Wogonoside Ameliorates Lipopolysaccharide-Induced Acute Lung Injury in Mice

Abstract

Wogonoside has been reported to have anti-inflammatory properties. In this study, we evaluated the effect of wogonoside on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Male BALB/c mice with ALI, induced by intranasal instillation of LPS, were treated with wogonoside 1 h prior to LPS exposure. Mice treated with LPS alone showed significantly increased TNF-α, IL-6, and IL-1β levels in the bronchoalveolar lavage fluid (BALF). When pretreated with wogonoside, the TNF-α, IL-6, and IL-1β levels were significantly decreased. Meanwhile, wogonoside significantly inhibited LPS-induced increases in the macrophage and neutrophil infiltration of lung tissues and markedly attenuated myeloperoxidase activity. Furthermore, wogonoside inhibited the TLR4 expression and the phosphorylation of NF-κB p65, and IκB induced by LPS. In conclusion, our results indicate that wogonoside exhibits a protective effect on LPS-induced ALI via suppression of TLR4-mediated NF-κB signaling pathways.

Full text

Wogonoside Ameliorates Lipopolysaccharide-Induced Acute
Lung Injury in Mice
Liang Zhang,
1
Yi Ren,
1
Chengliang Yang,
1
Yue Guo,
2
Xiaojing Zhang,
3
Gang Hou,
4
Xinjin Guo,
5
Nan Sun,
1
and Yongyu Liu
1,6
Abstract—Wogonoside has been reported to have anti-inflammatory properties. In this study, we eval-
uated the effect of wogonoside on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice.
Male BALB/c mice with ALI, induced by intranasal instillation of LPS, were treated with wogonoside
1 h prior to LPS exposure. Mice treated with LPS alone showed significantly increased TNF-α,IL-6,
and IL-1βlevels in the bronchoalveolar lavage fluid (BALF). When pretreated with wogonoside, the
TNF-α, IL-6, and IL-1βlevels were significantly decreased. Meanwhile, wogonoside significantly in-
hibited LPS-induced increases in the macrophage and neutrophil infiltration of lung tissues and mark-
edly attenuated myeloperoxidase activity. Furthermore, wogonoside inhibited the TLR4 expression and
the phosphorylation of NF-κB p65, and IκB induced by LPS. In conclusion, our results indicate that
wogonoside exhibits a protective effect on LPS-induced ALI via suppression of TLR4-mediated NF-κB
signaling pathways.
KEY WORDS: wogonoside; lipopolysaccharide (LPS); acute lung injury (ALI); nuclear factor-kappaB (NF-κB);
TLR4.
INTRODUCTION
Acute lung injury (ALI) and its severe form,
acute respiratory distress syndrome (ARDS), were
characterized by severe hypoxemia, pulmonary ede-
ma, and neutrophil accumulation in the lungs [1]. It
can be induced by many extreme conditions
including severe sepsis, severe bacterial pneumonia,
trauma, and burn [2–4]. Lipopolysaccharide (LPS)
has been referred to be an important risk factor of
acute lung injury [5]. LPS activates TLR4 signal
pathway and triggers an inflammatory response,
resulting in acute lung injury [6]. In the last decade,
ALI is a major clinical problem that has a high
mortality rate of 30 % to 40 % [7]. There are few
effective therapies in clinic. Therefore, the develop-
ment of novel therapies for acute respiratory distress
syndrome is urgently needed.
Wogonoside, one flavonoid derived from the root of
Scutellaria baicalensis Georgi, has been shown to have
antioxidant, anti-tumor, and antithrombotic activities [8–
10]. Recently, wogonoside has been reported to have anti-
inflammatory effects. Wogonoside was found to inhibit
TNF-αand IL-6 production in LPS-activated RAW264.7
cells [11], to inhibit LPS-induced angiogenesis in vitro and
in vivo [12]. However, the effect of wogonoside on LPS-
induced acute lung injury remains unclear. In this study, we
sought to assess the preventive effects of wogonoside on
LPS-induced mouse acute lung injury and elucidated the
potential anti-inflammatory mechanism.
1
Department of Thoracic Surgery, Liaoning Cancer Hospital and Institute,
Shenyang, 110042, Liaoning Province, People’s Republic of China
2
Shenyang Maternity Hospital, Shenyang, 110042, Liaoning Province,
People’sRepublicofChina
3
Department of Bone and Soft tissue Surgery, Liaoning Cancer Hospital
and Institute, Shenyang, 110042, Liaoning Province, People’s Republic
of China
4
Department of Respiratory Medicine, The First Affiliated Hospital of
China Medical University, Shenyang, 110042, Liaoning Province, Peo-
ple’s Republic of China
5
School of Chinese Medicine, Hong Kong Baptist University, Hong
Kong, SAR 999077, People’sRepublicofChina
6
To whom correspondence should be addressed at Department of Tho-
racic Surgery,Liaoning CancerHospital and Institute, Shenyang, 110042,
Liaoning Province, People’s Republic of China. E-mail:
liuyongyugz@163.com
0360-3997/14/0600-2006/0 #2014 Springer Science+Business Media New York
Inflammation, Vol. 37, No. 6, December 2014 (#2014)
DOI: 10.1007/s10753-014-9932-z
2006

MATERIALS AND METHODS
Materials
Wogonoside (purity >99 %) was purchased from
Shanghai Winherb Medical S&T Development Co. Ltd.
(Shanghai, China). Enzyme-linked immunosorbent assay
(ELISA) kits of TNF-α, IL-6, and IL-1βwere purchased
from BioLegend (CA, USA). LPS was purchased from
Sigma (St. Louis, MO, USA). Anti-pNF-κB p65, anti-
NF-κB p65, anti-TLR4, and anti-β-actin monoclonal anti-
bodies were purchased from Santa Cruz Biotechnology
Inc. (Santa Cruz, CA, USA). The myeloperoxidase
(MPO) determination kit was provided by the Jiancheng
Bioengineering Institute of Nanjing (Nanjing, Jiangsu,
China). All other reagents were of analytical grade.
Animals
Male BALB/c mice, weighing approximately 18 to
22 g, were purchased from the Experimental Animal Cen-
ter of the China Medical University (Shenyang, China).
The mice were housed in a room maintained at 24± 1 °C
with 40–80 % humidity. All mice received food and water
ad libitum. All animal experiments were performed in
accordance with the guide for the Care and Use of Labo-
ratory Animals published by the US National Institutes of
Health.
Experimental Design
After adjustment to the environment, seventy-two
mice were randomly divided into six groups and each
group contained twelve mice: control group, LPS group,
wogonoside (10, 20, and 40 mg/kg) + LPS group, DEX +
LPS group. Wogonoside (10, 20, and 40 mg/kg) and DEX
(5 mg/kg) were given with an intraperitoneal injection (i.p.)
1 h before LPS administration. Then, the mice were slight-
ly anesthetized with an inhalation of diethyl ether; 10 μgof
LPS in 50 μl PBS was instilled intranasal (i.n.) to induce
lung injury. Control mice were given 50 μl PBS without
LPS. After infusion of LPS 7 h, the mice were killed using
Fig. 1. Effects of wogonoside on the lung W/D ratio of LPS-induced ALI
mice. The values presented are the means ± SEM (n=6 in each group).
#
p<0.01 vs. controlgroup;*p<0.05 and **p<0.01vs. LPS group.
Fig. 2. Effects of wogonoside on the number of total cells, neutrophils,
and macrophages in the BALF of LPS-induced ALI mice. The values p-
resented are the mean ± SEM (n=4-6 in each group).
#
p<0.01 vs. control
group; *p<0.05 and **p<0.01 vs. LPS group.
2007Wogonoside Ameliorates LPS-Induced Acute Lung Injury

CO
2
inhalation. The lungs were received. Collection of
bronchoalveolar lavage fluid (BALF) was performed three
times through a tracheal cannula with autoclaved PBS,
instilled up to a total volume of 1.3 ml.
Lung Wet to Dry Weight (W/D) Ratio
After mice were euthanized, lungs were excised. Each
lung was blotted dry, weighed, and then placed in an oven
at 60 °C for 24 h to obtain the “dry”weight. The ratio of the
wet lung to the dry lung was calculated to assess tissue
edema.
Cytokine Assays
The levels of TNF-α, IL-6, and IL-1βwere measured
using sandwich enzyme-linked immunosorbent assay
(ELISA) kits (BioLegend, USA) according to the manu-
facturer’sinstructions.
Inflammatory Cell Counts of BALF
The BALF samples were centrifuged (4 °C,
3,000 rpm, 10 min) to pellet the cells. The sediment cells
were resuspended in 50 μl PBS. The total BALF cells were
counted double-blindly using a hemocytometer followed
by the differential counting of leukocytes (Giemsa staining;
two counts per slide, 300 cells per count).
MPO Activity Assay
MPO activity was determined using test kits pur-
chased from Jiancheng Bioengineering Institute of Nanjing
according to the instructions. The sample including 0.9 ml
homogenate and 0.1 ml of reaction buffer was heated to
37 °C in water, then the enzymatic activity was detected by
measuring the change in absorbance at 460 nm.
Histopathologic Evaluation
Histopathologic examination was performed on mice
that were not subjected to BALF collection. The lungs
were removed and stored in the fixative containing 10 %
paraformaldehyde in 0.1 M PBS (pH7.4) for 48 h at 4 °C.
The hematoxylin and eosin staining was carried out ac-
cording to the regular staining method.
Fig. 3. Effects of wogonoside on the productionof inflammatory cytokine
TNF-α, IL-1ß, and IL-6 in the BALF of LPS-induced ALI mice. BALF
was collected at 7 h following LPS challenge to analyze the inflammatory
cytokines TNF-α(a), IL-1ß (b), and IL-6 (c). The values presented are
mean ± SEM (n=6 in each group).
#
p<0.01 vs. control group; *p<0.05
and **p<0.01 vs. LPS group.
Fig. 4. Effects of wogonoside on MPO activity in lung tissues of LPS-
induced ALI. The values presented are the mean ± SEM (n=4–6ineach
group).
#
p<0.01 vs. control group; *p<0.05 and **p<0.01 vs. LPS group.
2008 Zhang, Ren, Yang, Guo, Zhang, Hou, Guo, Sun, and Liu

Western Blot Analysis
The lung tissue samples were frozen inliquid nitrogen
until homogenization. Then, the homogenate was centri-
fuged at 14,000×gfor 10 min at 4 °C. Protein concentra-
tions were determined by BCA protein assay kit. Equal
amounts of protein were loaded per well on a 10 % sodium
dodecyl sulfate polyacrylamide gel (SDS-PAGE) and
transferred onto polyvinylidene difluoride membrane.
The membranes were treated with 5 % skim milk for 2 h
at room temperature. Then, the membranes were incubated
with primary antibody (1:1,000) for 12 h at 4 °C. Subse-
quently, the membrane was incubated with the secondary
antibody at room temperature for 2 h.
Statistical Analysis
All data are expressed as means ± S.E.M. Statistically
significant differences between groups were determined by
ANOVA followed by Student’sttest. Statistical signifi-
cance was accepted p<0.05 or p<0.01.
RESULTS
Wogonoside Inhibited LPS-Induced Lung W/D Ratio
To investigate the effect of wogonoside on LPS-in-
duced lung edema, lung W/D ratio was detected. As shown
in Fig. 1, the W/D ratio was obviously increased after LPS
administration compared with control group. However,
administrations of wogonoside or DEX significantly re-
duced the increase of the lung W/D ratio.
Wogonoside Inhibited the Inflammatory Cell Count in
the BALF of LPS-Induced ALI Mice
The number of inflammatory cells, such as neutro-
phils and macrophages, in BALF was analyzed at 7 h after
LPS challenge. As shown in Fig. 2, LPS challenge signif-
icantly increased the number of total cells, neutrophils, and
macrophages compared with the control group (p<0.01).
Meanwhile, pretreatment with wogonoside (10, 20, and
40 mg/kg) and DEX (5 mg/kg) was found to significantly
decrease the number of total cells (p<0.01), neutrophils
(p<0.01), and macrophages (p<0.01).
Wogonoside Suppressed the Production of Cytokines in
the BALF of LPS-Treated ALI Mice
The effect of wogonoside on TNF-α, IL-1ß, and IL-6
production was analyzed at 7 h after LPS challenge by
ELISA. As shown in Fig. 3, the concentrations of TNF-α,
IL-6, and IL-1βin BALF were significantly increased after
LPS administration. Wogonoside (10, 20, and 40 mg/kg)
and DEX significantly reduced TNF-α(*p<0.05), IL-6
(*p<0.05 or **p<0.01), and IL-1ß (*p<0.05 or
**p<0.01) production induced by LPS.
Fig. 5. Effects of wogonoside on histopathological changes in lung tissues in LPS-induced ALI mice. Lungs from each experimental group were processed
for histological evaluation at 7 h after LPS challenge. Representative histological changes of lung obtained from mice of different groups. aControl group. b
LPS group. cLPS + DEX group. dLPS + wogonoside (10 mg/kg) group. eLPS + wogonoside (20 mg/kg) group. fLPS + wogonoside (40 mg/kg) group
(hematoxylin and eosin staining, magnification ×200).
2009Wogonoside Ameliorates LPS-Induced Acute Lung Injury

Effects of Wogonoside on the MPO Activity in ALI
Mice Induced by LPS
To assess the neutrophil accumulation within pulmo-
nary tissues, MPO activity was measured. After LPS ad-
ministration, the MPO activity in lung tissues was signifi-
cantly increased compared with the control group
(p<0.01). However, this increase was apparently reduced
by wogonoside (10, 20, and 40 mg/kg) (p<0.01) or DEX
(p<0.01) (Fig. 4).
Effects of Wogonoside on LPS-Mediated Lung
Histopathologic Changes
As shown in Fig. 5, lung sections obtained from mice
in LPS group showed characteristic histological changes,
including alveolar wall thickening, inflammatory cell infil-
tration, and pulmonary congestion (Fig. 5b). However,
LPS-induced pathological changes were significantly at-
tenuated by wogonoside (10, 20, and 40 mg/kg) and DEX
(5 mg/kg) treatment (Fig. 5c).
Effect of Wogonoside on TLR4 Expression and NF-κB
Activation in ALI Mice Induced by LPS
After LPS administration, the expression of TLR4 and
NF-κB was increased. Pretreatment with wogonoside (10,
20, and 40 mg/kg) inhibits the phosphorylation of IκB-α
and NF-κB p65 and the expression of TLR4 (Fig. 6).
DISCUSSION
Wogonoside, one flavonoid derived from the root of
Scutellaria baicalensis Georgi, has been shown to have
Fig. 6. Wogonoside pretreatment inhibited LPS-inducedTLR4 expression and NF-κB activation with Western blotting.
#
p<0.01 vs. control group; *p<0.05
and **p<0.01 group vs. LPS group.
2010 Zhang, Ren, Yang, Guo, Zhang, Hou, Guo, Sun, and Liu

anti-inflammatory activities [13]. The present study was to
examine the protective effect of wogonoside on LPS-in-
duced ALI. The results demonstrated that pretreatment
with wogonoside could notably inhibit lung edema, and
attenuate the pulmonary histologic changes. It also
inhibited TNF-α,IL-1β, and IL-6 production in BALF.
Furthermore, expressions of TLR4 and p65 NF-κBwere
also obviously reduced by wogonoside pretreatment when
compared with the LPS group. Wogonoside may be an
agent for preventing and treating LPS-induced ALI.
ALI/ARDS is characterized by the influx of serous
fluid into the air spaces, which leads to alveolar edema [14,
15]. To quantify the magnitude of pulmonary edema, the
lung W/D ratio was detected. Pretreatment with
wogonoside decreased the lung W/D ratio, which indicates
that wogonoside could attenuate the development of pul-
monary edema. MPO activity, a marker of neutrophil in-
flux into tissues, is assessed to quantify neutrophil accu-
mulation in tissues [16]. LPS-induced ALI is characterized
by neutrophil infiltration into lungs, exhibiting increased
MPO activity [17]. In this study, our results showed that
pretreatment with wogonoside significantly decreased
LPS-induced increases in MPO activity in the lung tissues.
Furthermore, histological analysis indicates that
wogonoside significantly attenuated tissue injury.
Cytokines play an important role in the initiation and
development of inflammation [18]. Reports demonstrated
that pro-inflammatory cytokines production played impor-
tant roles in the pathogenesis of ALI [19]. Elevated TNF-α,
IL-1ß, and IL-6 levels in BALF were observed in patients
with ALI or ARDS [20,21]. These cytokines play critical
roles in ALI and contribute to the severity of lung injury
[22]. TNF-αand IL-1βnot only amplify the inflammatory
cascade and cause inflammatory injury but also recruit
neutrophils into the lung and exhibit increased MPO activ-
ity [23]. Plasma IL-6 has been found to be a significant
predictor of morbidity and mortality in patients with ARDS
[24]. To detect the protective effect of wogonoside on LPS-
induced ALI, cytokines productions were detected by
ELISA. Our results showed that wogonoside significantly
inhibited the production of TNF-α, IL-1β, and IL-6 in
BALF induced by LPS.
Lipopolysaccharide (LPS) is a major component
of the outer membrane of Gram-negative bacteria,
which is recognized by TLR4. LPS binds to TLR4
and leads to the activation of NF-κB which induce
the transcription of a series of cytokine/chemokine
genes that are involved in the initiation or regulation
of the inflammatory response [25]. NF-κB is a dom-
inant transcription factor responsible for inflammation
[26]. Normally, NF-κB is sequestered in the cyto-
plasm by its inhibitors of NF-κB(IκBs). Once stim-
ulated by LPS, NF-κB is activated and regulates
TNF-α,IL-1β, and IL-6 expression [27]. To charac-
terize the anti-inflammatory mechanism of
wogonoside on LPS-induced ALI, the effects of
wogonosideonTLR4expressionandNF-κB activa-
tion were detected. The results showed that
wogonoside could inhibit TLR4 expression and NF-
κB activation induced by LPS.
In conclusion, our results provided the evidence that
pretreatment of wogonoside have a protective effect on
LPS-induced ALI in mice. The anti-inflammatory mecha-
nism of wogonoside may be attributed to inhibition of
TLR4 expression, which subsequently inhibits NF-κBac-
tivation and pro-inflammatory cytokines production.
Conflict of Interest. All authors declare that they have no
conflict of interest.
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