Inflammation, Vol. 37, No.

6, December 2014 ( # 2014)

DOI: 10.1007/s10753-014-9942-x

Anti-Inflammatory Effects of Apigenin in Lipopolysaccharide-

Induced Inflammatory in Acute Lung Injury by Suppressing
COX-2 and NF-kB Pathway

Jing Wang,1 Yu-Tao Liu,2 Lu Xiao,1 Lingpeng Zhu,1 Qiujuan Wang,1 and Tianhua Yan1,3

Abstract—This study aims to evaluate the possible mechanisms responsible for the anti-inflam-
matory effects of apigenin lipopolysaccharide (LPS)-induced inflammatory in acute lung injury. In
this study, the anti-inflammatory effects of apigenin on lipopolysaccharide (LPS)-induced acute
lung injury (ALI) in mice and the possible mechanisms involved in this protection were inves-
tigated. Pretreatment with apigenin prior to the administration of intratracheal LPS significantly
induced a decrease in lung wet weight/dry weight ratio in total leukocyte number and neutrophil
percent in the bronchoalveolar lavage fluid (BALF) and in IL-6 and IL-1β, the tumor neurosis
factor-α (TNF-α) in the BALF. These results showed that anti-inflammatory effects of apigenin
against the LPS-induced ALI may be due to its ability of primary inhibition of cyclooxygenase-2
(COX-2) gene expression and nuclear factor kB (NF-kB) gene expression of lung. The results
presented here suggest that the protective mechanism of apigenin may be attributed partly to d-
ecreased production of proinflammatory cytokines through the inhibition of COX-2 and NF-kB
activation. The results support that use of apigenin is beneficial in the treatment of ALI.

KEY WORDS: apigenin; inflammation; lung injury; COX-2; NF-kB.

INTRODUCTION showed as hypoxemia, alveolar-capillary barrier damage,

Acute lung injury (ALI) and its more severe form,
acute respiratory distress syndrome (ARDS), represent a
clinical syndrome that results from complex responses of
the lung to a multitude of direct and indirect insults [1].
Although the causes of ALI and ARDS are numerous,
endotoxin is thought to be the most pathogen that leads
to the development of ALI and ARDS. Endotoxin or
lipopolysacchride (LPS), derived from the cell wall of
gram-negative bacteria, induces a sepsis syndrome accom-
panied by key features of ALI, including the recruitment of
inflammatory cells into the lung with subsequent increases
in capillary permeability and alveolar edema [2]. It often
1
Department of Physiology and Pharmacology, China Pharmaceutical
University, Tongjiaxiang 24, Nanjing, Jiangsu 210009, China
2
Department of Pharmacy, Yantaishan Hospital, Yantai, Shandong
264000, China
3
To whom correspondence should be addressed at Department of Physi-
ology and Pharmacology, China Pharmaceutical University,
Tongjiaxiang 24, Nanjing, Jiangsu 210009, China. E-mail:
machunhuabest@126.com
and pulmonary inflammation, and often associated with
multiple organ failure in later stage. There is no specific
medicine to control ALI or ARDS as yet, so searching for
the new therapy target and exploring the new drug to treat
ARDS become the work of scientific research. Animal
models of ALI are important tools for studying mecha-
nisms relevant to the acute respiratory distress syndrome
(ARDS) in humans. Many methods, in a variety of differ-
ent animal species, can generate ALI, each with its own set
of advantages and limitations. The most common cause of
ALI is bacterial sepsis; many investigators have used in-
traperitoneal or intratracheal administration of lipopolysac-
charide (LPS) as a method of provoking ALI.
Cyclooxygenase (COX), the prostaglandin H syn-
thase enzyme, has been implicated in the mechanisms of
a wide variety of inflammatory diseases, including lung
injury [3]. COX-2, as the inducible isoform of COX, is
found to be increased markedly during inflammation [4].
It has been proven that COX-2 could be upregulated
by proinflammatory cytokines and further aggravates the
inflammatory immune response in lung injury [5]. In

2085
0360-3997/14/0600-2085/0 # 2014 Springer Science+Business Media New York
2086
addition, pharmacologic inhibition of COX-2 has been
proven to be able to decrease the proinflammatory cyto-
kines and chemokines and thus protect against ALI [6].
Nuclear factor-kappaB (NF-κB), a nuclear transcrip-
tion 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 [7, 8]. 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) [9]. 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 anti-inflammatory agents [10]. As an oriental medicine,
the root and stem bark of Magnolia officinalis has been
widely used in thrombotic stroke, gastrointestinal com-
plaints, anxiety and nervous stroke, etc.
Apigenin is a member of the flavone family and is
found in many fruits, vegetables, and nuts, including on-
ion, orange, and tea [11]. Many reports have shown that
apigenin possesses various pharmacological effects such as
antioxidative, antiviral, antitumor, and anti-inflammatory
activities [12–14]. However, no available study has evalu-
ated the effects of apigenin treatment on LPS-induced
acute lung injury.
MATERIALS AND METHODS
Experiment Design
In this study, total 50 ICR mice (male, 18–22 g) were
used. Mice were routinely bred in a specific pathogen-free
(SPF) laboratory in the animal center of China Pharmaceu-
tical University, with controlled temperature at 23±2 °C
and relative humidity of 50 % with 12 h light/dark cycle.
All experimental protocols were approved by the Animal
Studies Committee of China Pharmaceutical University.
The method of LPS-induced lung injury model was de-
scribed previously [15]. Mice were anesthetized by intra-
peritoneal injection of 10 % chloral hydrate 4 mg/kg and
then acutely cannulated for administration of LPS (4 mg/
kg, Escherichia coli O127:B8; Sigma) 40 μl in sterile
saline. At 6 h post-LPS infusion, animals were euthanized
by an overdose of ethyl carbamate, and blood sample
(100–500 μl) was removed via the femoral artery for
analysis of serum SOD levels. After death at the end of
the protocol, the right lung was removed to investigate the
histology of lung, MPO activity, and cytokine level. The
left lung was lavaged using 500 μl aliquots of sterile saline
three times for differential cytology and protein leakage.
Wang, Xiao, Zhu, Wang, and Yan
Other clusters were injected with Evans blue via the tail
vein 30 min before being euthanized for the determination
of permeability and wet/dry weight.
Experimental Groups
Apigenin (pure, 98 %) was purchased from National
Institutes for Food and Drug Control (Beijing, China).
Dexamethasone was purchased from Xiansheng Drug
Store (Nanjing, China). In general, five groups of mice
were studied at a time. These group clusters included the
control group (saline), LPS group, apigenin group 20 mg/
kg, 10 mg/kg intraperitoneally administered apigenin (1 h
before and 3 h after LPS instillation), and Dex (2 mg/kg)
group intraperitoneally administered dexamethasone 1 h
before LPS instillation.
Bronchoalveolar Lavage
The lungs were lavaged with 500 μl of saline three
times (total volume, 1.5 ml). Retrieval volume was maxi-
mized by compression of the thorax following the last
lavage. The total leukocyte count was determined using a
hemocytometer. Bronchoalveolar lavage fluid (BALF)
samples were centrifuged at 1,500 rpm for 10 min at
4 °C, the supernatants were stored in −80 °C for analysis
of cytokine concentrations, SOD activity (Nanjing
Jiancheng Bioengineering Institute, Nanjing, China), and
the pellet was resuspended in 100 μl of saline, centrifuged
onto slides, and stained for 8 min with Wright–Giemsa
staining (Nanjing Jiancheng Bioengineering Institute). The
slides were quantified for macrophages, neutrophils, and
lymphocytes by counting a total of 200 cells/slide at×40
magnification as the differential cell count.
Cytokine in BALF
BALF levels of IL-6, IL-1β, and TNF-α were mea-
sured by ELISA according to the manufacturer’s instruc-
tions (R&D, Minneapolis, MN, USA). All measurements
were performed in duplicate.
Lung Wet-to-Dry Weight Ratios
The right lungs were removed at the end of the
experiment. The trachea and esophagus were separated
from the lungs by blunt dissection, and the wet weight of
the latter was determined. Subsequently, the lungs were
incubated at 60 °C for 3 to 4 days to remove all moisture,
then the dry weight was measured and the ratio of wet-to-
dry weight calculated.
Anti-Inflammatory Effects of Apigenin
Histological Assessment
In order to evaluate tissue inflammation, hematoxylin
and eosin ((HE) staining was performed on paraffin-em-
bedded sections). The hematoxylin and eosin staining pro-
cess as previously described [16].
RNA Extraction and Real-Time PCR Analysis
The right lung was removed to investigate the COX-2
and NF-κB activities. The right lungs of each group were
homogenized in 1 ml of TRIzol (GIBCO BCR Inc., Shang-
hai, China) using a glass homogenizer. The total RNA was
isolated according to the manufacturer’s protocol. The
primers for PCR were used as follows: COX-2: forward
5′-T C C A G C C G G T A C C A G A A G-3′ and reverse
5′-G C AT C C C G G C T G A A C T C A C A-3′; NF-kB:
forward 5′-G C G T A C A C A T T C T G G G G A G T-3′
and reverse 5′-C C G A A G C A G G A G C T A T C A A
C-3′; β-actin: forward 5′-AGC CAT GTA CGT AGC CAT
CC-3′ and reverse 5′-CTC TCA GCT GTG GTG GTG
AA-3′. The SYBR green PCR Master Mix was used for
real-time PCR analysis. The cycle time values of the inter-
ested genes were first normalized with β-actin of the same
sample, and then the relative difference between control
and each treatment group was calculated and expressed as
a relative reduction, setting the control group at 100 %.
Statistical Analysis
The data are expressed as mean values±SD. The
differences between groups were analyzed by one-way
ANOVA or Student’s t tests, with P<0.05 considered as
significant.
RESULTS
Effects of Apigenin in BALF
To confirm the efficacy of LPS exposure, total leuko-
cyte and neutrophil numbers in BALF were determined at
the end of the intratracheal LPS protocol. Instillation of
LPS into the lungs produced a significant recruitment of
neutrophils into the alveolar space compared with the
control group (Fig. 1). Total leukocyte number and neutro-
phil number in BALF rose rapidly by 3.1 and 8.1 times
compared with the control group (P<0.01), respectively.
Apigenin (10 and 20 mg/kg) could dose-dependently re-
duce the total leukocyte cell number and neutrophil num-
ber (P<0.01). After Dex treatment, total leukocyte cell
2087
number, neutrophil number, in BALF were also obviously
decreased (P<0.01).
Effects of Apigenin on Cytokine in BALF
The levels of the cytokines IL-6, IL-1β, and TNF-α
in BALF were dramatically increased by intratracheal LPS
administration (Fig. 2). Apigenin (10 and 20 mg/kg) can
downregulate the IL-6, IL-1β, and TNF-α level in dose-
dependent manners, respectively. Dex also regulated the
IL-6, IL-1β, and TNF-α level in BALF.
Effects of Apigenin on Lung Wet/Dry Weight Ratio
To independently evaluate LPS-induced changes in
pulmonary vascular permeability to water, lung wet/dry
weight ratios was used. LPS instilled for 6 h caused a
significant increase in lung wet/dry weight ratio compared
with control (P<0.01). As shown in Fig. 3, both apigenin
and Dex exhibited variability on lung wet/dry weight ratio
when compared with the LPS group—those differences
achieved statistical significance (P<0.05).
Effects of Apigenin on Lung Histology
Histological evaluation of lungs (Fig. 4) by light
microscopy demonstrated a large number of neutrophil
sequestration and infiltration around the pulmonary vessel,
distributed in the alveolar and interstitial after intratracheal
LPS. Treatment groups significantly reduced the degree of
inflammatory cell infiltration (P<0.05). The results indi-
cate that apigenin can reduce the degree of pathological
inflammation in lung tissues in acute lung injury (P<0.05).
Effect of Apigenin on LPS-Induced COX-2 and NF-κB
Genes Expression in Mice
The expressions of inflammation-related genes COX-
2 and NF-κB were changed by LPS in lung. As shown in
Fig. 5, compared with the control group, the levels of
COX-2 and NF-κB in model group were significantly
increased (P<0.05) in pigenin-treated groups (10 and
20 mg/kg), the levels of COX-2 and NF-κB were signifi-
cantly decreased in a dose-dependent manner compared to
the model group, respectively (P<0.01).
DISCUSSION
One of the most important components of the initial
innate immune response in the lung against bacterial
2088 Wang, Xiao, Zhu, Wang, and Yan

Fig. 1. Instillation of LPS into the lungs

infection is the vigorous recruitment of neutrophils. In ALI, the site of injury and express multiple cytotoxic products
neutrophils are the earliest immune cells to be recruited to [17]. In LPS-induced ALI, the neutrophils accumulated in
the lungs, express proinflammatory cytokines, such as IL-
1β and TNF-α, and finally lead to the pulmonary injury

Fig. 2. The levels of the cytokines IL-6, IL-1β, and TNF-α in BALF Fig. 3. Apigenin and Dex exhibited variability on lung wet/dry weight ratio
Anti-Inflammatory Effects of Apigenin 2089

Fig. 4. Histological evaluation of lungs by light microscopy

[18]. In this study, we found the neutrophils increased inflammation not only in systemic inflammation, but also
evidently in lung tissues after LPS exposure. As expected, in local inflammation [23]. In the present study, it was
apigenin pretreatment significantly decreased the neutro- found that apigenin could decrease the LPS-induced lung
phils in lung tissues. In addition, histopathological study W/D ratio. These results suggested that apigenin has a
also indicated that apigenin pretreatment markedly attenu- protective effect on LPS-induced ALI.
ated the neutrophil infiltration in lungs. LPS is known to NF-κB comprises a family of transcription factors
induce the production of several inflammatory and chemo- that act as regulators of proinflammatory mediators. It is
tactic cytokines. TNF-α, IL-1β, and IL-6 are characterized well known that NF-κB is key signaling pathways account-
cytokines involved in the inflammatory process of acute ing for the expressions of proinflammatory cytokines in-
lung injury [19–21]. These cytokines, as well as other duced by LPS. Therefore, we investigated the possibility
proinflammatory compounds, initiate, amplify, and perpet- that apigenin inhibits the production of TNF-α and IL-6 by
uate the inflammatory response in acute lung injury. TNF- interfering with the activation of NF-κB. The NF-κB
α is the earliest and primary endogenous mediator of the
process of an inflammatory reaction. TNF-α, mainly pro-
duced by monocytes/macrophages, can elicit the inflam-
matory cascade, cause damage to the vascular endothelial
cells, and induce alveolar epithelial cells to produce other
cellular factors, such as IL-6 [22]. In this study, we found
the TNF-α, IL-1β, and IL-6 increased evidently in BALF
after LPS exposure. As expected, apigenin pretreatment
significantly decreased the TNF-α, IL-1β, and IL-6 in
BALF. Lung W/D ratio was evaluated as an index of
pulmonary edema, which is a typical symptom of Fig. 5. The levels of COX-2 and NF-κB in model group
2090
pathway has been considered to play a pivotal role in the
pathogenesis of ALI. In the present study, LPS caused a
significant increase in NF-κB expression compared with
the control group; apigenin was found to downregulate
NF-κB. These results suggest the protective effects of
apigenin on LPS-induced ALI.
COX enzymes could convert arachidonic acid to
prostanoid, which play an important role in the develop-
ment of both early and late phases of endotoxemia [24].
COX-2, the inducible form of cyclooxygenases, plays an
important role in lung injury and is upregulated by several
proinflammatory stimuli, such as cytokines and growth
factors. In this study, apigenin has been shown to inhibit
the increase of COX-2 activity induced by LPS. Thus, the
results support that use of apigenin is beneficial in the
treatment of ALI and the exact mechanism and clinical
application needs further investigation.
CONCLUSION
In this paper, we have established that apigenin may
exert some of its anti-inflammatory and pharmacological
effects by affecting the activity of NF-κB and COX-2 LPS-
induced acute lung injury in mice.
ACKNOWLEDGMENTS
This work was supported by a Project Funded by the
Priority Academic Program Development of Jiangsu
Higher Education Institutions.
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