibition obtained at 1.0 mg/ear was 83 ± 3%
(P < 0.01; n = 3). The treatment of mouse skin with α-amyrin
(1.0 mg/ear) alone did not influence the constitutive expression
of COX-2 (data not shown).
3.2. Inhibition of TPA-induced activation of NF-κB by
α-amyrin in mouse skin
The 5′-flanking region of cox-2 gene promoter contains
binding sequences for various transcription factors including
NF-κB (Kim and Fischer, 1998). Thus, we attempted to
evaluate the effects of α-amyrin on TPA-stimulated DNA
binding of NF-κB in mouse skin. As depicted in Fig. 5, NF-κB

Fig. 5. Effect of α-amyrin on TPA-induced NF-κB pathway activation. (A) Nuclear factor-κB-DNA binding was measured 1 h after acetone (control, C) or TPA
(2.5 μg/ear) treatment in the nuclear extracts. (B) Densitometric analysis of nuclear factor-κB activation. (C) p65/RelA protein was immunoprecipitated in the ear
cytosol extracts 1 h after TPA treatment and western blot was performed for phosphoserine residue. Membranes were stripped and re-probed with anti-p65/RelA
antibody. (Bellow graph) Quantification of phosphoserine residue detection normalized by total p65/RelA. (D) Immunodetection of IκBα degradation after TPA
topical treatment in the cytosolic extract. β-actin was used as a loading control. (Bellow graph) Quantification of IκBα protein expression normalized by β-actin.
Vehicle (V), α-amyrin (0.1–1.0 mg/ear) or dexamethasone (0.05 mg/ear, Dexa) were administered topically in association with TPA. Each column represents the mean ±
S.E.M. of 3 independent experiments. The asterisks denote the significance levels: ⁎P < 0.05 and ⁎⁎P < 0.01, compared with control-treated mice, and #P < 0.05 and
##P < 0.01, compared with vehicle-treated mice (one-way ANOVA followed by post-hoc Newman–Keuls test).
232 R. Medeiros et al. / European Journal of Pharmacology 559 (2007) 227–235
activity was detectable in nuclear proteins obtained from
acetone-treated mouse ears (control). However, DNA binding
activity was found to be significantly increased (P < 0.01; n = 3)
in nuclear extracts 1 h after TPA treatment. When mice were
treated with α-amyrin (0.1–1.0 mg/ear) the TPA-induced NF-
κB activation was markedly inhibited (Fig. 5A and B). The
inhibition of NF-κB obtained with the dose of 1.0 mg/ear was
98 ± 5% (P < 0.01; n = 3). Topical application of dexamethasone
(0.05 mg/ear) also largely (70 ± 6%) inhibited TPA-induced NF-
κB activation (Fig. 5A and B). Recent studies have suggested
that p65/RelA phosphorylation may be necessary for transcrip-
tional competence of NF-κB (Ghosh and Karin, 2002). To
assess the phosphorylation state of p65/RelA, we have used the
technique of immunoprecipitation for the subunit p65/RelA
followed by immunodetection of phosphoserine. As shown in
Fig. 5C, the topical application of TPA for 1 h in the mouse ears
resulted in a phosphorylation of the serine residues in the p65/
RelA subunit. In addition, the treatment with α-amyrin
significantly reduced the p65/RelA-serine residue phosphoryla-
tion (P < 0.01; n = 3). Our data also demonstrated that treatment
of mouse skin with TPA, in the presence or absence of α-amyrin
(1.0 mg/ear), did not interfere with the cytosolic levels of p65/
RelA (Fig. 5C). Because the nuclear translocation of NF-κB is
dependent on the phosphorylation and subsequent degradation
of IκBα (Ghosh and Karin, 2002), we further examined whether
α-amyrin could block TPA-induced degradation of IκBα. The
topical treatment with α-amyrin (0.1–1.0 mg/ear) dose-
dependently reduced the degradation of IκBα (Fig. 5D). At
the dose 1 mg/kg, α-amyrin completely blocked the degradation
of IκBα (P < 0.05; n = 3).
3.3. Effects of α-amyrin on TPA-induced activation of MAPKs
The MAPKs are proteins that mediate the signal transduction
from the cell surface to the nucleus, and NF-κB family proteins
are the important target molecules of these protein kinases
(Janssen-Heininger et al., 2001; Schulze-Osthoff et al., 1997;
Chun et al., 2003). Results of Fig. 6A and B indicate that very
low levels of phosphorylated ERK and p38 MAPK are detected
under basal conditions in the mouse ear. In contrast, in the TPA
treated group, a marked activation of ERK (4.2-fold; Fig. 6A)
and p38 MAPK (3.1-fold; Fig. 6B) is detected 1 h after
treatment. The topical treatment with α-amyrin (0.1–1.0 mg/
ear) dose-dependently reduced the activation of the both
MAPKs. At the dose of 1.0 mg/kg, α-amyrin almost completely
blocked the activation of ERK (P < 0.01; n = 3) and p38 MAPK
(P < 0.01; n = 3) induced by TPA (Fig. 6). Under these
experimental conditions, the level of the total form of each
kinase remained constant.
Fig. 6. Effect of α-amyrin on TPA-induced protein kinase activation. Activation
of (A) p38 MAPK, (B) ERK and (C) PKCα was measured 1 h after acetone
(control) or TPA (2.5 μg/ear) treatment. Animals were topically treated, at same
time of TPA administration, with vehicle (V), α-amyrin (0.1–1.0 mg/ear) or
dexamethasone (0.05 mg/ear, Dexa). (Bellow graphs) Quantification of
cytosolic phospho-p38 MAPK (p-p38) or phospho-ERK (p-ERK) normalized
by total p38 MAPK and ERK, respectively. PKCα activation was verified by
migration of protein from cytosol (cPKCα) to membrane (mPKCα). Results
represent a fraction between mPKCα and cPKCα. Each column of the graphs
represents the mean ± S.E.M. of 3 independent experiments. The asterisks denote
the significance levels: ⁎⁎P < 0.01, compared with control-treated mice, and
#P < 0.05 and ##P < 0.01, compared with vehicle-treated mice (one-way
ANOVA followed by post-hoc Newman–Keuls test).
 R. Medeiros et al. / European Journal of Pharmacology 559 (2007) 227–235
3.4. Inhibitory effect of α-amyrin on TPA-induced activation of
PKC
PKC isoforms are major regulators of cutaneous homeostasis
and mediate inflammation in response to TPA (Mueller, 2006).
The results shown in Fig. 6C indicate that topical treatment with
TPA for 1 h in the mouse ear was capable of activating PKCα
isoform, as charged by its translocation from cytosol- to
membrane-rich homogenates achieved in treated tissues. The
application of α-amyrin (0.1–1.0 mg/ear) in the mouse ear
resulted in a dose-dependent inhibition of the TPA-induced
PKCα activation (Fig. 6C). At the dose of 1.0 mg/kg, the
calculated percentage of inhibition caused by α-amyrin was
87 ± 6% (P < 0.01; n = 3).
4. Discussion
Several reports of the literature have shown the effectiveness
of α-amyrin or plants that contain this triterpene in rodent
models of inflammation, namely TPA-induced ear edema (Recio
et al., 1995; Otuki et al., 2005b), carrageenan-induced paw
edema (Recio et al., 1995) and arthritis-induced by complete
Freund's adjuvant (Kweifio-Okai et al., 1994). However, the
molecular mechanisms underlying these effects remain largely
unidentified. A better understanding of the mode of action of α-
amyrin would enhance the potential therapeutic interest on this
naturally-occurring pentacyclic triterpene. In the present study
we have greatly extended our previous findings (Otuki et al.,
2005b), by demonstrating that the anti-inflammatory effects of
α-amyrin appear to rely on the down regulation of COX-2
expression and the inhibition of NF-κB and some protein
kinases.
The COX product of arachidonic acid, PGE2, has long been
known to have proinflammatory properties. Intradermal injec-
tions of PGE2 into human skin cause erythema, while higher
doses produce edema. Of note, PGE2 can amplify the vascular
permeability changes induced by other mediators (Fogh and
Kragballe, 2000). Our present results show that at the same
range of dose that α-amyrin inhibited TPA-induced ear oedema
and polymorphonuclear cell influx (Otuki et al., 2005b) it
caused a dose-dependent inhibition of ear levels of PGE2 in the
mouse skin treated with TPA. Such results could explain, at
least in part, the topical and systemic anti-inflammatory and
antinociceptive properties of α-amyrin reported in previous
works (Recio et al., 1995; Otuki et al., 2005a,b). A growing
number of studies suggest that PGE2 is associated with
pathogenesis of inflammatory skin disease, for instance atopic
dermatitis (Fogh and Kragballe, 2000; Neisius et al., 2002).
Therefore, our data showing significant reduction of the tissue
levels of PGE2 after TPA application in the mouse ears caused
by topical application of α-amyrin might be potentially relevant
in the research for new therapeutic agents which have
prostanoids as a target.
The well known enzymes cyclooxygenase COX-1 and
COX-2 are responsible for the transformation of the arachidonic
acid in prostaglandins, and have an important function in the
inflammatory processes. In contrast to the constituent form
233
COX-1, COX-2 is generally, but not exclusively, induced in
reply to stimulators such as growth factors, cytokines, tissue
injury and ultraviolet radiation (Fogh and Kragballe, 2000; Lee
et al., 2003; Mitchell and Warner, 2006). Of note, the deletion of
one of the genes responsible for the production of COX-2 in
mice is capable of reducing the inflammatory process in animal
models of cutaneous inflammation, indicating that arachidonic
acid metabolites derived from COX-2 exert a critical role in the
inflammatory skin diseases (Muller-Decker et al., 2002). The
results of the present study clearly show that the inhibition of
the levels of PGE2 in the mouse ear after application of α-
amyrin is unrelated with its ability to inhibit the activity of either
COX-1 or COX-2 enzymes. This conclusion derives from the
view that in contrast to that reported for the selective inhibitor of
COX-1 SC560 or COX-2 rofecoxib, α-amyrin, even at high
concentration in vitro, failed to interfere with their activity.
Next, we assessed whether the inhibition of ear levels of PGE2
after topical application of α-amyrin could be associated with its
ability to interfere with the COX-2 expression. Our data show
for the first time that the topical application of the triterpene α-
amyrin, at the same dose range where it causes the skin's anti-
inflammatory property, dose-dependently inhibited the expres-
sion of the COX enzyme. These observations could explain the
relatively strong and rapid onset topical anti-inflammatory
property reported for this plant triterpene.
NF-κB has emerged as one of the most promising and
ubiquitous molecular targets in the prevention of cancer and
inflammation (Ghosh and Karin, 2002; Karin, 2006).
Degradation of IκB allows the NF-κB to translocate to the
nucleus, where it regulates the transcription of several genes,
including the COX-2 and several cytokine genes (Kim and
Fischer, 1998; Ghosh and Karin, 2002). Notably, several
studies have indicated that the increase in the activation of
factor NF-κB has an important role in the development and
maintenance of cutaneous inflammatory diseases, including
psoriasis, contact dermatitis and atopic dermatitis (Bell et al.,
2003). Therefore, drugs that inhibit the activation of factor
NF-κB are in principle, of potential interest for the treatment
cutaneous inflammatory diseases (Courtois, 2005). In the
present study, we have shown that TPA produces an increase
in NF-κB DNA-binding activity, through a mechanism
involving the degradation of IκBα, phosphorylation of p65/
RelA and the subsequent translocation of NF-κB into the
nucleus. Of interest, we found that topical application of α-
amyrin to the mouse skin at the same doses where it was
effective in inhibiting COX-2 expression after TPA topical
application (0.1–1.0 mg/ear), consistently and dose-depen-
dently inhibited TPA-induced NF-κB activation. This effect
seems to be related to its ability of reducing IκBα degradation
and p65/RelA phosphorylation. Therefore, the inhibition of
NF-κB activation by α-amyrin could explain its effects on the
expression of COX-2 enzyme, as well as the previously
reported capability of α-amyrin to diminish IL-1β levels in
the mouse skin following application of TPA (Otuki et al.,
2005b).
MAPKs and PKC are critical components of cellular signal
transduction cascades. They are directly involved in many
234 R. Medeiros et al. / European Journal of Pharmacology 559 (2007) 227–235
diseases, including cancer and inflammation, and have become
one of the most important target classes for drug development
(Cohen, 2002; Dancey and Sausville, 2003). Thus, in the
present study we characterized the effect of topical administra-
tion of α-amyrin in TPA-induced PKC, as well as ERK and p38
MAPK activation. Our data demonstrated that topical treatment
with α-amyrin greatly inhibited the TPA-induced PKCα, ERK
and p38 MAPK activation at the same range of doses necessary
to prevent the IκBα degradation and p65/RelA phosphorylation
after TPA application. Several studies have indicated the
participation of these protein kinases in the regulation of
inflammatory gene expression, acting both at transcriptional
and posttranscriptional levels (Whitmarsh and Davis, 1996;
Ono and Han, 2000; Yang et al., 2003; Clark et al., 2003).
Recent reports have implicated both MAPKs and PKC in the
up-regulation of COX-2 expression by TPA, through a
mechanism dependent on NF-κB activation (Surh, 2003).
Likewise, the activation of these protein kinases has been
implicated in the regulation of the half-lives of many
inflammatory mediator RNAs, including COX-2, TNF-α, and
IL-1β (Clark et al., 2003; Winzen et al., 1999; Bevilacqua et al.,
2003). In this context, α-amyrin regulating the p38 MAPK,
ERK and PKCα activation could decrease COX-2 expression
by reducing the activity of transcriptional factors (eg, NF-κB),
by modulating the stability of some pro-inflammatory proteins
mRNAs (eg, IL-1β and TNF-α), or even by diminishing the
stability of COX-2 mRNA. However, additional studies are
necessary to confirm these hypotheses.
In summary, we have provided in vivo evidence that the
plant-derived pentacyclic triterpene α-amyrin exhibits strong
and rapid onset topical anti-inflammatory actions in the mouse
ear edema induced by TPA through a mechanism that seems to
involve its ability to inhibit the levels of PGE2, via inhibition of
the COX-2 expression. Based on our results and in the previous
investigations we might infer that α-amyrin exerts its anti-
inflammatory properties through regulation of PKCα and
MAPKs activation probably by inhibiting the transcriptional
activity of NF-κB. Collectively, the present findings suggest
that α-amyrin might constitute a potential and relevant
alternative for the treatment of inflammatory diseases.
Acknowledgements
We are grateful to the Merck and Co., Inc. for donating the
Rofecoxib used in this work. This study was supported by the
Conselho Nacional de Desenvolvimento Científico (CNPq), by
the Programa de Apoio aos Núcleos de Excelência (PRONEX)
(Brazil) and by the Fundação de Apoio a Pesquisa de Santa
Catarina (FAPESC). M.F.O and R.M. are PhD students in
pharmacology, and they thank CNPq (Brazil) for fellowship
support.
References
Bell, S., Degitz, K., Quirling, M., Jilg, N., Page, S., Brand, K., 2003.
Involvement of NF-kappaB signalling in skin physiology and disease. Cell.
Signal. 15, 1–7.
Bevilacqua, A., Ceriani, M.C., Capaccioli, S., Nicolin, A., 2003. Post-
transcriptional regulation of gene expression by degradation of messenger
RNAs. J. Cell. Physiol. 195, 356–372.
Chen, C.C., Sun, Y.T., Chen, J.J., Chiu, K.T., 2000. TNF-alpha-induced
cyclooxygenase-2 expression in human lung epithelial cells: involvement of
the phospholipase C-gamma 2, protein kinase C-alpha, tyrosine kinase, NF-
kappa B-inducing kinase, and I-kappa B kinase 1/2 pathway. J. Immunol.
165, 2719–2728.
Chun, K.S., Keum, Y.S., Han, S.S., Song, Y.S., Kim, S.H., Surh, Y.J., 2003.
Curcumin inhibits phorbol ester-induced expression of cyclooxygenase-2 in
mouse skin through suppression of extracellular signal-regulated kinase
activity and NF-kappaB activation. Carcinogenesis 24, 1515–1524.
Chun, K.S., Kim, S.H., Song, Y.S., Surh, Y.J., 2004. Celecoxib inhibits phorbol
ester-induced expression of COX-2 and activation of AP-1 and p38 MAP
kinase in mouse skin. Carcinogenesis 25, 713–722.
Clark, A.R., Dean, J.L., Saklatvala, J., 2003. Post-transcriptional regulation of
gene expression by mitogen-activated protein kinase p38. FEBS Lett. 546,
37–44.
Cohen, P., 2002. Protein kinases—the major drug targets of the twenty-first
century? Nat. Rev., Drug Discov. 1, 309–315.
Courtois, G., 2005. NF-kappaB in skin homeostasis. Exp. Dermatol. 14,
781–782.
Dancey, J., Sausville, E.A., 2003. Issues and progress with protein kinase
inhibitors for cancer treatment. Nat. Rev., Drug Discov. 2, 296–313.
do Vale, A.E., David, J.M., Brandão, H.N., David, J.P., 2005. A new flavonol
glycoside derivative from leaves of Moldenhawera nutans. Z. Naturforsch.
[C] 60, 45–49.
Ferreira, J., Triches, K.M., Medeiros, R., Calixto, J.B., 2005. Mechanisms
involved in the nociception produced by peripheral protein kinase c
activation in mice. Pain 117, 171–181.
Fogh, K., Kragballe, K., 2000. Eicosanoids in inflammatory skin diseases.
Prostaglandins Other Lipid Mediat. 63, 43–54.
Ghosh, S., Karin, M., 2002. Missing pieces in the NF-kappaB puzzle. Cell 109,
S81–S96.
Homey, B., 2005. Chemokines and inflammatory skin diseases. Adv. Dermatol.
21, 251–277.
Janssen-Heininger, Y.M., Poynter, M.E., Baeuerle, P.A., 2001. Recent advances
towards understanding redox mechanisms in the activation of nuclear factor
kappaB. Free Radic. Biol. Med. 28, 1317–1327.
Karin, M., 2006. Nuclear factor-kappaB in cancer development and progression.
Nature 441, 431–436.
Kim, Y., Fischer, S.M., 1998. Transcriptional regulation of cyclooxygenase-2 in
mouse skin carcinoma cells. Regulatory role of CCAAT/enhancer-binding
proteins in the differential expression of cyclooxygenase-2 in normal and
neoplastic tissues. J. Biol. Chem. 273, 27686–27694.
Kweifio-Okai, G., De Munk, F., Rumble, B.A., Macrides, T.A., Cropley, M.,
1994. Antiarthritic mechanisms of amyrin triterpenes. Res. Commun. Mol.
Pathol. Pharmacol. 85, 45–55.
Kyriakis, J.M., Avruch, J., 2001. Mammalian mitogen-activated protein kinase
signal transduction pathways activated by stress and inflammation. Physiol.
Rev. 81, 807–869.
Lee, J.L., Mukhtar, H., Bickers, D.R., Kopelovich, L., Athar, M., 2003.
Cyclooxygenases in the skin: pharmacological and toxicological implica-
tions. Toxicol. Appl. Pharmacol. 192, 294–306.
Medeiros, R., Cabrini, D.A., Ferreira, J., Fernandes, E.S., Mori, M.A., Pesquero,
J.B., Bader, M., Avellar, M.C., Campos, M.M., Calixto, J.B., 2004.
Bradykinin B1 receptor expression induced by tissue damage in the rat
portal vein: a critical role for mitogen-activated protein kinase and nuclear
factor-kappaB signaling pathways. Circ. Res. 94, 1375–1382.
Mitchell, J.A., Warner, T.D., 2006. COX isoforms in the cardiovascular system:
understanding the activities of non-steroidal antiinflammatory drugs. Nat.
Rev., Drug Discov. 5, 75–86.
Mueller, M.M., 2006. Inflammation in epithelial skin tumours: old stories and
new ideas. Eur. J. Cancer 42, 735–744.
Muller-Decker, K., Neufang, G., Berger, I., Neumann, M., Marks, F.,
Furstenberger, G., 2002. Transgenic cyclooxygenase-2 overexpression
sensitizes mouse skin for carcinogenesis. Proc. Natl. Acad. Sci. U. S. A.
99, 12483–12488.
 R. Medeiros et al. / European Journal of Pharmacology 559 (2007) 227–235
Neisius, U., Olsson, R., Rukwied, R., Lischetzki, G., Schmelz, M., 2002.
Prostaglandin E2 induces vasodilation and pruritus, but no protein
extravasation in atopic dermatitis and controls. J. Am. Acad. Dermatol.
47, 28–32.
Numerof, R.P., Dinarello, C.A., Asadullah, K., 2005. Cytokines as potential
therapeutic targets for inflammatory skin diseases. Eur. Cytokine Netw. 16,
101–103.
Okayama, Y., 2005. Oxidative stress in allergic and inflammatory skin diseases.
Curr. Drug Targets Inflamm. Allergy 4, 517–519.
Oliveira, F.A., Lima-Junior, R.C., Cordeiro, W.M., Vieira-Junior, G.M., Chaves,
M.H., Almeida, F.R., Silva, R.M., Santos, F.A., Rao, V.S., 2004a. Pentacyclic
triterpenoids, alpha, beta-amyrins, suppress the scratching behavior in a mouse
model of pruritus. Pharmacol. Biochem. Behav. 78, 719–725.
Oliveira, F.A., Vieira-Junior, G.M., Chaves, M.H., Almeida, F.R., Santos, K.A.,
Martins, F.S., Silva, R.M., Santos, F.A., Rao, V.S., 2004b. Gastroprotective
effect of the mixture of alpha- and beta-amyrin from Protium heptaphyllum:
role of capsaicin-sensitive primary afferent neurons. Planta Med. 70,
780–782.
Oliveira, F.A., Chaves, M.H., Almeida, F.R., Lima, R.C., Silva, R.M., Maia,
J.L., Brito, G.A., Santos, F.A., Rao, V.S., 2005. Protective effect of alpha-
and beta-amyrin, a triterpene mixture from Protium heptaphyllum (Aubl.)
March. trunk wood resin, against acetaminophen-induced liver injury in
mice. J. Ethnopharmacol. 98, 103–108.
Ono, K., Han, J., 2000. The p38 signal transduction pathway: activation and
function. Cell. Signal. 12, 1–13.
Otuki, M.F., Ferreira, J., Lima, F.V., Meyre-Silva, C., Malheiros, A., Muller,
L.A., Cani, G.S., Santos, A.R., Yunes, R.A., Calixto, J.B., 2005a.
Antinociceptive properties of mixture of alpha-amyrin and beta-amyrin
235
triterpenes: evidence for participation of protein kinase C and protein
kinase A pathways. J. Pharmacol. Exp. Ther. 313, 310–318.
Otuki, M.F., Vieira-Lima, F., Malheiros, A., Yunes, R.A., Calixto, J.B.,
2005b. Topical antiinflammatory effects of the ether extract from Protium
kleinii and alpha-amyrin pentacyclic triterpene. Eur. J. Pharmacol. 507,
253–259.
Pivarcsi, A., Homey, B., 2005. Chemokine networks in atopic dermatitis: traffic
signals of disease. Curr. Allergy Asthma Rep. 5, 284–290.
Recio, M.C., Giner, R.M., Manez, S., Rios, J.L., 1995. Structural requirements
for the antiinflammatory activity of natural triterpenoids. Planta Med. 61,
182–185.
Rossner, M., Yamada, K.M., 2004. What's in a picture? The temptation of image
manipulation. J. Cell Biol. 166, 11–15.
Schulze-Osthoff, K., Ferrari, D., Riehemann, K., Wesselborg, S., 1997.
Regulation of NF-κB activation by MAP kinase cascades. Immunobiology
198, 35–49.
Surh, Y.J., 2003. Cancer chemoprevention with dietary phytochemicals. Nat.
Rev., Cancer 3, 768–780.
Whitmarsh, A.J., Davis, R.J., 1996. Transcription factor AP-1 regulation by
mitogen-activated protein kinase signal transduction pathways. J. Mol. Med.
74, 589–607.
Winzen, R., Kracht, M., Ritter, B., Wilhelm, A., Chen, C.Y., Shyu, A.B., Muller,
M., Gaestel, M., Resch, K., Holtmann, H., 1999. The p38 MAP-kinase
pathway signals for cytokine-induced mRNA stabilization via MAP-kinase-
activated protein kinase 2 and an AU-rich region-targeted mechanism.
EMBO J. 18, 4969–4980.
Yang, S.H., Sharrocks, A.D., Whitmarsh, A.J., 2003. Transcriptional regulation
by the MAP-kinase signaling cascades. Gene 320, 3–21.