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183
Korean J Physiol Pharmacol
Vol 19: 183
－
189, March, 2015
http://dx.doi.org/10.4196/kjpp.2015.19.2.183
pISSN 1226-4512
eISSN 2093-3827
ABBRE VI ATI ON S: fennel, Foeniculum vulgare Mill.; LPS, lipopoly-
saccharide; BALF, bronchoalveolar lavage fluid; NO, nitric oxide;
ALI, acute lung injury; ROS, reactive oxygen species; MDA, malon-
dialdehyde; DEX, dexamethasone; LDH, lactate dehydrogenase;
H&E, hematoxylin and eosin; ECL, enhanced chemiluminescence;
ELISA, enzyme-linked immunosorbent assay.
Received December 31, 2014, Revised January 7, 2015,
Accepted January 7, 2015
Corresponding to: Geun Hee Seol, Department of Basic Nursing
Science, School of Nursing, Korea University, 145 Anam-ro, Seong-
buk-gu, Seoul 136-701, Korea. (Tel) 82-2-3290-4933, (Fax) 82-2-927-
4676, (E-mail) ghseol@korea.ac.kr
*The two authors equally contributed to this paper.
This is an Open Access article distributed under the terms of the
Creat ive Co mmons Attribut ion Non-Commerc ial Licens e (http://
creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial
use, distribution, and reproduction in any medium, provided the original work
is properly cited.
Foeniculum vulgare Mill. Protects against Lipopolysaccharide-induced
Acute Lung Injury in Mice through ERK-dependent NF-kB Activation
Hui Su Lee*, Purum Kang*, Ka Young Kim, and Geun Hee Seol
Department of Basic Nursing Science, School of Nursing, Korea University, Seoul 136-701, Korea
Foeniculum vulgare Mill. (fennel) is used to flavor food, in cosmetics, as an antioxidant, and to treat
microbial, diabetic and common inflammation. No study to date, however, has assessed the anti-inflam-
matory effects of fennel in experimental m odels of inflamm ation. The aim s of this study were to
investigate the anti- inflam matory effects of fennel in m odel of lipopolysaccharide (LPS )-induced acute
lung injury. Mice were randomly assigned to seven groups (n=7∼10). In five groups, the mice were
intraperitoneally injected with 1% Tween 80-saline (vehicle), fennel (125, 250, 500μl/kg), or dexame-
thas one (1 m g/ kg ), followed 1 h l ater by intrat ra cheal ins till ation of L PS (1 . 5 m g/ kg ). In two grou ps ,
the mice were intraperitoneally injected with vehicle or fennel (250 μl/kg), followed 1 h later by intra-
tracheal instillation of sterile saline. M ice were sacrificed 4 h later, and bronchoalveolar lavage fluid
(BA LF) and lung tis sues were obtained. Fennel significantly and dose-dependently reduced LDH
activity and immune cell numbers in LPS treated m ice. In addition fennel effectively suppressed the
LPS-induced increases in the production of the inflamm atory cytokines interleukin-6 and tum or
necrosis fact or- a lpha , wit h 50 0 μl/kg fennel showing m axim al reduction. Fennel also significantly and
dose-dependently reduced the activity of the proinflamm atory mediator matrix metalloproteinase 9 and
the imm une m odulator nitric oxide (NO). Assessm ents of the involvement of the MA PK signaling
pathway showed that fennel significantly decreased the LPS-induced phosphorylation of ERK. Fennel
effectively blocked the inflam matory processes induced by LPS, by regulating pro-inflammatory cytokine
produ ction, tran script ion fact ors , and N O .
Key Words: ERK, Foeniculum vulgare Mill., LPS, TNF-α
INTRODUCTION
Acute lung injury (ALI), characterized by unbalanced in-
flammatory responses, is a leading cause of acute respira-
tory failure and multiple organ dysfunctions [1,2]. ALI is
associated with neutrophilic inflammation, which can be ac-
celerated by endotoxins such as lipopolysaccharide (LPS)
from Gram-negative bacteria [3]. Experimental models of
LPS-induced ALI have therefore been used to explore in-
flammatory responses in the lung. LPS-induced ALI has
been associated with the production of reactive oxygen spe-
cies (ROS) in alveolar macrophages and to involve NF-κB
signaling pathways, including the MAPK/JNK/p38/ERK
pathways [4].
Foeniculum vulgare Mill. (fennel) is used to flavor foods,
in cosmetics, and to treat microbial, diabetic and common
inflammation. Evaluation of fennel seed extracts using a
DPPH radical scavenging assay also indicated that fennel
may have antioxidant activity effect [5]. Oral administra-
tion of a methanolic extract of F. vulgare fruit decreased
malondialdehyde (MDA) level, suggesting that this extract
has inflammation-relieving effects in experimental animals
[6]. Moreover, fennel decreased ROS and MDA in mouse
tumor tissue [7]. Trans-anethole, the major component of
fennel, was found to reduce paw edema and inflammatory
pain [8], with the anti-inflammatory effects of trans-anet-
hole reported to derive from its regulation of NF-κB signal-
ing pathways [9].
Because fennel contains several components, which can
affect each other, there is a need to confirm that this essen-
tial oil shows consistent effects. To date, however, no study
has analyzed the anti-inflammatory effects of fennel in a
mouse model of LPS-induced ALI. This study therefore ex-
plored the anti-inflammatory effects of fennel in LPS-in-
184 HS Lee, et al
duced ALI in mice, and investigated the signaling pathways
involved.
METHODS
Animals and M aterials
Male BALB/C mice, aged five weeks and weighing 19 to
21 g, were obtained from Orient Bio (Sungnam, Korea) and
acclimatized to standard laboratory conditions for 3 to 5
days. All experimental procedures were conducted in ac-
cordance with guidelines relevant to the care of ex-
perimental animals, as approved by the Animal Research
Committee of Korea University (approval no. KUIACUC-
2012-181), informed by the Guide for the Care and Use of
Laboratory Animals published by the US National
Institutes of Health (NIH publication No. 85-23; revised
1996). Mice were randomly assigned to seven groups (n=7∼
10) and were anesthetized by intraperitoneal injection of
a mixture of 0.3 mg/kg tiletamine-zolazepam (Zoletil 50,
Virbac Laboratories, Carros, France) and 0.2 mg/kg xyla-
zine (Rompun, Bayer Korea, Ansan, Korea). In five groups,
the mice were intraperitoneally injected with 1% Tween
80-saline (vehicle), fennel (125, 250, 500μl/kg), or dex-
amethasone (DEX) (1 mg/kg), followed 1 h later by intra-
tracheal instillation of LPS (1.5 mg/kg). In the remaining
two groups, the mice were intraperitoneally injected with
1% Tween 80-saline (vehicle) or fennel (250μl/kg), followed
1 h later by intratracheal instillation of sterile saline. The
dose of fennel was based on a previous study of trans-anet-
hole, the main component of fennel [9]. The mice were sacri-
ficed 4 h later, and their bronchoalveolar lavage fluid
(BALF) and lung tissues were obtained. Lipopolysaccharide
(LPS, from E. coli 0.55:B5), Tween 80 and DEX were ob-
tained from Sigma-Aldrich (St. Louis, MO, USA). Pure fen-
nel essential oil was purchased from Aromarant Co. Ltd.,
Rottingen, Germany and came from locally cultivated
plants. The fennel essential oil that we used (batch No.
091119; Aromarant Co. Ltd) was analyzed by gas chroma-
tography/mass spectrometry (GC/MS). The main compo-
nents of fennel essential oil detected by GC/MS analysis
were 75.81% trans-anethole, 5.93% fenchonem, 5.82% limo-
nene, 4.30% methyl chavicol, 3.52% α-pinene and 0.39%
α-phellandrene.
Lactate dehydrogenase (LDH) assay
The activity of LDH, an enzyme used as a marker for
cytotoxicity, was measured using a commercial LDH assay,
according to the manufacturer’s instructions (Takara Bio
Inc., Otsu, Japan). BALF samples were mixed 1：1 with
freshly prepared reaction mixture and incubated in the
dark for 30 min at room temperature. Absorbance was
measured at 490 nm and at a reference wavelength of 620
nm using a microplate reader (BMG Labtech, Ortenberg,
Germany).
Cell counting
BALF samples were centrifuged at 500×g for 10 min at
4oC, and the sedimented cells were resuspended in PBS.
The cells were stained with Diff-Quick (International
Reagents Co., Kobe, Japan), and total and differential leu-
kocyte counts were determined using a Countess automated
cell counter (Invitrogen Life Technologies, Carlsbad, CA,
USA). Results are expressed as the number of each cell type
per milliliter of BALF.
Histopathology
Lung tissues were fixed in 10% paraformaldehyde, em-
bedded in paraffin, and cut into 4μm thick sections. The
sections were stained with hematoxylin and eosin (H&E),
and viewed under a light microscope (200×).
Enzyme-linked immunosorbent assay (ELIS A)
The concentrations of the inflammatory cytokines IL-6 and
TNF-α in BALF were measured using commercially avail-
able ELISA kits, in accordance to the manufacturer’s in-
structions (PeproTech, London, UK).
M easurem ent of nitric oxide (N O)
Since NO has a short half-life, we measured nitrite level,
an indirect measure of NO production [10]. The amount of
nitrite in BALF was measured using the Griess reaction.
Griess reagent included 0.1% N-(1-naphthyl) ethylenedi-
amine dihydrochloride and 1% sulfanilamide. Briefly, Griess
reagent was added to 100μl of BALF supernatant, and the
solutions were mixed and incubated for 10 min at room
temperature. Optical density at 540 nm was measured in
a microplate reader (BMG Labtech, Ortenberg, Germany).
Zy mographic analysis
The secretion of matrix metalloproteinase-9 (MMP-9)
protein was measured by gelatin zymography. A volume of
BALF sample was mixed with an equal volume of non-
reducing sample buffer, and the samples were electro-
phoresed in 8% sodium dodecyl sulfate polyacrylamide elec-
trophoresis gels (SDS-PAGE) containing 1 mg/ml gelatin.
The gels were washed with 2.5% Triton X-100 for 2 h and
subsequently incubated for 20 h at 37oC in 50 mM Tris-Cl
buffer (pH 7.4) containing 10 mM CaCl2 and 0.02% NaN3.
The gels were subsequently stained for 1 h with 0.5%
Coomassie Brilliant Blue G250 in 7.5% acetic acid/10%
propanol-2 and destained to visualize the protein bands.
Relative densities of MMP-9 were analyzed with Bio-Rad
Quantity One software (Bio-Rad, Hercules, CA, USA).
Extraction of lung nuclear proteins
Lung tissues obtained at sacrifice were immediately fro-
zen in liquid nitrogen, and 50 mg samples of frozen lung
tissue were subsequently homogenized with a Precellys 24
bead-based tissue homogenizer in 0.5 ml ice-cold buffer A
(10 mM HEPES with pH 7.9, 1.5 mM MgCl2, 10 mM KCl,
0.1 mM Na2EDTA, 0.5 mM DTT, 1% Nonidet P-40, 0.5 mM
PMSF, 0.5μg/ml leupeptin, 125μg/ml aprotinin, 25μg/ml
pepstatin A). Cell debris was removed by centrifugation at
2,000 rpm for 30 sec; the supernatants were incubated on
ice for 5 min and again centrifuged for 10 min at 5,000
rpm. Cytoplasmic proteins in the supernatant were col-
lected and the pellet was resuspended in 50μl of cold buffer
B (20 mM HEPES with pH 7.9, 1.5 mM MgCl2, 0.42 M
NaCl, 20% glycerol, 0.5 mM DTT, 0.5 mM PMSF, 0.5μg/ml
leupeptin, 125μg/ml aprotinin, 25μg/ml pepstatin A) and
incubated on ice for 30 min. The nuclear fraction was col-
Anti-inflammatory Effects of Foeniculum vulgare Mill. 185
Fig. 1. Effect of fennel on lactate dehydrogenase (LDH) activity in
BALF of LPS-treated mice. Mice were intratracheally administered
LPS (1.5 mg/kg) 1 h after intraperitoneal injection of 1% Tween
80-saline (vehicle), fennel (125, 250, 500μl/kg), or DEX (1 mg/kg).
The activity of LDH in BALF was measured to evaluate cell
damage. Data are reported as mean±S.E.M. (n=7∼10 per group).
#
##p＜0.001 compared with vehicle group; **p＜0.01, ***p＜0.001
compared with the vehicle+LPS group.
Fig. 2. Effects of fennel on cell numbers in BALF of LPS-treated
mice. The numbers of total cells, neutrophils, macrophages, and
lymphocytes in BALF were analyzed. Data are reported as mean±
S.E.M. (n=7∼10 per group). #p＜0.05, ##p＜0.01, ###p＜0.001
compared with the vehicle group; *p＜0.05, **p＜0.01, ***p＜0.001
compared with the vehicle+LPS group.
lected by centrifugation at 12,000 rpm for 2 min.
Western blot analysis
Lung tissue homogenate samples were separated on 10%
SDS-PAGE. The proteins were electrophoretically trans-
ferred onto nitrocellulose membranes, which were blocked
for 30 min at room temperature. The membranes were in-
cubated overnight at 4oC with primary antibodies to NF-κB
p65, Lamin B, IκB-α, GAPDH, p-ERK, ERK, p-p38, p38,
p-JNK, and JNK, followed by incubation with horseradish
peroxidase-conjugated secondary antibody for 1 h at room
temperature. Bands were visualized by enhanced chemilu-
minescence (ECL) reagents according to the manufacturer’s
protocol. Relative densities were analyzed using Bio-Rad
Quantity One software (Bio-Rad, Hercules, CA, USA).
Statistical analysis
All data were expressed as mean±S.E.M. and compared
using one-way analysis of variance, followed by the Tukey
HSD post hoc test. All statistical analyses were performed
using SPSS 20 software, with results considered statisti-
cally significant at p＜0.05.
RESULTS
Effect of fennel on LDH activity in BALF of mice with
LPS-induced ALI
The activity of LDH was significantly higher in BALF
of mice with LPS-induced ALI than in mice treated with
vehicle alone (63.02±27.28 U/L vs. 19.90±8.13 U/L, p＜0.001)
(Fig. 1), whereas pretreatment with DEX, which has been
shown to protect against LPS-induced ALI, significantly re-
duced LDH activity (25.66±4.35, p=0.004). Mice pretreated
with 125 (16.24±4.43, p＜0.001), 250 (11.07±2.21, p＜0.001),
and 500 (9.57±1.05, p＜0.001)μl/kg fennel, followed by
LPS, showed significantly decreased LDH activity com-
pared with mice treated with vehicle plus LPS. LDH level
was similar in mice treated with fennel (250μl/kg) and ve-
hicle without LPS.
Effect of fennel on inflamm atory cell count in BALF
Recruitment of excess numbers of inflammatory cells is
necessary for the pathogenesis of ALI. Compared with ve-
hicle alone, treatment with vehicle+LPS significantly in-
creased the numbers of total cells (8.84×105 cells/ml, p＜
0.001), neutrophils (3.67×105 cells/ml, p＜0.001), macro-
phages (2.84×105 cells/ml, p＜0.001), and lymphocytes
(2.44×105 cells/ml, p=0.005) in BALF (Fig. 2). In LPS-treat-
ed mice, pretreatment with fennel 125μl/kg (total cells,
6.49×105 cells/ml, p=0.204; neutrophils, 2.57×105 cells/ml,
p=0.099; macrophages, 1.47×105 cells/ml, p=0.001; lympho-
cytes, 1.64×105 cells/ml, p=0.236), 250μl/kg (total cells,
5.71×105 cells/ml, p=0.032; neutrophils, 2.16×105 cells/ml,
p=0.007; macrophages, 1.34×105 cells/ml, p＜0.001; lympho-
cytes; 1.95×105 cells/ml, p=0.769), and 500μl/kg (total cells,
2.41×105 cells/ml, p＜0.001; neutrophils, 0.85×105 cells/ml,
p＜0.001; macrophages, 0.74×105 cells/ml, p＜0.001; lym-
phocytes, 0.72×105 cells/ml, p＜0.001) significantly and
dose-dependently reduced the total numbers of cells, sim-
ilar to DEX, as well as decreasing the numbers of neu-
trophils, macrophages, and lymphocytes (Fig. 2).
Effect of fennel on lung histopathology of LPS -treated
mice
Hematoxylin and eosin (H&E) staining showed that LPS
treatment (Fig. 3B) was characterized by neutrophil se-
questration, infiltration around the pulmonary vessels, and
alveolar wall thickening in lung tissue compared with ve-
hicle (Fig. 3A). However neutrophil sequestration, infiltra-
tion around the pulmonary vessels, and alveolar wall thick-
ening were significantly alleviated by pretreatment with
500μl/kg fennel (Fig. 3C), as well as by DEX (Fig. 3D).
Effect of fennel on IL-6 and TN F-
α
in BA LF
LPS significantly increased the concentrations in BALF
of the inflammatory cytokines IL-6 (0.97±0.09 vs. 0.18±0.04
ng/ml, p＜0.001; Fig. 4A) and TNF-α (7.18±0.53 vs.
186 HS Lee, et al
Fig. 3. Effect of fennel on the histo-
pathology of lung tissues in LPS-
treated mice. Fennel (500μl/kg) or
DEX (1 mg/kg) was administered in-
traperitoneally to mice 1 h prior to
LPS treatment. Lung sections from
each group were stained with hema-
toxylin and eosin (H&E) (×200). (A)
Vehicle group, (B) Vehicle+LPS gro-
up, (C) Fennel+LPS group, (D) DEX+
LPS group.
Fig. 4. Effects of fennel on (A) IL-6 and (B) TNF-α expression in the BALF of LPS-treated mice. IL-6 and TNF-α in BALF were analyzed
by ELISA. Data are reported as mean±S.E.M. (n=7∼10 per group). ##p＜0.01, ###p＜0.001 compared with the vehicle group; *p＜0.05, ***p
＜0.001 compared with the vehicle+LPS group.
0.10±0.01 ng/ml, 0＜0.001; Fig. 4B) compared with vehicle.
Pretreatment with fennel 125μl/kg (IL-6, 0.76±0.10 ng/ml,
p=0.468; TNF-α, 5.27±0.30 ng/ml, p=0.010), 250μl/kg (IL-6,
0.77±0.07, p=0.517; TNF-α, 5.36±0.58 ng/ml, p=0.016), and
500μl/kg (IL-6, 0.58±0.11, p=0.017; TNF-α, 4.29±0.29
ng/ml, p＜0.001), however, significantly and dose-depend-
ently suppressed the production of IL-6 and TNF-α, with
500μl/kg fennel showing maximum reduction.
Effect of fennel on MM P-9 activity in LPS -treated mice
MMP-9, a representative proinflammatory mediator that
plays an essential role in lung inflammation, was analyzed
in BALF by gelatin zymography. BALF from mice treated
with vehicle+LPS showed a 10-fold increase in a gelati-
nolytic band at 92 kDa, the molecular weight of MMP-9
(p＜0.001), compared with vehicle-treated mice (Fig. 5).
Pretreatment with 250 and 500μl/kg fennel dose-depend-
ently reduced MMP-9 activity, and pretreatment with DEX
also reduced MMP-9 activity.
Effect of fennel on nitric oxide (N O) production in BALF
NO is a critical immune modulator in the proinflam-
Anti-inflammatory Effects of Foeniculum vulgare Mill. 187
Fig. 5. Effect of fennel on MMP-9 activity in LPS-treated mice.
Relative MMP-9 activity in BALF was analyzed by zymography
followed by scanning densitometry. Data are reported as
mean±S.E.M. (n=7∼10 per group). ##p＜0.01, ###p＜0.001 compared
with the vehicle group; *p＜0.05, **p＜0.01, ***p＜0.001 compared
with the vehicle+LPS group.
Fig. 6. Effect of fennel on NO production in the BALF of
LPS-treated mice. NO concentrations in BALF were measured by
nitrite assays. Data are reported as mean±S.E.M. (n=7∼10 per
group). ###p＜0.001 compared with the vehicle group; **p＜0.01,
***p＜0.001 compared with the vehicle+LPS group.
Fig. 7. Effect of fennel on NF-κB
activation in LPS-treated mice. Nu-
clear and cytosolic extracts in lung
tissue were fractionated and the ex-
pression of NF-κB p65 (A) and IκB-α
(B) proteins in nuclear and cytosolic
extracts, respectively, were assessed
by western blotting. Lamin B and
GAPDH were used as internal controls.
Data are reported as mean±S.E.M.
(n=7∼10 per group). ##p＜0.01, ###p＜
0.001 compared with the vehicle group;
*p＜0.05, ***p＜0.001 compared with
the vehicle+LPS group.
matory cytokine response associated with ALI. Treatment
with LPS significantly enhanced the production of NO com-
pared with vehicle (2.98±0.45μM vs. 1.09±0.24μM, p=
0.001; Fig. 6). However, this increase was significantly and
dose-dependently reduced by pretreatment with fennel 125
μl/kg (0.52±0.27μM, p＜0.001), 250μl/kg (0.56±0.73μM,
p＜0.001), and 500μl/kg (0.67±0.23μM, p＜0.001).
Effect of fennel on activation of NF-
κ
B in LPS -induced
ALI mice
NF-κB activation was assessed by western blotting to
determine the anti-inflammatory pathways by which fennel
reduced LPS-induced ALI in mice. Treatment with ve-
hicle+LPS increased the level of expression of NF-κB p65
2.13-fold (p=0.007) compared with vehicle alone (Fig. 7A).
However, in LPS-treated mice, pretreatment with 500μl/kg
fennel reduced the expression of NF-κB p65 1.90-fold com-
pared with pretreatment with vehicle alone (p=0.019). Mice
treated with vehicle+LPS showed 4.05-fold lower IκB-α
expression compared with those treated with vehicle alone
(p＜0.001), whereas mice treated with 500μl/kg fennel plus
LPS showed 2.79-fold higher IκB-α expression compared
with those treated with vehicle+LPS (p=0.023) (Fig. 7B).
This finding indicated that fennel suppressed NF-κB acti-
vation by blocking IκB-α degradation.
Effect of fennel on the M APK signaling pathw ay
The effect of fennel on the MAPK signaling pathway was
analyzed to determine its anti-inflammatory mechanism of
action. LPS increased the levels of expression levels of phos-
pho-ERK (5.11-fold, p＜0.001) (Fig. 8A and 8B), phospho-p38
(1.27-fold, p=0.474) (Fig. 8C and 8D), and phospho-JNK
(1.97-fold, p=0.036) (Fig. 8E and 8F). In contrast, 250μl/kg
(2.86-fold, p=0.004) and 500μl/kg (2.07-fold, p=0.021) fen-
nel significantly reduced the level of LPS-induced ERK
phosphorylation.
DISCUSSION
Although inflammation is a normal immune reaction, un-
controlled inflammation can lead to organ dysfunction or
disease [11]. Clinical ALI involves neutrophilic inflam-
188 HS Lee, et al
Fig. 8. Effect of fennel on the MAPK
signaling pathway in LPS-treated
mice. Lung tissues were analyzed by
western blotting with antibodies to
p-ERK (A), p-p38 (C), and p-JNK (E),
and quantitative protein expression
was normalized to ERK (B), p38 (D),
and JNK (F), respectively. Data are
reported as mean±S.E.M. (n=7∼10
per group). #p＜0.05, ###p＜0.001 com-
pared with the vehicle group; *p＜
0.05, **p＜0.01 compared with the
vehicle+LPS group.
mation and is a common complication of other conditions
[12]. Because LPS from Gram-negative bacteria evokes in-
flammatory responses and endotoxic symptoms [13], LPS
is used in experimental models of inflammation. In agree-
ment with previous findings, we found that LPS-treated
mice showed inflammatory responses, including elevations
in immune system cells and proinflammatory cytokines, as
well as alterations in lung histology. Fennel, which has
been shown to have anti-inflammatory effects, protected
mice against LPS-induced ALI. Fennel reduced lung dam-
age, the numbers of pro-inflammatory cells, and the pro-
duction of pro-inflammatory mediators induced by LPS.
NF-κB, an important transcription factor in inflam-
matory responses, has been shown to regulate the pro-
duction of pro-inflammatory cytokines [14]. Although NF-κB
activation is important in normal inflammatory responses,
its overproduction is closely associated with inflammatory
diseases, such as sepsis [14]. In the absence of stimuli, NF-
κB is located in the cytoplasm, where it binds to IκB-α
and remains inactive. Thus, regulating IκB-α may control
the NF-κB signaling pathway. Trans-anethole, the main
constituent of fennel, has been reported to reduce NF-κB
concentrations in mice with hepatic ischemia/reperfusion
injury [15], as well as to reduce NF-κB levels, while slight-
ly increasing IκB-α levels, in LPS-treated BALB/C mice
[9]. Similarly, we found that treatment with fennel not only
reduced p65 expression, but increased IκB-α level. Thus,
fennel may directly suppress NF-κB activation, perhaps
by enhancing the expression of its inhibitor, IκB-α.
Calcium signaling plays an important role in inflam-
matory conditions [16]. Administration of LPS has been
found to transiently elevate intracellular calcium level,
leading to ERK phosphorylation and the expression of TNF-α
[17]. Fennel was reported to significantly reduce the ex-
pression of TNF-α in response to S. aureus [18], suggesting
that it modulates intracellular calcium concentration.
Fennel induced the relaxation of guinea pig tracheal chains
via hyperpolarization, thus inhibiting calcium influx [19].
In addition, high doses of trans-anethole were reported to
modify calcium channels on isolated rat aortas [20], further
suggesting that the protective effect of fennel on TNF-α
and ERK expression was due, at least in part, to calcium
modulation.
The MAP kinase pathway, which includes ERKs, JNKs
and p38, is also involved in the endotoxic effects of LPS,
leading to inflammation. This pathway and TNF-α ex-
pression are both upstream and downstream of each other
[21]. NO is another important signaling molecule, which
regulates physiological functions, including vascular con-
traction, neuronal signal and inflammation [22]. NO may
derive from inducible NO synthase (iNOS) associated path-
ophysiological processes related to inflammation. Unlike
JNK and p38, ERKs negatively activate iNOS. We found
that fennel significantly reduced nitrate levels, suppressing
LPS-induced ERK expression but having no effect on JNK
or p38 levels.
Fennel contains mainly trans-anethole, limonene, and
anisole [18]. Trans-anethole was shown to have anti-inflam-
matory effects, substantially similar to those of fennel, on
pro-inflammatory cytokines, NO, and transcription factors
[9]. Moreover, d-limonene has shown anti-inflammatory ef-
fects in rat kidney by modulating NF-κB and iNOS [23].
Oral administration of limonene to rats suppressed both
NF-κB and IL-6 [24]. Taken together, these findings sug-
gest that limonene and trans-anethole, the main compo-
nents of fennel, are responsible for the anti-inflammatory
effects of fennel.
In conclusion, this study confirmed that fennel effectively
blocked LPS-induced inflammation, by regulating pro-in-
flammatory cytokines, transcription factors, and NO. These
findings suggest that fennel may have clinical activity in
mitigating inflammatory conditions.
Anti-inflammatory Effects of Foeniculum vulgare Mill. 189
ACKNOWLEDGEMENTS
This work was supported by the National Research
Foundation of Korea (NRF) grant funded by the Korean
government (MSIP) (No.2012R1A2A2A02007145).
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