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Magnolol Inhibits the Inflammatory Response in Mouse Mammary Epithelial Cells and a Mouse Mastitis Model

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Mastitis comprises an inflammation of the mammary gland, which is almost always linked with bacterial infection. The treatment of mastitis concerns antimicrobial substances, but not very successful. On the other hand, anti-inflammatory therapy with Chinese traditional medicine becomes an effective way for treating mastitis. Magnolol is a polyphenolic binaphthalene compound extracted from the stem bark of Magnolia sp., which has been shown to exert a potential for anti-inflammatory activity. The purpose of this study was to investigate the protective effects of magnolol on inflammation in lipopolysaccharide (LPS)-induced mastitis mouse model in vivo and the mechanism of this protective effects in LPS-stimulated mouse mammary epithelial cells (MMECs) in vitro. The damage of tissues was determined by histopathology and myeloperoxidase (MPO) assay. The expression of pro-inflammatory cytokines was determined by enzyme-linked immunosorbent assay (ELISA). Nuclear factor-kappa B (NF-κB), inhibitory kappa B (IκBα) protein, p38, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and Toll-like receptor 4 (TLR4) were determined by Western blot. The results showed that magnolol significantly inhibit the LPS-induced TNF-α, IL-6, and IL-1β production both in vivo and vitro. Magnolol declined the phosphorylation of IκBα, p65, p38, ERK, and JNK in LPS-stimulated MMECs. Furthermore, magnolol inhibited the expression of TLR4 in LPS-stimulated MMECs. In vivo study, it was also observed that magnolol attenuated the damage of mastitis tissues in the mouse models. These findings demonstrated that magnolol attenuate LPS-stimulated inflammatory response by suppressing TLR4/NF-κB/mitogen-activated protein kinase (MAPK) signaling system. Thereby, magnolol may be a therapeutic agent against mastitis.
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Magnolol Inhibits the Inflammatory Response in Mouse
Mammary Epithelial Cells and a Mouse Mastitis Model
Wang Wei,
1
Liang Dejie,
1
Song Xiaojing,
1
Wang Tiancheng,
1
Cao Yongguo,
1
Yang Zhengtao,
1
and Zhang Naisheng
1,2
AbstractMastitis comprises an inflammation of the mammary gland, which is almost always linked
with bacterial infection. The treatment of mastitis concerns antimicrobial substances, but not very suc-
cessful. On the other hand, anti-inflammatory therapy with Chinese traditional medicine becomes an
effective way for treating mastitis. Magnolol is a polyphenolic binaphthalene compound extracted from
the stem bark of Magnolia sp., which has been shown to exert a potential for anti-inflammatory activity.
The purpose of this study was to investigate the protective effects of magnolol on inflammation in
lipopolysaccharide (LPS)-induced mastitis mouse model in vivo and the mechanism of this protective
effects inLPS-stimulated mouse mammary epithelial cells (MMECs) in vitro. The damage of tissues was
determined by histopathology and myeloperoxidase (MPO) assay. The expression of pro-inflammatory
cytokines was determined by enzyme-linked immunosorbent assay (ELISA). Nuclear factor-kappa B
(NF-κB), inhibitory kappa B (IκBα) protein, p38, extracellular signal-regulated kinase (ERK), c-Jun N-
terminal kinase (JNK), and Toll-like receptor 4 (TLR4) were determined by Western blot. The results
showed that magnolol significantly inhibit the LPS-induced TNF-α, IL-6, and IL-1βproduction both
in vivo and vitro. Magnolol declined the phosphorylation of IκBα, p65, p38, ERK, and JNK in LPS-
stimulated MMECs. Furthermore, magnolol inhibited the expression of TLR4 in LPS-stimulated
MMECs. In vivo study, it was also observed that magnolol attenuated the damage of mastitis tissues
in the mouse models. These findings demonstrated that magnolol attenuate LPS-stimulated inflamma-
tory response by suppressing TLR4/NF-κB/mitogen-activated protein kinase (MAPK) signaling system.
Thereby, magnolol may be a therapeutic agent against mastitis.
KEY WORDS: magnolol; lipopolysaccharide (LPS); mastitis; cytokine; nuclear factor-kappaB (NF-κB);
mitogen-activated protein kinases (MAPKs).
INTRODUCTON
Mastitis refers to inflammation which is the normal
defense reaction of the host immune system. Many differ-
ent bacteria and fungi are known to cause mastitis, and
Staphylococcus aureus and Streptococcus agalactiae are
two of the most common species that have infected udder
as main reservoir [1]. Moreover, Escherichia coli is con-
sidering correlation with mastitis, particularly around par-
turition or during early lactation [2]. Lipopolysaccharide
(LPS), a major component of the outer membrane of gram-
negative bacteria induces mouse or bovine mastitis models
and causes the production of pro-inflammatory cytokines,
which have proven to be invaluable tools for the study of
coliform mastitis [3]. Mammary gland epithelial cells have
shown the capacity to increase an inflammatory or active
defense reaction in their own right mediated by cytokines
[4]. While interacting with invading bacteria, mammary
epithelial cells are able to generate multiple inflammatory
cytokines as well as other immune cells [5]. Therefore,
acute inflammatory response may cause tissue damages,
and anti-inflammatory therapy is a crucial way to alleviate
symptoms in mastitis.
The innate immune system is poised to rapidly re-
spond to the earliest stages in infection through Toll-like
1
Department of Clinical Veterinary Medicine, College of Veterinary
Medicine, Jilin University, Changchun, Jilin Province 130062,
Peoples Republic of China
2
To whom correspondence should be addressed at Department of Clin-
ical Veterinary Medicine, College of Veterinary Medicine, Jilin
University, Changchun, Jilin Province 130062, Peoples Republic
of China. E-mail: zhangns@jlu.edu.cn
0360-3997/15/0100-0016/0 #2014 Springer Science+Business Media New York
Inflammation, Vol. 38, No. 1, February 2015 (#2014)
DOI: 10.1007/s10753-014-0003-2
16
receptors (TLRs) and other pattern recognition receptors
(PRRs) [6]. TLR4, a key PRRs, ligated with LPS and
recruited the downstream signaling pathway: NF-κBand
mitogen-activated protein kinases (MAPKs) through re-
ceptor dimerization [7]. Activated NF-κB and MAPK sig-
naling pathways regulated the inflammatory process by
promoting the production of pro-inflammatory cytokines,
such as tumor necrosis factor-α(TNF-α) and interleukin-6
(IL-6) [8,9]. Since severe inflammatory responses are
representative features of mastitis and play a key role in
the pathogenesis of tissue damage [10], any substances that
inhibit the activation of TLR4 signaling pathways are
considered as potential anti-inflammatory agents to prevent
tissue damage during the development of mastitis.
Magnolol (5, 59-diallyl-2, 29-dihydroxybiphenyl) is a
major component isolated from the stem bark of Magnolia
sp. (Fig. 1), including Magnolia obovate and Magnolia
officinalis [11]. It has been used to treat cough, diarrhea,
and allergic rhinitis in China, Korea, and Japan [12].
Magnolol has been reported to suppress the overproduction
of nitric oxide (NO) and TNF-αin LPS-stimulated macro-
phages [13], to decrease inflammatory cytokines produc-
tion in THP-1 cells [14]. Recently, the mechanisms under-
lying the anti-inflammatory effect of magnolol have been
reported. Fu et al.[15] confirmed that magnolol reveals an
anti-inflammatory property by downregulating the activa-
tion of NF-κB and MAPK signaling pathways and the
release of pro-inflammatory cytokines in LPS-stimulated
macrophages RAW 264.7 cells. Thus, the objective of this
study is to evaluate anti-inflammatory effect of magnolol
using LPS-induced mastitis in mice then to investigate
whether the effects are through the control of TLR4-
mediated NF-kB and MPAK signaling pathways in LPS-
stimulated mouse mammary epithelial cells (MMECs).
MATERIALS AND METHODS
Chemicals and Reagents
Magnolol (purity >98 %) was purchased from the
National Institute for the Control of Pharmaceutical and
Biological Products (Beijing, China). Dimethyl sulfoxide
(DMSO), LPS (E. coli 055:B5), and 3-(4, 5-dimethylthia-
zol-2-y1)-2, 5-diphenyltetrazolium bromide (MTT) were
purchased from Sigma Chemical Co. (St. Louis, MO,
USA). Dulbeccos modified Eaglesmedium(DMEM
F12/1:1) and fetal calf serum (FCS) and trypsin/EDTA
were purchased from Hyclone (Logan, UT, USA).
Collagenase I and II were purchased from Invitrogen
Corp. (Carlsbad, California, USA). Epidermal growth fac-
tor (EGF), transferrin, and T3 were purchased from
PeproTech. Dexamethasone (DEX) sodium phosphate in-
jection (no. H41020055) was purchased from Changle
Pharmaceutical Co. (Xinxiang, Henan, China). The mye-
loperoxidase (MPO) determination kit was purchased from
the Jiancheng Bioengineering Institute of Nanjing
(Nanjing, Jiangsu Province, China). Mouse TNF-αand
IL-6 enzyme-linked immunosorbent assay (ELISA) kits
were purchased from Biolegend (San Diego, CA, USA).
All of the rabbit monoclonal antibodies and mouse mono-
clonal antibodies were purchased from Cell Signaling
Technology Inc. (Beverly, MA, USA). Horseradish
peroxidase-conjugated goat anti-rabbit and goat-mouse
antibodies were provided by GE Healthcare
(Buckinghamshire, UK). All other chemicals were of
reagent grade.
In Vivo Study
Animals
BALB/c mice, 68 weeks old (18 male and 36 fe-
male), were purchased from the Center of Experimental
Animals of Baiqiuen Medical College of Jilin University
(Jilin, China). Mice were fed routinely for 23daystofit
the environment, and then one male and two female mice
were housed in each cage with water and food supplied ad
libitum. All mice were kept in a pathogen-free condition,
with a 12-h light/dark cycle. The experiments followed the
guidelines for the care and use of laboratory animals pub-
lished by the US National Institutes of Health.
Fig. 1. Chemical structure of magnolol.
17Magnolol Inhibits the Inflammatory Response
Mastitis Mouse Model and Grouping Design
The lactating mice, 57 days after birth of the off-
spring, were randomly divided into six groups: blank con-
trol group, LPS group, magnolol (5, 10, and 20 mg/kg)+
LPS groups, and dexamethasone (DEX)+ LPS group. Each
group contained six mice. The establishment of mastitis
mouse model was followed by Li et al.[16]. Briefly, the
pups were removed 1 h before inducing inflammation of
the mammary gland, then magnolol (5, 10, and 20 mg/kg)
was given by intraperitoneal (i.p.) injection, and DEX
(0.5 mg/kg) was used as a positive control. Blank control
and LPS group mice were given an equal volume of sterile
water intraperitoneally. After that, the lactating mice were
anesthetized by urethane (15 g urethane dissolved in
150 ml physiological saline, 1.5 g/kg i.p.). A 100-μlsy-
ringe with a 30-gauge blunt needle was used to inoculate
both L4 (on the left) and R4 (on the right) abdominal
mammary glands. The anesthetized mice were laid on their
backs under a binocular microscope. The teats and the
surrounding area were prepped with 70 % ethanol. Each
udder canal was exposed by a small cut at the near end of
the teat and then challenged via teat canal catheterization
with 10 μg of LPS dissolved in 50 μl non-pyrogenic
phosphate-buffered saline (PBS). At 12 h after LPS infu-
sion, the mice were killed using CO
2
inhalation and mam-
mary tissues were collected and stored at 80 °C until
analysis.
Histopathological Examination
Mammary glands for histopathological examination
were fixed in 10 % formalin for 4872 h, dehydrated with
graded alcohol and embedded in paraffin, and then stained
with hematoxylin and eosin (H&E).
Myeloperoxidase Assay
The whole right mammary gland tissues were
weighed and homogenized with PBS (1:9, w/v) on ice
and then centrifuged (2,000g×40 min at 4 °C). The super-
natant was collected and centrifuged again (2,000g×40
min at 4 °C) to remove any remaining lipid. MPO activity
in homogenates was determined by the manufacturers
instruction.
Enzyme-Linked Immunosorbent Assay
The preparation of tissue homogenates was the same
to MPO assay, then the homogenate of mammary gland
tissue samples was centrifuged to obtain the supernatant for
analyzing the level of TNF-α,IL-1β, and IL-6 by ELISA
kits in accordance with the manufacturers instructions.
In Vitro Study
Cell Culture and Treatment
Primary cultured MMECs were prepared as previous-
ly described by Smalley [17]. Briefly, mammary tissues
were removed aseptically from 68 week-old gravid
BALB/c mice and minced into pasties. The minced tissues
were digested by collagenase I/II/trypsin mixture
(Invitrogen, Carlsbad, California, USA) and shaken at
37 °C. After filtration, to remove unassociated tissue and
debris, the cells were collected by centrifugation at 250g
for 5 min three times. Cell pellets were resuspended in
DMEM/F12 containing 10 % FCS and incubated for 1 h at
37 °C, then collected the supernatant. This step was repeat-
ed three times to clear away fibroblasts. After the last
incubation, cells were resuspended in DMEM/F12 contain-
ing 10 % FCS, 0.5 % Transferrin, 0.1 % T3, and 0.5 %
EGF and cultured at 37 °C with 5 % CO
2
. The medium was
exchanged every day. For experiments, MMECs were
stimulated with 1 μg/ml LPS alone or in the presence of
various concentrations (12.5, 25, or 50 μg/ml) of magno-
lol. The magnolol stock solution was prepared with
DMSO. The control cells were incubated for 24 h in the
absence of LPS or magnolol.
MTT Assay for Cell Viability
Cell viability was measured using a standard MTT
assay. MMECs were plated and incubated for 3 days in 96-
well plate. Magnolol was dissolved in DMSO and the
DMSO concentration in the assay did not exceed 0.1 %.
Then various concentrations of magnolol (0, 12.5, 25, 50,
100, and 200 μg/ml) were added to the wells, followed by
stimulation with 1 μg/ml of LPS for 18 h. Then, 20 μl
MTT (5 mg/ml) was added to each well, and the cells were
further incubated for an additional 4 h. The supernatants
were removed and the formation of formazan was resolved
with 150 μl/well of DMSO. The optical density was mea-
sured at 570 nm on a microplate reader (TECAN, Austria).
Enzyme-Linked Immunosorbent Assay
MMECs were seeded in 24-well plates and incubated
in the presence of either LPS 1 μg/ml alone or LPS plus
magnolol 12.5, 25, or 50 μg/ml for 18 h. Cell-free super-
natants were subsequently employed for the proinflamma-
tory cytokine assays using a mouse ELISA kit, according
18 Wei, Dejie, Xiaojing, Tiancheng, Yongguo, Zhengtao, and Naisheng
to the manufacturers instructions (BioLegend, Inc, San
Diego, CA, USA).
Wester n Blo t Analy si s
MMECs were incubated, then pretreated with various
concentrations of magnolol for 1 h and then stimulated
with LPS (1 μg/ml) for 1 h. The control and magnolol-
treated cells were washed triple with cold PBS and total
proteins from cells were extracted by M-PER Mammalian
Protein Extraction Reagent (Thermo Scientific, USA).
Protein concentration was determined by bicinchoninic
acid (BCA) method. The protein concentrations in super-
natants were determined, and aliquots of protein (20 μg)
were separated by sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) and transferred onto a
nitrocellulose membrane. The membrane was blocked with
5 % skim milk in Tris-buffered saline with Tween 20 (TBS-
T) for 1 h and then incubated with various primary anti-
bodies: phospho-IκBα,NF-κB p65, phospho-extracellular
signal-regulated kinase (ERK)1/2, phospho-c-Jun N-
terminal kinase (JNK), or phospho-p38 and β-actin at
4 °C overnight. Subsequently, the membrane was washed
five times with TBS-T for 10 min and incubated with the
secondary antibody conjugated with horseradish peroxi-
dase at room temperature for 1 h. The membrane was again
washed three times for 10 min with TBS-T, and finally, the
results were visualized by using an enhanced chemilumi-
nescence (ECL) Western blotting kit (Thermo Scientific,
USA) and tested by ECL Plus Western Blotting Detection
System (Amersham Life Science, UK).
Statistical Analysis
All of the data are expressed as the mean± SEM. The
differences among the various experimental groups were
analyzed by a one-way ANOVA (Dunnettsttest) and a
two-tailed Studentsttest. P<0.05 was considered to be
statistically significant.
RESULTS
In Vivo Study
Histopathological Findings of Mammary Gland Tissues
Mammary gland tissues in different groups were har-
vested at 12 h after LPS challenge for evaluating the
pathological changes. The mammary gland sections were
subjected to H&E staining. There was no pathological
change observed in blank control group (Fig. 2a). In LPS
group (Fig. 2b), the mice exhibited obvious increase in
inflammatory cell infiltration, containing neutrophils, mac-
rophages, etc., and the construction of mammary gland
tissues were significantly damaged, including interstitial
edema and thickening of the alveolus wall. However, these
pathological changes were relieved in DEX group (Fig. 2c)
and magnolol treatment groups with the doses of 5 mg/kg
(Fig. 2d), 10 mg/kg (Fig. 2e), and 20 mg/kg (Fig. 2f).
Effect of Magnolol on MPO Activity
MPO activity is a measure of mammary gland paren-
chymal phagocyte infiltration [18]aswellascytokines.
The MPO activity of mammary glands in LPS group was
obviously raised, compared with the blank control group.
Treatment of magnolol at a dose of 5 mg/kg could not
noticeably reduce the MPO activity, while significantly
decline of MPO activity was appeared at doses of 10 and
20 mg/kg, compared with the LPS group. Meanwhile, the
MPO activity of mammary gland tissue in DEX treatment
group was significantly decreased, compared with the LPS
group (Fig. 3).
Magnolol Dose-Dependently Suppresses
Pro-inflammatory Cytokine Production in LPS-Induced
Mouse Mastitis
To evaluate the levels of cytokines in the tissue ho-
mogenate of LPS-induced mammary gland, the mammary
gland was collected 12 h after LPS challenge. The level of
TNF-α,IL-1β, and IL-6 were measured by ELISA. The
above pro-inflammatory cytokines in LPS group were
significantly increased compared with those in blank con-
trol group. However, magnolol inhibited the expression of
TNF-α,IL-1β, and IL-6 in a dose-dependent manner, and
DEX significantly reduced the expression of those com-
pared to that in the LPS group (Fig. 4).
In Vitro Study
Effects of Magnolol on Cell Viability of MMECs
The potential cytotoxicity of magnolol was examined
by MTT assay in the presence or absence of LPS (1 μg/ml)
to determine the effective concentration for treatment.
Cells were treated with various magnolol concentrations
(0, 12.5, 25, 50, 100, and 200 μg/ml) and co-treated LPS
(1 μg/ml) for 18 h. The results showed that LPS (1 μg/ml)
and the concentrations of magnolol at 12.5, 2,5 and
50 μg/ml were not attributable to cytotoxic effects (Fig. 5).
19Magnolol Inhibits the Inflammatory Response
Magnolol Dose-Dependently Represses Pro-inflammatory
Cytokine Production in LPS-Stimulated MMECs
To determine the effects of magnolol on TNF-αand
IL- 6 production by LPS-stimulated MMECs, ELISA anal-
yses were conducted. Cells were pretreated with magnolol
at different concentrations (12.5, 25, or 50 μg/ml), then
stimulated with LPS, and the levels of TNF-αand IL-6
were measured by ELISA. As shown in Fig. 6, the group
treatment with LPS showedsignificantly increased produc-
tion of pro-inflammatory cytokines in culture supernatants
of MMECs compared with the control group, while treat-
ment with magnolol at concentrations of 12.5, 25, or
50 μg/ml showed markedly inhibited production of
TNF-α,IL-1β, and IL-6 in LPS-stimulated MMECs
(Fig. 6). Specially, magnolol showed a strong suppres-
sive effect on the production of TNF-α,IL-1β, and IL-6 at
concentration of 50 μg/ml, respectively, which indicated
that magnolol inhibits the production of these proinflam-
matory cytokines involved in the inflammation process in a
dose-dependent manner.
Magnolol Inhibits the Degradation and Phosphorylaton
of NF-κB Pathway in LPS-Stimulated MMECs
NF-κB regulates both innate and adaptive immune
responses. It is activated rapidly in response to a wide
range of stimuli, including pathogens, stress signals, and
proinflammatory cytokines, such as LPS and TNF-α[19].
In order to evaluate the inhibitory effect of magnolol on
NF-κB signaling pathway, the expression of
phosphorylated-p65 and degraded-IκBαin LPS-
stimulated MMECs, which pretreated with magnolol, were
examined by Western blot analysis. In Fig. 7, the phos-
phorylation of p65 after LPS treatment was dramatically
inhibited by magnolol in a dose-dependent manner when
Fig. 2. Histopathological findings. Histopathological histopathology of mammary tissue after infusion with LPS (×100). Mammary tissue of blank control
group (a), LPS group (b), and treatment groups administered DEX 5 mg/kg (c), magnolol 5 mg/kg (d), 10 mg/kg (e), and 20 mg/kg (f).
Fig. 3. Myeloperoxidase (MPO) activity assay. MPO activity in mamma-
ry tissue from blank control group, LPS group, and treatment groups
administered 5, 10, and 20 mg/kg magnolol and 5 mg/kg DEX. Data are
presented as mean±SEM (n=6).
#
p<0.01 significantly different from
blank control group; *p<0.05; ** p<0.01 significantly different from
LPS group.
20 Wei, Dejie, Xiaojing, Tiancheng, Yongguo, Zhengtao, and Naisheng
compared with the control group. Furthermore, IκBαwas
markedly degraded after treatment with LPS, whereas
treatment with magnolol prevented this degradation in a
dose-dependent manner. These data suggest that magnolol
may block the activation of NF-κB signaling in LPS-
stimulated MMECs.
Magnolol Suppresses the Phosphorylation of ERK, JNK,
and p38 MAPKs in LPS-Stimulated MMECs
MAPK proteins play critical roles in the induction
of pro-inflammatory mediators, such as TNF-αand IL-
6 as well as in the activation of transcription factors,
such as NF-κB[20]. Thus, MAPK signaling pathways
provide specific targets for inflammatory responses. To
confirm whether MAPKs are involved in the inhibition
of proinflammatory cytokines production by magnolol,
phosphorylation of ERK, JNK, and p38 MAPKs in
LPS-stimulated MMECs (pretreatment with magnolol)
was examined by Western blot. As shown in Fig. 8,LPS
treatment caused a strong increase in the phosphoryla-
tion of ERK, JNK, and p38MAPKs. However, co-
treatment with various concentrations of magnolol re-
duced the levels of all MAPK phosphorylations.
Especially, magnolol significantly inhibited the activi-
ties of ERK MAPKs in a dose-dependent manner
(Fig. 8). These suggests that magnolol decrease the
phosphorylation of ERK, JNK, and p38 MAPKs in
LPS-stimulated MMECs.
Magnolol Reduces the Expression of TLR4
in LPS-Stimulated MMECs
TLR4 signaling plays a vital role in inflammatory
response. Activation of TLR4 signaling by LPS induces
the production of inflammatory cytokines through the ac-
tivation of NF-κB and MAPK signaling pathways. To
investigate whether magnolol affect TLR4 signaling, the
expression of TLR4 was determined by Western blot. The
results showed that magnolol downregulated the expres-
sionofTLR4inLPS-stimulatedMMECsinadose-
dependent manner (Fig. 7).
Fig. 4. Cytokine assay. TNF-α,IL-1β, and IL-6 levels in mammary tissue
from blank control group, LPS group and treatment groups administered
magnolol 5, 10, and 20 mg/kg and 5 mg/kg DEX. Data represent the
contents of 1 ml supernatant of mammary homogenate and are presented
as mean±SEM (n=6).
#
p<0.01 significantly different from blank control
group; *p<0.05; **p<0.01 significantly different from LPS group.
Fig. 5. Effects of magnolol on LPS-stimulated cell viability in MMECs.
MMECs were treated with the different concentrations of magnolol (0,
12.5, 25, 50, 100, and 200 μg/ml) in the absence or presence of 1 μg/ml
LPS for 18 h. Cell viability was assessed by MTT assay. The values are
presented as mean±SEM of three independent experiments.
21Magnolol Inhibits the Inflammatory Response
Fig. 6. Magnolol inhibits LPS-stimulated cytokine production in a dose-dependent manner. Cells were treated with 1 μg/ml LPS in absence or presence of
magnolol (12.5, 25, or 50 μg/ml) for 18 h. Levels of TNF-αand IL-6 in culture supernatants were measured by ELISA. The values presented are the
mean±SEM of three independent experiments.
#
p<0.01 vs. the control group; *p<0.05; **p<0.01 vs. the LPS group.
Fig. 7. Magnolol inhibits LPS-stimulated activation of NF-κB (p65), the degradation of IκBα, and the expression of TLR4. Cells were treated with various
concentrations (12.5, 25, or 50 μg/ml) of magnolol for 1 h, followed by continuous incubation with or without LPS (1 μg/ml) for the next 1 h. Cell lysates
were analyzed by the Western blot analysis using specific antibodies. β-actin was used as a control. The values presented are the mean± SEM of three
independent experiments.
#
p<0.01 vs. the control group; *p<0.05; **p<0.01 vs. the LPS group.
22 Wei, Dejie, Xiaojing, Tiancheng, Yongguo, Zhengtao, and Naisheng
DISCUSSION
Mastitis is defined as an inflammation of the mam-
mary gland. It usually occurs primarily in response to
intramammary bacterial infection, but also to intramam-
mary mycoplasmal, fungal, or algal infections [21]. Bovine
mastitis is a major disease affecting dairy cattle worldwide
and is a costly disease for dairy producers [22]. But in
recent years, conventional methods have frequently been
unable to prevent intramammary infection, antibiotics are
not very effective in the treatment of mastitis and have a
negative impact on human health [23]. Mammary epithe-
lial cells, the most numerous cells in the mammary gland,
are parts of the functional unit of the udder, which are
responsible for the synthesis of many components in milk
that provide nutritional and immunological support to the
offspring [24]. Moreover, experiments with LPS-
stimulated mastitis in rats suggest that large-scale produc-
tion of proinflammatory cytokines, such as TNF-α,might
contribute to tissue damage [25]. Many traditional Chinese
medicines (TCM) extracts has been verified to have anti-
inflammatory properties on mastitis [26,27]. Magnolol has
been reported the anti-inflammatory effect [28]andourlab
further found that the role of magnolol mainly by down-
regulated the expression of TLR4 and thus attenuated
TLR4-mediated activation of NF-κB and MAPK signaling
and release of proinflammatory cytokines in LPS-
stimulated macrophages RAW 264.7 cells [15]. Thus, in
the present study, we evaluated the anti-inflammatory
effects of magnolol in LPS-induced mastitis and elucidated
the potential anti-inflammatory mechanism.
In vivo, histopathological observation indicated that
magnolol markedly inhibited the infiltration of inflamma-
tory cells and decreased mammary damage. Some
Fig. 8. Magnolol inhibits LPS-stimulated MAPK activation. Cells were pretreated with magnolol (12.5, 25, or 50 μg/ml) for 1 h and then treated with
1μg/ml LPS for 1 h. Protein samples were analyzed by Western blot with specific antibodies. β-actin was used as a control. The values presented are the
mean±SEM of three independent experiments.
#
p<0.01 vs. the control group; *p<0.05; **p<0.01 vs. the LPS group.
23Magnolol Inhibits the Inflammatory Response
cytokines were responsible for the damage of mammary
gland tissues, such as TNF-αand IL-1β[29], and the
results of cytokines assay were proved that in this study,
magnolol inhibited the secretion of TNF-α, IL-6, and IL-
1βin LPS-induced mastitis mouse model. The above
results were consistent with MPO activity assay. MPO
activity, a marker of neutrophil influx into tissue, is directly
proportional to the number of neutrophils in the tissue [30].
As shown in Fig. 3, we confirmed that pretreatment with
magnolol significantly reduced the MPO activity of mam-
mary gland and decreased mammary neutrophilia.
Accordingly, it is proposed that magnolol could reduce
the inflammatory cell infiltration and decrease mammary
damage through inhibiting the production of pro-
inflammatory cytokines.
In vitro, we further confirmed the effects of magnolol
in anti-inflammation by LPS-stimulated MMECs.
Proinflammatory cytokines, such as IL-1β, IL-2, IL-6,
IL-10, and TNF-α, are crucial mediators in a range of acute
and chronic responses to inflammatory diseases, while
TNF-αand IL-6, as the principal proinflammatory cyto-
kines, are involved in the pathophysiology of endotoxin-
induced mastitis, increase the expression of adhesion mol-
ecules, and trigger the production of reactive oxygen spe-
cies (ROS) [3134]. Accordingly, in this study, we first
investigated the suppressive effect of magnolol on the
production of TNF-αandIL-6inLPS-stimulated
MMECs by ELISA. Moreover, we checked the potential
cytotoxicity of magnolol using MTT assay; magnolol did
not affect the viability of MMECs, up to a concentration of
50 μg/ml (Fig. 5). The following experiments were in
accordance with this concentration. The ELISA results
showed that the production of TNF-αand IL-6 in LPS-
stimulated MMECs was decreased by various concentra-
tions of magnolol (12.5, 25, or 50 μg/ml) in a dose-
dependent manner (Fig. 6). These findings indicated that
the inhibitory effect of magnolol on TNF-αand IL-6 have
important implications for decreasing the inflammatory
response.
NF-κB, a main regulatory transcription factor, exists
as homo-dimeric or hetero-dimeric complexes of p50 and
p65 subunits bound to IκB and plays a key role in cellular
responses to various stimuli such as stress, cytokines, free
radicals, ultraviolet radiation, oxidized LDL, and bacterial
or viral antigens [35]. Upon stimulation, activated IκBα
kinase phosphorylates IκBαand phosphorylated IκBαare
ubiquitinated and degraded through proteasome pathway
and made the phosphorylation of NF-κBp65.Activated
NF-κB induces the expression ofnumerousgenes involved
in innate and adaptive immune regulation, inflammatory
responses, cell adhesion, osteogenesis, and anti-apoptosis
[36,37]. Thus, it is not surprising that the inhibition of NF-
κB regulation is closely associated with inflammatory
responses. In addition, it is well known that various natural
herbs are responsible for anti-inflammatory responses via
the suppression of NF-κB signaling pathway [38,39].
Thus, in this study, we investigated whether magnolol
inhibits the phosphorylation of NF-κB p65 and degrada-
tion of IκBαprotein by Western blot. The data showed that
LPS-stimulated phosphorylation of p65 was blocked by
magnolol in a dose-dependent manner through the inhibi-
tion of IκBαdegradation (Fig. 7). The results demonstrat-
ed that magnolol may inhibit the production of TNF-αand
IL-6 via inhibition of NF-κB by blocking IκBαdegrada-
tion and p65 phosphorylation.
Mitogen-activated protein kinase (MAPK) signaling
pathways, comprised of three MAP kinase cascades (ex-
tracellular signal-regulated kinases (ERKs), c-Jun NH2-
terminal kinases (JNKs,) and p38 MAP kinase), present
in all eukaryotic cells. Upon activation of the MAP kinases,
transcription factors present within the cytoplasm or nucle-
uses are phosphorylated and activated, leading to expres-
sion of target genes resulting in a biological response,
including the regulation of proinflammatory mediator ex-
pression [40]. Thus, we investigated the effect of magnolol
on the phosphorylation of ERK, JNK, and p38MAPKs in
LPS-stimulated MMECs by Western blot. The results
showed that magnolol reduced the production of proin-
flammatory cytokines in MMECs maybe by suppressing
the MAPK signaling pathway, including the inhibition of
the phosphorylation of ERK, JNK, and p38MAPKs
(Fig. 8).
TLR4 is a pattern recognition receptor; it responds to
LPS and triggers the activation of NF-κB and MAPK
signaling pathways. The latter regulates the release of
proinflammatory cytokines [41]. We investigated whether
the anti-inflammatory activity of magnolol exerted though
TLR4-mediated signaling in LPS-stimulated MMECs by
Western blot. Our results showed that magnolol inhibits the
expression of TLR4 in LPS-stimulated MMECs (Fig. 7).
Based on all of the above, our results suggested that the
magnolol suppresses the production of pro-inflammatory
cytokine by decreasing the expression of TLR4 and inhib-
iting the activation of NF-κB and MAPKs in LPS-
stimulated MMECs.
In conclusion, in vivo, magnolol could alleviate the
inflammation reaction from mastitis caused by LPS. In
vitro, magnolol suppressed the expression of TLR4 upre-
gulated by LPS and reduced the production of pro-
inflammatory mediators regulated by the NF-κBand
24 Wei, Dejie, Xiaojing, Tiancheng, Yongguo, Zhengtao, and Naisheng
MAPK in LPS-stimulated MMECs. These findings sug-
gested that magnolol may be a useful agent for preventing
and treating LPS-induced mastitis.
ACKNOWLEDGMENTS
This work was supported bya grant fromthe National
Natural Science Foundation of China (Nos. 31272622,
31201925), the Research Fund for the Doctoral Program
of Higher Education of China (Nos. 20110061130010,
20120061120098), and Jilin Province Science Foundation
for Youths (No. 20130522087JH).
AUTHORSCONTRIBUTIONS
Naisheng Zhang and Zhengtao Yang conceived and
designed the paper. Wei Wang, Xiaojing Song, and Tian-
cheng Wang executed the experiment and analyzed the
samples. Dejie Liang and Yongguo Cao analyzed the data.
All authors interpreted the data, critically revised the man-
uscript for important intellectual contents, and approved
the final version.
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26 Wei, Dejie, Xiaojing, Tiancheng, Yongguo, Zhengtao, and Naisheng
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