Inflammation ( # 2012)
DOI: 10.1007/s10753-012-9571-1
Suppression of MAPK and NF-κB Pathways by Limonene
Contributes to Attenuation of Lipopolysaccharide-Induced
Inflammatory Responses in Acute Lung Injury
Gefu Chi,1 Miaomiao Wei,2,3 Xianxing Xie,2 L. W. Soromou,2 Fang Liu,2 and Shuhua Zhao3,4
Abstract—The present study aimed to investigate the protective role of limonene in lipopolysaccharide (LPS)-induced acute lung injury (ALI). ALI was induced in mice by intratracheal instillation
of LPS (0.5 mg/kg), and limonene (25, 50, and 75 mg/kg) was injected intraperitoneally 1 h prior to
LPS administration. After 12 h, bronchoalveolar lavage fluid (BALF) and lung tissue were collected. Limonene pretreatment at doses of 25, 50, and 75 mg/kg decreased LPS-induced evident lung
histopathological changes, lung wet-to-dry weight ratio, and lung myeloperoxidase activity. In addition, pretreatment with limonene inhibited inflammatory cells and proinflammatory cytokines including tumor necrosis factor-α, interleukin-1β, and interleukin-6 in BALF. Furthermore, we
demonstrated that limonene blocked the phosphorylation of IκBα, nuclear factor-κB (NF-κB) p65,
p38 mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase, and extracellular signalregulated kinase in LPS-induced ALI. The results presented here suggest that the protective mechanism of limonene may be attributed partly to decreased production of proinflammatory cytokines
through the inhibition of NF-κB and MAPK activation.
KEY WORDS: limonene; lipopolysaccharide (LPS); nuclear factor-κB (NF-κB); mitogen-activated protein
kinase (MAPK).
INTRODUCTION
Acute lung injury (ALI) and acute respiratory
distress syndrome (ARDS) are common complications
in the intensive care unit and take responsibility for
significant morbidity and mortality. A recent epidemiological study revealed approximately 190,000 cases per
year in the USA, with an associated 74,500 patients
dying from ALI [1, 2]. ALI often showed as hypoxemia,
alveolar-capillary barrier damage, and pulmonary in1
College of Animal Science and Technology, Inner Mongolia National
University, Tongliao, 028042, People’s Republic of China
2
Key Laboratory of Zoonosis Research, Ministry of Education,
Institute of Zoonosis, College of Animal Science and Veterinary
Medicine, Jilin University, 5333 Xi’an Road, Changchun, 130062,
People’s Republic of China
3
The Second Hospital of Jilin University, ZiqiangStreet 218#,
Changchun, 130041, People’s Republic of China
4
To whom correspondence should be addressed at The Second
Hospital of Jilin University, ZiqiangStreet 218#, Changchun,
130041, People’s Republic of China. E-mail: [email protected]
flammation and is associated with multiple organ failure
at a later stage [3]. Although great advances in
understanding the pathophysiology of ALI had been
achieved, the available therapies have not reduced the
mortality or increased the quality of life of survivors.
Lipopolysaccharide (LPS) is a component of the Gramnegative bacterial cell wall, which can induce inflammatory responses and cause disturbance in the immune
system function [4]. Intratracheal administration of LPS
has gained wide acceptance as a clinically relevant
model of severe lung injury [5]. Thus, we use this model
to determine whether limonene could prevent ALI
induced by LPS (intranasal—i.n.) administration in
mice.
Nuclear factor-kappaB (NF-κB), a nuclear transcription factor, is a regulator of inflammatory processes.
Chen et al. have reported that NF-κB plays an important
role in the pathogenesis of lung diseases [6, 7]. NF-κB is
required for maximal transcription of numerous cytokines, including tumor necrosis factor-α (TNF-α),
interleukin-1β (IL-1β), and interleukin-6 (IL-6) [8].
0360-3997/12/0000-0001/0 # 2012 Springer Science+Business Media New York
Chi, Wei, Xie, Soromou, Liu, and Zhao
These cytokines are thought to be important in the
generation of ALI. Therefore, it has been suggested that
inhibitors of NF-κB function may be useful as antiinflammatory agents [9]. Mitogen-activated protein
kinases (MAPK) are also involved in inflammation
[10]. Many lines of evidence have suggested that
members of MAPKs, including p38 MAPK, extracellular regulated kinase (ERK), and c-Jun NH2-terminal
kinase (JNK), are important players in signal transduction pathways activated by a range of stimuli and
mediate a number of physiological and pathological
changes in cell function [11]. It has been shown that
inhibition of any of these three MAPK pathways (JNK,
p38 MAPK, and ERK pathways) is sufficient to block
induction of TNF-α by LPS [12]. Therefore, treatments
aimed at inhibition of NF-κB and MAPKs may have
potential therapeutic advantages in curing inflammatory
diseases.
Limonene is a 10-carbon cyclohexanoid monoterpene derivative found in oils of lemon, orange, and
grape fruit. Numerous pharmacological effects, such as
anti-inflammation, antitumor, and anti-asthma activities,
have been attributed to limonene [13–15]. It has been
shown that limonene could inhibit LPS-induced production of nitric oxide, prostaglandin E2, and proinflammatory cytokines in RAW264.7 macrophages [13]. In
addition, Hirota et al. have reported that limonene may
have potential anti-inflammatory efficacy for the treatment of bronchial asthma by inhibiting cytokines, ROS
production, and inactivating eosinophil migration [15].
However, no available study has evaluated the effects of
limonene treatment on LPS-induced acute lung injury in
a mouse model. Therefore, we sought to investigate
whether an intraperitoneal (i.p.) injection of limonene
could protect against nonspecific pulmonary inflammation in mice. Our results might provide a pharmacological basis on its folkloric use for the treatment of ALI.
MATERIALS AND METHODS
Chemicals and Reagents
Limonene and dexamethasone (DEX) were purchased from the National Institutes for Food and Drug
Control (Beijing, China). LPS (Escherichia coli 055:B5)
was purchased from Sigma Co. Mouse TNF-α, IL-1β,
and IL-6 enzyme-linked immunosorbent assay (ELISA)
kits were purchased from Biolegend, Inc. (San Diego,
CA, USA). Rabbit mAb IκBα, p-NF-κB p65, SAPK/
JNK, P-p38 MAPK, P-ERK, ERK, rabbit Ab P-SAPK/
JNK, p38 MAPK, and mouse mAb p-IκBα were
purchased from Cell Signaling Technology Inc (Beverly,
MA, USA). Horseradish peroxidase-conjugated goat
antirabbit and goat mouse antibodies were purchased
from GE Healthcare (Buckinghamshire, UK). Phosphatase inhibitor and PVDF membrane were purchased
from Roche. All other chemicals were of reagent grade.
Animals
BALB/c mice (male, 8–10 weeks old, 18–20 g
each) were purchased from the Center of Experimental
Animals of Baiqiuen Medical College of Jilin University
(Jilin, China). Mice were allowed to acclimatize to the
laboratory for at least 7 days under climate-controlled
conditions. All procedures were in accordance with the
guide for the Care and Use of Laboratory Animals
published by the US National Institute of Health.
Experimental Protocol for Acute Lung Injury Model
All mice were randomly divided into six groups:
control, model, dexamethasone (0.5 mg/kg), and limonene (75, 50, and 25 mg/kg). Before inducing acute lung
inflammation, limonene (25, 50, and 75 mg/kg) and
dexamethasone (0.5 mg/kg) were given by i.p. injection.
DEX was injected as a positive control. One hour later,
10 μg of LPS was instilled i.n. in 50 μl PBS to induce
lung injury. Twelve hours after LPS administration, all
animals were sacrificed by diethyl ether asphyxiation.
Bronchoalveolar Lavage
At 12 h after LPS challenge, mice were sacrificed
by diethyl ether asphyxiation. The lungs were lavaged
with 500 μl of sterile PBS (7.2) three times (total
volume 1.5 ml). The recovery ratio of the fluid was
about 87±2 %. Then, the bronchoalveolar lavage fluid
(BALF) was immediately centrifuged at 3,000 rpm for
10 min at 4 °C, and the cell-free supernatants were
stored at −80 °C for cytokine analysis. The sediment
cells were resuspended in PBS for total cell and
neutrophil counts using hemacytometer and cytosine by
staining with the Wright–Giemsa staining method.
Cytokine Assay
Levels of TNF-α, IL-1β, and IL-6 in BALF were
determined by ELISA kits according to the instructions
recommended by the manufacturers. The optical density
of each well was read at 450 nm.
Role of Limonene in LPS-Induced Acute Lung Injury
Measurement of Wet-to-Dry Ratio of the Lungs
The mice were euthanized at 12 h after LPS
challenge. The right lungs were excised and the “wet”
weight was recorded. The lungs were then placed in an
incubator at 80 °C for 48 h to obtain the “dry” weight.
The ratio of the wet lung to the dry lung was an index of
lung edema.
Myeloperoxidase Assay in Lung Tissues
Myeloperoxidase (MPO) is an important marker of
polymorphonuclear leukocyte activation. The activity of
MPO was determined in the lung tissue by MPO kit
(Nanjing Jiancheng, China). Twelve hours after LPS
treatment, mice under diethyl ether anesthesia were
killed and the right lungs were excised. One hundred
milligrams of lung tissue was homogenized and fluidized in extraction buffer to obtain 5 % of the
homogenate. The sample including 0.9 ml homogenate
and 0.1 ml of reaction buffer was heated to 37 °C in a
water bath for 15 min, then the enzymatic activity was
determined by measuring the changes in absorbance at
460 nm using a 96-well plate reader.
Pulmonary Histopathology
Mouse left lungs were excised at 12 h after LPS
administration. The lungs were fixed in 4 % neutral
buffered formalin for 24 h, embedded in paraffin, and
cut into 3-μm sections. Hematoxylin–eosin stains were
performed using standard protocol. After staining,
pathological changes in the lung tissues were observed
under a light microscope.
Western Blot Analysis
Lung tissues were harvested at 12 h after LPS
administration and frozen in liquid nitrogen immediately
until homogenization. Proteins were extracted with lysis
buffer (RIPA with protease and phosphatase inhibitor)
for 15 min on ice. Proteins, which were extracted from
the lungs using Nuclear Protein Extraction Kit, were
used to analyze phosphor-NF-κB p65. Protein concentrations were determined by BCA protein assay kit.
Equal amounts of protein were loaded per well on a
10 % sodium dodecyl sulfate polyacrylamide gel and
transferred to PVDF membranes. The membranes were
blocked with 5 % bovine serum albumin (BSA) (5 g
BSA was dissolved in 100 ml TTBS) for 2 h at room
temperature and incubated with primary antibody diluted
1:1,000 in 5 % BSA overnight at 4 °C. After that, with
the use of peroxidase-conjugated secondary antimouse
and antirabbit antibodies (1:5,000 dilution), the bound
antibodies were detected by SuperSignal West Pico
chemiluminescent substrate. β-Actin was detected as
an internal control of protein loading.
Statistical Analysis
The data are expressed as mean values ± SEM.
Differences between groups were analyzed by one-way
ANOVA or Student’s t tests, with p<0.05 considered as
significant.
RESULTS
Effect of Limonene on LPS-Induced Cytokine
Production
To determine the effects of limonene on LPS-induced
cytokine production, we measured the contents of TNF-α,
IL-1β, and IL-6 in BALF by ELISA. As illustrated in
Fig. 1, TNF-α (p<0.01), IL-1β (p<0.05), and IL-6
(p<0.01) levels were found to be significantly increased
in the LPS group compared with the normal group.
Limonene (25, 50, and 75 mg/kg) and DEX (0.5 mg/kg)
pretreatment efficiently reduced the production of TNF-α
(p<0.01 or p<0.05), IL-1β (p<0.01 or p<0.05), and IL-6
(p<0.01 or p<0.05) in a dose-dependent manner.
Effects of Limonene on Inflammatory Cell Count
in BALF
Mice exposed to LPS showed an increase in the
number of total cells (p<0.05) and neutrophils (p<0.01) as
compared to the control group. Pretreatment with limonene
(50 and 75 mg/kg) and DEX (0.5 mg/kg) significantly
decreased the number of total cells (p<0.05) and neutrophils (p<0.05), compared to those in the LPS group
(Fig. 2). However, limonene (25 mg/kg) did not decrease
the number of total cells and neutrophils.
Effects of Limonene on LPS-Induced Lung
Wet-to-Dry Ratio
The lung wet-to-dry (W/D) ratio was evaluated to
indicate the pulmonary edema. Twelve hours after LPS
challenge, the lung W/D ratios (p<0.01) were significantly higher than those in the control group. In the
limonene groups (25, 50, and 75 mg/kg) and the DEX
(0.5 mg/kg) group, there were significant reductions in
the lung edema formation (p<0.05) (Fig. 3a).
Chi, Wei, Xie, Soromou, Liu, and Zhao
Fig. 1. Effects of limonene on the production of inflammatory cytokines TNF-α, IL-1β, and IL-6 in the BALF of LPS-induced ALI mice. Limonene
(25, 50, and 75 mg/kg) and DEX (0.5 mg/kg) were given by intraperitoneal injection 1 h prior to an instillation administration of LPS. a The
concentrations of TNF-α in BALF after LPS i.n. administration and the effect of limonene at varied doses. b The concentrations of IL-1β in BALF
after LPS i.n. administration and the effect of limonene at varied doses. c The concentrations of IL-6 in BALF after LPS i.n. administration and the
effect of limonene at varied doses. The values presented are the means ± SEM (n06 in each group). #p<0.05, ##p<0.01 vs the control group; *p<
0.05, **p<0.01 vs the LPS group.
Role of Limonene in LPS-Induced Acute Lung Injury
Fig. 2. Effect of limonene on the number of total cells and neutrophils in the BALF of LPS-induced ALI mice. Mice were given an intraperitoneal
injection of limonene (25, 50, and 75 mg/kg) and DEX (0.5 mg/kg) 1 h prior to an i.n. administration of LPS. BALF was collected at 12 h following
LPS challenge to measure the number of total cells (a) and neutrophils (b). The values presented are the mean ± SEM (n06 in each group). #p<0.05,
##p<0.01 vs the control group; *p<0.05, **p<0.01 vs the LPS group.
Effect of Limonene on LPS-Induced
Polymorphonuclear Leukocyte Infiltration
Effect of Limonene on LPS-Induced Pathological
Changes of the Lung
MPO activity increase reflects polymorphonuclear
leukocyte accumulation in the lung. Twelve hours
after LPS administration, the MPO (p<0.01) activity
in the lung tissues was significantly increased compared with the control group. Intraperitoneal injection
of limonene (25, 50, and 75 mg/kg) and DEX
(0.5 mg/kg) dramatically decreased MPO activity
(p<0.01 or p<0.05) compared with the LPS group
(Fig. 3b).
The lungs, harvested at 12 h after LPS injection,
were subjected to H&E staining. In the control group, no
evident histological alteration was observed in lung
specimens. In contrast, lung tissues from the experimental
group administered LPS alone showed significant pathological changes, such as inflammatory cell infiltration and
alveolar hemorrhage. However, LPS-induced pathological changes were significantly attenuated by limonene
(25, 50, and 75 mg/kg) and DEX (0.5 mg/kg) (Fig. 4).
Chi, Wei, Xie, Soromou, Liu, and Zhao
Fig. 3. Effects of limonene on the lung W/D ratio and lung MPO of LPS-induced ALI mice. Mice were given an intraperitoneal injection of limonene
(25, 50, and 75 mg/kg) and DEX (0.5 mg/kg) 1 h prior to an i.n. administration of LPS. The lung W/D ratio (a) and lung MPO (b) were determined at
12 h after LPS was given. The values presented are the mean ± SEM (n06 in each group). #p<0.05, ##p<0.01 vs the control group; *p<0.05, **p<
0.01 vs the LPS group.
Effect of Limonene on LPS-Induced MAPK
and NF-κB Activation
Activation of MAPKs and NF-κB signaling pathways plays key roles in the regulation of inflammatory
mediator production. Therefore, we performed western
blotting to investigate the activation of phosphor-ERK,
phosphor-JNK, phosphor-p38, phosphor-IκB-α, and
phosphor-NF-κB p65. LPS stimulation resulted in prominent phosphorylation of MAPKs (Fig. 5), phosphorIκBα, and phosphor-NF-κB p65 (Fig. 6) in mouse lung
tissues. Pretreatment with limonene (50 and 75 mg/kg)
and DEX (0.5 mg/kg) prevented the phosphorylation of
p38, ERK, JNK, IκBα, and NF-κB p65. However,
limonene (25 mg/kg) prevented the phosphorylation of
p38, JNK, IκBα, and NF-κB p65, but not the phosphorylation of ERK.
DISCUSSION
ALI and ARDS are the syndromes of acute respiratory failure that results from a disturbance of the alveolarcapillary barrier associated with several clinical disorders.
ALI associated with inflammation is a severe disease that
Role of Limonene in LPS-Induced Acute Lung Injury
Fig. 4. Effect of limonene on LPS-mediated lung histopathogic changes. Lungs (n03) from each experimental group were processed for histological
evaluation at 12 h after LPS challenge: a the lung section from the control mouse; b the lung section from LPS-induced ALI model mouse; c the lung
section from the mice exposed to LPS and administered with 0.5 mg/kg DEX; d the lung section from the mice exposed to LPS and administered with
75 mg/kg limonene; e the lung section from the mice exposed to LPS and administered with 50 mg/kg limonene; and f the lung section from the mice
exposed to LPS and administered with 25 mg/kg limonene. Representative histological section of the lungs was stained by hematoxylin and eosin
(H&E staining, magnification × 100).
presents high morbidity and mortality rates, and there are
no effective drugs in the clinic [16]. Therefore, prevention
of ALI is an important therapeutic goal. LPS is a principal
component of the outer membrane of Gram-negative
bacteria and can enter the blood stream and elicit
inflammatory responses that may lead to shock and
ultimately to death [17]. Intraperitoneal administration of
LPS is a widely used model of ALI in mice. The symptoms
of LPS-induced ALI expressed by the mouse model have
close resemblance to the observed pathology in humans
[18]. Thus, we used this model to study the prevention of
limonene on LPS-induced ALI in mice. To the best of our
knowledge, the current study is the first to demonstrate the
effects of limonene on LPS-induced ALI.
In ALI, the predominant inflammatory cells are the
neutrophils, which play an important role in the development of most cases of ALI [19]. Teder et al. reported that
mice that failed to clear apoptotic neutrophils might lead to
Chi, Wei, Xie, Soromou, Liu, and Zhao
Fig. 5. Effect of limonene on LPS-induced phosphorylation of ERK, JNK, and p38 MAPK in LPS-induced ALI mice. Mice were given different
concentrations of limonene (25, 50, and 75 mg/kg) and DEX (0.5 mg/kg) with an intraperitoneal injection 1 h prior to an i.n. administration of LPS.
Protein samples were analyzed by western blot with specific antibodies as described. Similar results were obtained in three independent experiments
and one of three representative experiments is shown. The values presented are the mean ± SEM. #p<0.05, ##p<0.01 vs the control group; *p<0.05,
**p<0.01 vs the LPS group.
exacerbated inflammation and increased mortality in ALI
[20]. Leukocyte activation produces reactive oxygen
species and protease that lead to alveolar barrier disruption,
permeability escalation, and direct tissue injury [21].
Widespread destruction of alveolar epithelium and flooding of the alveolar spaces with proteinaceous exudates
containing large amounts of neutrophils represent the
typical lesion in ALI [22]. MPO is an enzyme located
mainly in the primary granules of neutrophils and its main
function is to kill microorganisms, but under certain
conditions, it produces excess oxidant leading to tissue
damage [23]. In this study, we found that the recruitment of
neutrophils in the airways and MPO activity in the lungs
were dramatically increased after LPS administration. In
contrast, pretreatment of limonene significantly decreased
MPO activity and reduced neutrophil infiltration. Furthermore, limonene successfully abated lung tissue injury. This
result was consistent with histological analysis of the lung
and corroborated our findings in the airways. Lung W/D
ratio was evaluated as an index of pulmonary edema,
which is a typical symptom of inflammation not only in
systemic inflammation, but also in local inflammation [24].
In the present study, it was found that limonene could
decrease the LPS-induced lung W/D ratio. These results
suggested that limonene has a protective effect on LPSinduced ALI.
The character of ALI is the acute inflammatory
process in the airspaces and lung parenchyma injury
Role of Limonene in LPS-Induced Acute Lung Injury
Fig. 6. Effect of limonene on the activation of NF-κB in the lungs of LPS-induced ALI mice. To induce ALI, LPS was instilled intranasally 1 h after
intraperitoneal administration of limonene (25, 50, and 75 mg/kg) and DEX (0.5 mg/kg). Protein samples were analyzed by western blot with specific
antibodies as described. β-Actin was used as an internal control. Similar results were obtained in three independent experiments and one of three
representative experiments is shown. The values presented are the mean ± SEM. #p<0.05, ##p<0.01 vs the control group; *p<0.05, **p<0.01 vs the
LPS group.
involving the release of inflammatory mediators such as
TNF-α, IL-1β, and IL-6. Several lines of evidence from
several clinical studies indicated that proinflammatory
cytokines, notably TNF-α, IL-1β, and IL-6, participate
in the early development of inflammation; they have
been shown to play a crucial role in ALI and ARDS [25].
Increased concentrations of TNF-α, IL-1β, and IL-6 in
the BALF have been observed in patients with ALI and
were related to poor outcome [26, 27]. The first
endogenous mediator in the inflammatory process is
TNF-α, which is produced from LPS-stimulated monocytes and macrophages that elicit the inflammatory
cascade and contribute to the severity of lung injury
[28]. The binding of TNF-α with receptors in the lung
tissue leads to the leakage of enzymes out of the
organelle, which causes damage to the lung parenchyma
[29]. IL-6 is one of the most important mediators of fever,
and in ARDS, high plasma and BALF levels are
predictive of poor outcomes [30]. IL-1β can stimulate
the production of a variety of chemotactic cytokines such
as monocyte chemotactic peptide and macrophage inflammatory peptide-1a; they have earned a position of
prominence at the head of the inflammatory cytokine
cascade [31]. Both TNF-α and IL-6 induce adhesion
molecule expression in vascular endothelial cells, resulting in recruitment of leukocytes into the inflammatory site
[32]. Since cytokines have been considered as therapeutic
targets for LPS-induced ALI, many treatments have had
inhibitory effects on the gene expression of cytokines in
the lung [33]. Therefore, TNF-α, IL-1β, and IL-6 served
as predictive markers for ALI severity. In the present
study, LPS caused a significant increase in TNF-α, IL-1β,
and IL-6 expression in BALF compared with the control
group; limonene was found to downregulate TNF-α, IL1β, and IL-6 secretion at 12 h after LPS challenge. These
results suggest that the protective effects of limonene on
LPS-induced ALI were partly attributed to inhibition of
TNF-α, IL-1β, and IL-6.
To further characterize the nature of the inhibitory
effect of limonene on cytokine production, we examined
the effects of limonene on the activation of the MAPKs
and NF-κB signaling pathways. It is well known that
MAPKs (ERK, JNK, and p38) and NF-κB are key
signaling pathways accounting for the expressions of
proinflammatory cytokines induced by LPS. Therefore,
we investigated the possibility that limonene inhibits the
production of TNF-α, IL-1β, and IL-6 by interfering
with the activation of NF-κB and MAPK. The NF-κB
pathway has been considered to play a pivotal role in the
pathogenesis of ALI. The nuclear accumulation of NFκB p65 was observed in alveolar macrophages from
patients with acute lung injury caused by severe
infection, in contrast to alveolar macrophages from
control patients [34]. The heterodimers of NF-κB
components, which are mostly p50/p65, are normally
retained in the cytoplasm in an inactive form through
Chi, Wei, Xie, Soromou, Liu, and Zhao
being associated with an inhibitor of κB (IκB) protein [35].
A wide variety of stimuli can cause the phosphorylation of
IκB, a process that is followed by the protein’s ubiquitination and subsequent degradation. The phosphorylationinduced degradation of the IκB inhibitor enables the NFκB dimers to enter the nucleus and activate specific target
gene expression [36]. In the current study, we found that
increased phosphor-IκB expression and NF-κB p65
activity in LPS-induced tissue were detected. However,
pretreatment with limonene inhibited phosphor-IκB expression and prevented the translocation of NF-κB into the
nucleus of the lung. In addition, recently, much attention
has been given to the MAPK superfamily, especially p38
MAPK, due to its consistent activation by proinflammatory
cytokines and to its role in nuclear signaling [37]. Previous
lines of evidence have shown that inhibition of MAPKs
pathway is involved in the decrease of sepsis-induced
TNF-α, IL-1β, and IL-6 production [38]. LPS induces its
inflammatory effects through the activation of the MAPK
pathway. It has been shown that p38 MAPK induced by
LPS is critical for LPS-induced cytokine release [39].
There is also evidence suggesting that inhibition of p38
MAPK could effectively attenuate LPS-induced lung
injury [40]. Furthermore, some studies have found that
activation of the p38 MAPK and JNK contributed to
sepsis-induced organ injury, whereas inhibition of the p38
and JNK improved survival in septic mice [41, 42]. Schuh
and Pahl reported that the ERK pathway plays an important
role in LPS-mediated pulmonary inflammation [43]. Based
on all of the above, to clarify the mechanisms of limonene
in LPS-induced ALI, the expression of ERK, JNK, and p38
MAPK in lung tissue was examined by western blot
analysis. Here we found that LPS stimulation led to evident
phosphorylations of p38 MAPK, ERK, and JNK, which
were consistent with the increase of TNF-α after challenge
of LPS. Our results suggest that limonene attenuated the
activation of ERK, p38 MAPK, and JNK in LPS-induced
ALI. Therefore, we conclude that limonene may decrease
LPS-induced proinflammatory cytokines in LPS-induced
ALI by inhibiting the activation of NF-κB, ERK, p38
MAPK, and JNK signaling pathways.
In conclusion, the present study showed that
limonene played a potent anti-inflammatory role in
LPS-induced ALI in mice. Our results indicated that
limonene attenuated LPS-induced inflammatory cell
infiltration and reduced the activity of MPO. Administration of limonene reduced inflammatory cytokines
(TNF-α, IL-1β, and IL-6) in BALF, decreased W/D
ratio, and improved the pulmonary functions. Furthermore, we demonstrated that limonene inhibited ERK,
JNK, p38 MAPK, and NF-κB activation in LPS-induced
ALI. Therefore, limonene may be considered as a
potential agent for preventing ALI in the future. Also,
further and comprehensive studies are needed before
clinical application.
ACKNOWLEDGMENTS
This work was supported by the National Science
and Technology Supporting Plan of China (No.
2006BAD31B03-4).
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