SINERGIC-ANTINFLAMATORIO-ESCINA-GLICOCORT-PHYTOMEDICINE-2010

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Please cite
in vitro. Ph
ARTICLE IN PRESSGModelPHYMED-50932; No.of Pages6
Phytomedicine xxx (2010) xxx–xxx
Contents lists available at ScienceDirect
Phytomedicine
journa l homepage: www.e lsev ier
Escin exerts synergistic anti-inﬂammatory effect
glucoco
Wenyu X yin
Zhen Lib
a Department o
b Shandong Luy
a r t i c l
Keywords:
Escin
Aesculus hippocastanum
Glucocorticoids
Anti-inﬂammatory effects
Synergism
id sap
ted to
present study was designed to investigate whether escin exhibits synergistic anti-inﬂammatory effects
when combined with glucocorticoids. The carrageenan-induced paw edema and pleuritis in bilaterally
adrenalectomized rats were used to investigate the anti-inﬂammatory effects of escin and glucocorticoid
alone or combined. The carrageenan-induced paw edema was inhibited only when escin and corticos-
terone (Cort) were administered together. Co-administration of escin with Cort signiﬁcantly reduced the
volume of exudates and the number of white blood cells of exudates in bilaterally adrenalectomized rats
with pleuritis, but treatment with escin or Cort alone at a suboptimal concentration did not show any
Introductio
Glucoco
inﬂammato
anti-inﬂam
corticoids r
elucidated b
their outsta
severe and
mizing the
their admin
speciﬁc an
at synergis
∗ Correspon
E-mail add
1 These auth
authors.
0944-7113/$ –
doi:10.1016/j.
this article in press as: Xin, W., et al., Escin exerts synergistic anti-inﬂammatory effects with low doses of glucocorticoids in vivo and
ytomedicine (2010), doi:10.1016/j.phymed.2010.08.013
effect on the pleuritis rats. After the murine macrophagic RAW264.7 cells stimulated by lipopolysaccha-
ride (LPS), they were treated with escin, Cort or escin and Cort. Then nitric oxide (NO), tumor necrosis
factor-� (TNF-�) and interleukin 1� (IL-1�) of cell culture supernatants were analyzed. Escin or Cort
markedly reduced the content ofNO, TNF-� and IL-1� secretedby LPS-stimulatedRAW264.7macrophage
cells. The combination of suboptimal concentrations of escin with Cort, which alone could not markedly
inhibit the release of inﬂammatory factors, inhibited the secretion of NO, TNF-� and IL-1� in LPS-
stimulated RAW264.7 macrophage cells. The ﬁndings suggest escin can synergize with glucocorticoids
to enhance their anti-inﬂammatory effect.
© 2010 Elsevier GmbH. All rights reserved.
n
rticoids are the most commonly used anti-
ry and immunosuppressive drugs. The
matory and immunosuppressive effects of gluco-
ely on several molecular mechanisms, which have been
y basic research (Rhen and Cidlowski 2005). However,
nding therapeutic effects are often accompanied by
irreversible side effects. One of the methods of mini-
undesirable side effects of corticotherapy is through
istration with other pharmaceuticals, especially more
ti-inﬂammatories or non-immunosupressors, aiming
tic effects in order to avoid the use of glucocorti-
ding author. Tel.: +86 535 6706060; fax: +86 535 6706066.
ress: fufenghua@sohu.com (F. Fu).
ors contributed equally to thiswork and shouldbe considered co-ﬁrst
coids or to reduce the dosage and duration of corticotherapy
(Longui 2007).
Escin is a natural mixture of triterpene saponins (Fig. 1)
from Aesculus chinensis Bge. Up to now, at least three types of
pharmacodynamic actions have been attributed to escin: anti-
edematous properties, anti-inﬂammatory activities, and venotonic
properties (Sirtori 2001). It was reported that escin posses signiﬁ-
cant anti-inﬂammatory properties, well exempliﬁed by a reduced
inﬂammatory granuloma, and also by the inhibitionof foot swelling
and pleurisy induced by carrageenan in rats (Guillaume and
Padioleau 1994). The results from our laboratory show that escin
is a potent anti-inﬂammatory drug with long anti-inﬂammatory
effects and without immunosuppression (Wang et al. 2009).
In this study, we investigated whether the combination of escin
and glucocorticoids would produce synergistic effects in the inhi-
bition of carrageenan-induced inﬂammation in adrenalectomized
rats. In vitro, we investigated the possible mechanisms of the syn-
ergistic anti-inﬂammatory effects of escin and glucocorticoids on
the LPS-stimulated murine macrophage cell line, RAW264.7.
see front matter © 2010 Elsevier GmbH. All rights reserved.
phymed.2010.08.013
rticoids in vivo and in vitro
ina,1, Leiming Zhanga,1, Fang Suna, Na Jianga, Hua
, Jie Heb, Fenghua Fua,∗
f Pharmacology, School of Pharmacy, Yantai University, Yantai 264005, PR China
e Pharmaceutical Company Limited, Shandong 264005, PR China
e i n f o a b s t r a c t
Escin, a natural mixture of triterpeno
hippocastanum), had been demonstra
.de /phymed
s with low doses of
g Fana, Tian Wanga,
onins isolated from the seed of the horse chestnut (Aesculus
possess anti-edematous and anti-inﬂammatory effects. The
Please cite
in vitro. Ph
ARTICLE IN PRESSGModelPHYMED-50932; No.of Pages6
2 W. Xin et al. / Phytomedicine xxx (2010) xxx–xxx
esci
Materials a
Animals and
Animal
accordance
on the use
Sprague–Da
Experiment
the rats we
allowed fre
The esc
obtained as
by Luye Ph
O55:B5) an
Paw edema
Both adr
rats weighi
hydrate ane
ported with
3d. On the
ﬁve groups:
ment, escin
(2mg/kg) a
was induce
saline in th
given intrav
allow imme
ber. The vo
injection an
mometer, s
2004). Data
of swelling
ume×100%
y of a
adr
: th
escin
kg) a
of in
eena
n th
ibs a
arrag
Fig. 1. The structure of sodium
nd methods
agents
experimental procedures were carried out in strict
with the National Institutes of Health regulations
and care of animals for scientiﬁc purposes. Male
wley rats weighing 250–280g were obtained from the
al Animal Center of Luye Pharmaceutical Company. All
re housed in diurnal lighting conditions (12h/12h) and
e access to food and water.
Pleuris
The
groups
ment,
(2mg/
lation
carrag
given o
third r
after c
this article in press as: Xin, W., et al., Escin exerts synergistic anti-inﬂamma
ytomedicine (2010), doi:10.1016/j.phymed.2010.08.013
in used in the experiments was sodium aescinate
a lyophilized powder in a 5mg vial manufactured
armaceutical Company Limited. LPS (Escherichia coli
d corticosterone were obtained from Sigma Chemicals.
in the adrenalectomized rats
enal glands were removed frommale Sprague–Dawley
ng 250–280g through a dorsal incision under chloral
sthesia. After surgery, adrenalectomized ratswere sup-
1% NaCl solution instead of water and pellet food for
third day, the animals were divided randomly into
the vehicle control, sodium diclofenac (5mg/kg) treat-
(2mg/kg) treatment, Cort (1mg/kg) treatment, escin
nd Cort (1mg/kg) combined treatment. Paw edema
d by injection of 100�l of 0.5% carrageenan diluted in
e left hind foot pad, and the corresponding drugs were
enously 3h after carrageenan. The paw was marked to
rsion to the same extent in the measurement cham-
lume was measured immediately before carrageenan
d 3.5, 4, 5, 7, and 9h thereafter by using a hydroplethis-
pecially modiﬁed for small volumes (Posadas et al.
were expressed as the degree of swelling (degree
= (volume after edema−normal volume)/normal vol-
).
given intrav
administrat
the rib cag
the third an
wall was op
ration with
determined
exudates an
to determin
vital trypan
Cell culture
The mou
chased from
USA). The c
inactivated
streptomyc
humid atm
(5×104 cel
for 24h un
centrations
the plates w
tory factors
followed by
n.
drenalectomized rats
enalectomized rats were divided randomly into ﬁve
e vehicle control, dexamethasone (4mg/kg) treat-
(2mg/kg) treatment, Cort (1mg/kg) treatment, escin
nd Cort (1mg/kg) combined treatment. The accumu-
ﬂammatory exudate was induced by injection of 1%
n (2ml/kg) into the pleural cavity. The injections were
e right side of themediastinumbetween the second and
s previously described (Vinegar et al. 1973). Two hours
eenan administration, the corresponding drugs were
tory effects with low doses of glucocorticoids in vivo and
enously. The rats were sacriﬁced 6h after the last drug
ion, and the skin and pectoral muscle retracted to leave
e exposed. A longitudinal incision was made between
d ﬁfth ribs on each side of the mediastinum. The chest
ened and the inﬂammatory exudate collected by aspi-
a Pasteur pipette, and the total volume of exudate was
. The cavity was then rinsed with 2ml of saline and the
dwashingﬂuidswerepooled.A100�l aliquotwasused
e the total leukocyte count in a Burker’s chamber after
blue staining.
se monocyte-macrophage cell line Raw264.7 was pur-
the American Type Culture Collection (Rockville, MD,
ells were cultured in RPMI 1640 containing 10% heat-
fetal calf serum, 100U/ml penicillin and 100�g/ml
in. Cells were kept at 37 ◦C with 5% CO2 in a fully
osphere. For the measurement of cell viability, 100�l
l/ml) cells were seeded in a 96-well plate and incubated
der the above conditions. Subsequently, different con-
of escin or Cort were added to the respective wells and
ere incubated for a further 48h. Tomeasure inﬂamma-
, 200�l of cells (1×106) were seeded in 48-well plates,
the addition of 1�g/ml LPS and prepared escin and
Please cite mmatory effects with low doses of glucocorticoids in vivo and
in vitro. Ph
ARTICLE IN PRESSGModelPHYMED-50932; No.of Pages6
W. Xin et al. / Phytomedicine xxx (2010) xxx–xxx 3
Cort solution 2h later. Plates were then incubated for another 24h
until the respective measurement were taken.
SRB assay for measuring cell proliferation
Cell viab
Kirtikara 20
was added
were then i
by ﬂicking
(100�l, 0.4
were incuba
and repeate
to dry at roo
10mM) wa
shaken for 3
Analysis of N
The conc
medium co
using the G
with an equ
naphthylen
at 540nm w
tions of nitr
obtained fro
Measureme
The inhi
� and IL-1�
by sandwic
at the end
ELISA kits fr
(Wuhan, Ch
respectively
instruction.
Evaluation o
The inte
Berenbaum
synergistic,
E(da,db) >E
the doses o
effect of a co
effects (Ber
Statistical a
All data
was evaluat
ple t-tests. S
p<0.05.
Results
Effects of esc
The paw
after the ca
mation. In
for evaluati
because the
sodium dicl
Table 1
Effects of escin and Cort on carrageenan-induced paw edema in bilaterally
adrenalectomized rats (degree of swelling).
Group Degree of swelling (%) Inhibition rate (%)
l 15.93 ± 0.17 –
enac 2.95 ± 0.08* 81.48
17.19 ± 0.07 −7.91
15.82 ± 0.70 0.69
Cort 8.67 ± 0.54* 45.57a
me was measured 6h after the corresponding drug administration. Results
essed as means± SEM (n=10).
.05, compared with the control group.
,db) >E(da) +E(db).
f escin and Cort on the volume of exudate in bilaterally adrenalectomized
pleuritis.
l
Cort
me of
ults a
.05, co
,db) >
l gro
inh
they
ion i
fect
istrat
ede
of es
inje
renal
teriz
umbers of leukocytes (Table 3). When escin or Cort was
istered alone during the inﬂammation period, 2h after the
on of carrageenan, there was a slight reduction in exudate
e and the number of leukocytes 6h after its administration,
gh this was not signiﬁcant. As observed with carrageenan-
d paw edema, when escin and Cort were administered
er they signiﬁcantly inhibited the inﬂammatory response
d by intrapleural injection of carrageenan (p<0.05), as
strated by the signiﬁcant attenuation of exudate formation
2) and leukocyte inﬁltration (Table 3). This effect of escin
edwith Cort in preventing pleurisy induced by carrageenan
f escin and Cort on the number of leukocytes in exudates of bilaterally
ctomized rats with pleuritis.
Leukocytes (×109 l−1) Inhibition rate (%)
l 18.66 ± 3.98 –
9.32 ± 3.74** 50.03
18.15 ± 1.42 2.71
15.66 ± 2.87 16.03
Cort 9.06 ± 3.73** 51.45a
ber of leukocytes was counted in a Burker’s chamber 6h after the corre-
g drug administration. Results are expressed as means± SEM (n=10).
.01, compared with the control group.
,db) >E(da) +E(db).
this article in press as: Xin, W., et al., Escin exerts synergistic anti-inﬂa
ytomedicine (2010), doi:10.1016/j.phymed.2010.08.013
ility was evaluated using the SRB protocol (Vichai and
06). Fifty microliters of cold trichloroacetic acid (50%)
to each well at the end of the treatment. The plates
ncubated at 4 ◦C for 1h. The supernatant was removed
the plates and repeatedly rinsed with water. SRB dye
% in 1% acetic acid) was then added to the plates, which
ted at 37 ◦C. The SRBwas removedbyﬂicking theplates
dly washing with acetic acid. The plates were allowed
m temperature before Tris–HCl buffer (pH 10.5, 150�l,
s added to each well and the plates were covered and
0min. The absorbance was then measured at 490nm.
O
entration of nitrite in the supernatant of the cell culture
rresponds to the production of NO and was measured
reiss reagent. Brieﬂy, 100�l of medium was mixed
al volume of Griess reagent (1% sulfanilamide and 0.1%
ediamine in 5% phosphoric acid). Absorbance readings
ere recorded using an ELISA plate reader. Concentra-
ite were calculated in accordance to the standard curve
m sodium nitrite.
nt of TNF-˛ and IL-1ˇ
bitory effects of escin andCort on theproductionof TNF-
from LPS-stimulated RAW264.7 cells were determined
h ELISA. The cell culture supernatant was harvested
of treatment and used for the TNF-� and IL-1� assay.
om Bender Medsystem (Vienna, Austria) and Uscnilife
ina)wereused todetermine the TNF-� and IL-1� levels,
, in the supernatants according to the manufacturer’s
f drug interactions
raction between escin and Cort was analyzed using the
’s method to determine whether the combination was
which is performed based on the following equation:
(da) +E(db), where E is the observed effect, da and db are
f agents a and b. Synergism is indicated when the total
mbination is greater than expected from the sum of its
enbaum 1989).
nalysis
were expressed asmeans± SEM. Statistical signiﬁcance
ed using one-way ANOVA with post hoc test for multi-
tatistical signiﬁcance was considered signiﬁcant when
in and Cort on paw edema in adrenalectomized rats
volume of the rats increased approximately 15.93%
rrageenan injection, indicating the induction of inﬂam-
addition, this model was conﬁrmed to be effective
ng acute inﬂammation and anti-inﬂammatory effects
edema in the positive control group administeredwith
ofenac was signiﬁcantly reduced in comparison to the
Contro
Diclof
Escin
Cort
Escin +
The volu
are expr
* p<0
a E(da
Table 2
Effects o
rats with
Group
Contro
Dex
Escin
Cort
Escin +
The volu
tion. Res
* p<0
a E(da
contro
was no
when
reduct
This ef
admin
ing the
Effects
The
ally ad
charac
large n
admin
injecti
volum
althou
induce
togeth
induce
demon
(Table
combin
Table 3
Effects o
adrenale
Group
Contro
Dex
Escin
Cort
Escin +
The num
spondin
** p<0
a E(da
Exudate volume (ml) Inhibition rate (%)
2.33 ± 0.97 –
1.23 ± 0.52* 47.35
1.97 ± 0.57 15.51
2.30 ± 0.70 1.43
1.10 ± 0.54* 52.86a
exudate was measured 6h after the corresponding drug administra-
re expressed as means± SEM (n=10).
mpared with the control group.
E(da) +E(db).
up. When escin or Cort was administered alone there
ibition of the paw edema induced by carrageenan, but
were administered together, there was a signiﬁcant
n the edema 6h after administration (52.86%, p<0.05).
was also shown to be synergistic, demonstrating that
ion of escin combinedwith Cort was effective at reduc-
ma induced by carrageenan (Table 1).
cin and Cort on exudate volume in pleuritis
ction of carrageenan into the pleural cavity of bilater-
ectomized rats elicited an acute inﬂammatory response
ed by the accumulation of ﬂuid (Table 2) that contained
Please cite
in vitro. Ph
ARTICLE IN PRESSGModelPHYMED-50932;
No.of Pages6
4 W. Xin et al. / Phytomedicine xxx (2010) xxx–xxx
Fig. 2. Escin
Dose-depende
dependent inh
means± SEM
trol group, *p<
was also sy
The effects o
secretion in
For an in
typemacro
line has pre
anti-inﬂam
2004). To st
inﬂammato
stimulated
results sho
of NO, TNF-
cells in a d
test, cell via
than 95%w
This indicat
macrophag
factor relea
Stimulat
increase in
compared
10mg/l) or
dependent
by escin wa
cantly inhib
Extensiv
cytokines li
matory cell
(Cheung et
vention for
and IL-1� (
Furthermor
the anti-inﬂ
scin and Cort dose-dependently inhibit LPS-induced TNF-� release. (A) The
f escin on LPS-induced TNF-� secretion in macrophages. (B) The effects
n LPS-induced TNF-� secretion in macrophages. Values are means± SEM
independent experiments. ††p<0.01 compared with the control group,
compared with the LPS alone treated group.
ects of escin combined with Cort on LPS-induced
atory factor secretion in macrophages
combination of doses of escin with Cort, which alone could
arkedly inhibit secretion of inﬂammatory factors, greatly
ed th
64.7
mg/l
on o
n an
and Cort dose-dependently inhibit LPS-induced NO release. (A)
nt inhibition of LPS-induced nitric oxide secretion by escin. (B) Dose-
ibition of LPS-induced nitric oxide secretion by Cort. Values are
of three independent experiments. ††p<0.01 compared with the con-
0.05, **p<0.01 compared with the LPS alone treated group.
nergistic.
f escin or Cort on LPS-induced inﬂammatory factor
macrophages
vitro model of macrophage activation, we used wild
phage-likeRAW264.7cells stimulatedwithLPS. This cell
viously been used to characterize the action of various
matory components at the molecular level (Wu et al.
Fig. 3. E
effects o
of Cort o
of three
*p<0.05
The eff
inﬂamm
The
not m
inhibit
RAW2
(0.001
secreti
of esci
this article in press as: Xin, W., et al., Escin exerts synergistic anti-inﬂamma
ytomedicine (2010), doi:10.1016/j.phymed.2010.08.013
udy whether escin or Cort can effect as an inhibitor of
ry factor release in this system, RAW264.7 cells were
with 1�g/ml LPS with or without escin or Cort. Our
w that escin or Cort markedly inhibited the secretion
� and IL-1� in LPS-stimulated RAW264.7 macrophage
ose-dependent manner (Figs. 2–4). Based on the SRB
bility after 24h of escin or Cort treatment was greater
hen comparedwith the control group (data not shown).
es that the level of escin or Cort used was not toxic to
es. The results imply that the inhibition of inﬂammatory
se by escin or Cort was not due to cell death.
ion of cells with 1�g/ml LPS for 24h induced a 1.8-fold
nitrite production compared to the basal level (13.1�M
to 23.9�M). In these experiments, escin (0.1, 1 and
Cort (0.001, 0.01 and0.03mg/l) evoked a concentration-
inhibitionof nitrite release (Fig. 2). Signiﬁcant inhibition
s observed at 10mg/l (p<0.05), and Cort could signiﬁ-
it NO secretion at 0.03mg/l (p<0.01).
e studies have been conducted and concluded that
ke TNF-� and IL-1� are the major products of inﬂam-
s and contribute to the progression of inﬂammation
al. 2009). Following LPS stimulation, escin or Cort inter-
24h signiﬁcantly reduced the secretion of TNF-� (Fig. 3)
Fig. 4) compared to a single LPS stimulation (p<0.05).
e, there was a dose-dependent relationship between
ammatory effects of escin and Cort.
Fig. 4. Escin a
effectsof escin
on LPS-induce
independent
**p<0.01 com
e secretion of NO, TNF-� and IL-1� in LPS-stimulated
macrophage cells (Table 4). Escin (0.1mg/l) or Cort
) alone slightly, but did not signiﬁcantly inhibit the
f nitrite oxide induced by LPS. However, combination
d Cort at these concentrations signiﬁcantly inhibited
tory effects with low doses of glucocorticoids in vivo and
nd Cort dose-dependently inhibit LPS-induced IL-1� release. (A) The
onLPS-induced IL-1� secretion inmacrophages. (B)TheeffectsofCort
d IL-1� secretion in macrophages. Values are means± SEM of three
experiments. ††p<0.01 compared with the control group, *p<0.05,
pared with the LPS alone treated group.
Please cite
in vitro. Ph
ARTICLE IN PRESSGModelPHYMED-50932; No.of Pages6
W. Xin et al. / Phytomedicine xxx (2010) xxx–xxx 5
Table 4
Effect of escin combined with Cort on LPS-induced nitric oxide synthesis in macrophages.
Group Concentration (mg/l) NO (�mol l−1) Inhibition rate (%)
LPS Escin Cort
Control – – – 13.1 ± 0.57 –
LPS 1.0 – – 23.9 ± 1.32†† –
Escin 1.0 0.1 – 22.2 ± 0.79 16.2
Cort 1.0 – 0.001 22.7 ± 3.65 11.0
Escin +Cort 2.0 0.1 0.001 20.5 ± 0.97* 31.8a
Results are expressed as means± SEM of three independent experiments.
†† p<0.01 compared with the control group.
* p<0.05 compared with the LPS alone treated group.
a E(da,db) >E(da) +E(db).
Table 5
Effect of escin combined with Cort on LPS-induced TNF-� synthesis in macrophages.
Group −1
Control
LPS
Escin
Cort
Escin +Cort
Results are exp
†† p<0.01 co
* p<0.05 co
a E(da,db) >E
nitric oxide
as a synergi
These re
which was
ited by 46.7
alone had li
as a synerg
can be com
Discussion
Drug co
choice for t
outcomes f
the therape
maintaining
slowing do
viding selec
versus host
The pre
with glucoc
tively. The
glucocortico
eena
arra
lecto
rCor
e of e
entw
t inh
This
num
ally a
ivatio
Table 6
Effect of escin
Group
Control
LPS
Escin
Cort
Escin +Cort
Results are exp
†† p<0.01 co
* p<0.05 co
a E(da,db) >E
Concentration (mg/l)
LPS Escin Cort
– – –
1.0 – –
1.0 0.1 –
1.0 – 0.001
2.0 0.1 0.001
ressed as means± SEM of three independent experiments.
mpared with the control group.
mpared with the LPS alone treated group.
(da) +E(db).
secretion (31.8%; p<0.05). This was again conﬁrmed
stic effect of escin and Cort.
sults were also conﬁrmed for the secretion of TNF-�,
inhibited by 24% (Table 5), and IL-1�, which was inhib-
% (Table 6). Thiswas remarkable as the individual drugs
ttle effect on IL-1� secretion. This was again conﬁrmed
istic effect of escin and Cort, demonstrating that they
bined to inhibit inﬂammation
carrag
using c
adrena
dose)o
volum
treatm
niﬁcan
found.
in the
bilater
Act
this article in press as: Xin, W., et al., Escin exerts synergistic anti-inﬂamma
ytomedicine (2010), doi:10.1016/j.phymed.2010.08.013
mbination has been widely used and has become the
reating inﬂammation. There is a long list of favorable
or synergism, including (1) increasing the efﬁcacy of
utic effect, (2) decreasing the dosage, but increasing or
the same efﬁcacy to avoid toxicity, (3) minimizing or
wn the development of drug resistance, and (4) pro-
tive synergism against target (or efﬁcacy synergism)
(or toxicity antagonism) (Chou 2006).
sent study is to evaluate if the combination of escin
orticoid (low dose) can treat inﬂammation more effec-
results show that the co-administration of escin with
id synergistically inhibited inﬂammation induced by
propagation
or injury, m
1, IL-6, IL-1
component
into the cir
2002). LPS
inﬂammato
increasing s
the level o
1995). TNF-
T and B cel
cal role in b
combined with Cort on LPS-induced IL-1� synthesis in macrophages.
Concentration (mg/l)
LPS Escin Cort
– – –
1.0 – –
1.0 0.1 –
1.0 – 0.001
2.0 0.1 0.001
ressed as means± SEM of three independent experiments.
mpared with the control group.
mpared with the LPS alone treated group.
(da) +E(db).
TNF-� (ngml ) Inhibition rate (%)
0.89 ± 0.09 –
11.70 ± 1.45†† –
11.66 ± 0.92 0.36
11.97 ± 1.40 −2.46
9.06 ± 0.93* 24.43a
n. In vivo, the anti-inﬂammatory effects were assayed
geenan-induced paw edema and pleuritis in bilaterally
mized rats. It shows that treatment with escin (low
t (lowdose) alonedidnot inhibit thepawedemaand the
xudate in bilaterally adrenalectomized rats, but when
ith escin (lowdose) and Cort (lowdose) together, a sig-
ibition of paw edema and the volume of exudate was
synergistic effect
was also observed by the reduction
ber of white blood cells in the exudates of pleuritis in
drenalectomized rats.
n of macrophages is a major event in the initiation and
tory effects with low doses of glucocorticoids in vivo and
of inﬂammation. When stimulated by microorganism
acrophages release NO, prostaglandin-E2, TNF-�, IL-
2, and other proinﬂammatory cytokines. LPS, a major
of the cell wall of Gram-negative bacteria, is released
culation following bacterial lysis (Raetz and Whitﬁeld
is widely used to study the mechanism of the anti-
ry effect. NO synthesis is greatly ampliﬁed by LPS and
tudies demonstrated that inﬂammation correlateswith
f NO (Kuo and Schroeder 1995; Miller and Grisham
� is produced mainly by monocyte/macrophage cells,
ls. TNF-� is a pleiotropic cytokine which plays a criti-
oth acute and chronic inﬂammation. When the effect
IL-1� (pgml−1) Inhibition rate (%)
0 –
50.5 ± 14.80†† –
40.3 ± 11.81 20.1
49.5 ± 22.22 2.0
26.8 ± 1.76* 46.9a
Please cite mma
in vitro. Ph
ARTICLE IN PRESSGModelPHYMED-50932; No.of Pages6
6 W. Xin et al. / Phytomedicine xxx (2010) xxx–xxx
of TNF-� is speciﬁcally blocked, the severity of inﬂammation is
reduced (Weaver 2004). IL-1� is another potent proinﬂammatory
cytokine. It mediates a wide range of reactions involved in the
acute-phase response in inﬂammation. Minimal levels of IL-1�
evoke fever, hypotension, release of adrenocorticotropic hormone
and the production of cytokines such as IL-6. These, in turn, will
induce the synthesis of hepatic acute-phase proteins such as serum
amyloid A and C-reactive protein and stimulate the synthesis of
adhesion molecules in endothelial cells and leucocytes, leading
to leukocytosis and thrombocytosis (Ferrero-Miliani et al. 2007).
Although the release of inﬂammatory mediators is essential for
host survival from infection and is also required for the repair of
tissue injury, these beneﬁcial effects are critically dependent on
the magnitude of the immune response because large amounts of
macrophage-derived inﬂammatory mediators can also cause col-
lateral damage to normal cells and are potentially lethal shock
when the release is sufﬁcient to cause systemic exposure (Wu et al.
2004).
In our study, escin or Cort dose-dependently reduced the
content of NO, TNF-� and IL-1� in LPS-induced RAW264.7
macrophages. Based on the previous ﬁndings, we select the sub-
optimal doses which did not inhibit the levels of NO, TNF-� and
IL-1� in LPS-induced RAW264.7 macrophages treated by escin or
Cort alone. The following results showed that the combination of
suboptimal dose of escin with suboptimal dose of Cort markedly
inhibited the synthesis of NO, TNF-� and IL-1� in LPS-stimulated
RAW264.7 macrophages.
Our results demonstrate that escin and glucocorticoid have
synergistic anti-inﬂammatory effect. In addition, the molecular
mechanisms on synergistic anti-inﬂammatory effect of escin and
glucocortic
and IL-1�. T
dose of gluc
coid sugges
studied furt
Conﬂict of
The auth
Disclosur
Acknowledgements
This study was supported by the National Natural Science
Foundation of China (Grant 30772760); the 11th Five Years Key
Programs for Science and TechnologyDevelopment of China (Grant
2008ZX09202-008); and Shandong Province Natural Science Foun-
dation (Grant Y2008C51).
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Escin exerts synergistic anti-inflammatory effects with low doses of glucocorticoids in vivo and in vitro
Introduction
Materials and methods
Animals and agents
Paw edema in the adrenalectomized rats
Pleurisy of adrenalectomized rats
Cell culture
SRB assay for measuring cell proliferation
Analysis of NO
Measurement of TNF-α and IL-1β
Evaluation of drug interactions
Statistical analysis
Results
Effects of escin and Cort on paw edema in adrenalectomized rats
Effects of escin and Cort on exudate volume in pleuritis
The effects of escin or Cort on LPS-induced inflammatory factor secretion in macrophages
The effects of escin combined with Cort on LPS-induced inflammatory factor secretion in macrophages
Discussion
Conflict of interest
Acknowledgements
References