Inflammation ( # 2013)

DOI: 10.1007/s10753-013-9732-x

Thymol Inhibits LPS-Stimulated Inflammatory Response via

Down-Regulation of NF-κB and MAPK Signaling Pathways
in Mouse Mammary Epithelial Cells

Dejie Liang,1 Fengyang Li,1 Yunhe Fu,1 Yongguo Cao,1 Xiaojing Song,1 Tiancheng Wang,1
Wei Wang,1 Mengyao Guo,1 Ershun Zhou,1 Depeng Li,1 Zhengtao Yang,1,2 and Naisheng Zhang1,2

Abstract—Thymol is a natural monoterpene phenol primarily found in thyme, oregano, and

tangerine peel. It has been shown to possess anti-inflammatory property both in vivo and in
vitro. In the present paper, we studied the anti-inflammatory effect of thymol in lipopolysac-
charide (LPS)-stimulated mouse mammary epithelial cells (mMECs). The mMECs were stim-
ulated with LPS in the presence or absence of thymol (10, 20, 40 μg/mL). The concentrations
of tumor necrosis factor α (TNF-α), interleukin (IL)-6, and IL-1β in the supernatants of culture
were determined using enzyme-linked immunosorbent assay. Cyclooxygenase-2 (COX-2), ind-
ucible nitric oxide synthase (iNOS), extracellular signal-regulated protein kinase (ERK), c-Jun
N-terminal kinase (JNK), nuclear factor-κB (NF-κB), and inhibitor protein of NF-κB (IκBα)
were measured using western blot. The results showed that thymol markedly inhibited the
production of TNF-α and IL-6 in LPS-stimulated mMECs. The expression of iNOS and COX-2
was also suppressed by thymol in a dose-dependent manner. Furthermore, thymol blocked the
phosphorylation of IκBα, NF-κB p65, ERK, JNK, and p38 mitogen-activated protein kinases
(MAPKs) in LPS-stimulated mMECs. These results indicate that thymol exerted anti-inflam-
matory property in LPS-stimulated mMECs by interfering the activation of NF-κB and MAPK
signaling pathways. Thereby, thymol may be a potential therapeutic agent against mastitis.

KEY WORDS: thymol; lipopolysaccharide (LPS); mammary epithelial cells (MECs); nuclear factor-kappaB
(NF-κB); mitogen-activated protein kinases (MAPKs).

INTRODUCTION
Inflammation is a complex pathophysiological pro-
cess mediated by a variety of signaling molecules, which
were produced by leukocytes, macrophages, mast cells,
and many other cell types [1]. In the mammary gland of
mammals, macrophages are the prime candidates for the
first line of defense against invaded bacteria and their
products. Mammary alveolar epithelial cells not only come
in direct contact with the invading bacteria and act as more
1 effects of mastitis [2, 4, 5]. LPS-induced inflammation
Department of Clinical Veterinary Medicine, College of Veterinary
Medicine, Jilin University, Changchun, Jilin Province 130062,
People’s Republic of China
2
To whom correspondence should be addressed at Department of
Clinical Veterinary Medicine, College of Veterinary Medicine, Jilin
University, Changchun, Jilin Province 130062, People’s Republic of
China. E-mail: zhangns@jlu.edu.cn; yangzhengtao01@sina.com
than a physical barrier for pathogens and allergens but also
are capable of producing inflammatory mediators and
phagocytizing cellular or acellular debris in response to
these microbes [2, 3]. However, excessive inflammatory
response in the mammary gland may cause mastitis, which
costs lots of expenses [4]. Lipopolysaccharide (LPS), a
structural component of the outer membrane of gram-
negative bacteria, is a potent agent widely used to model
bacterial challenges of mammalian cells and induces pro-
inflammatory cytokine production. In recent years, there
have been reports that LPS could effectively induce
mastitis and is recognized as a valuable tool to study the
is characterized by the release of pro-inflammatory
cytokines, such as tumor necrosis-α (TNF-α), interleukin
(IL)-6, IL-1β, and other mediators, those of which play
important roles during the inflammation process [6, 7]. The
expression of inducible nitric oxide synthase (iNOS) and
cyclooxygenase 2 (COX-2) was also reported to be

0360-3997/13/0000-0001/0 # 2013 Springer Science+Business Media New York

 Liang, Li, Fu, Cao, Song, Wang, Wang, Guo, Zhou, Li, Yang, and Zhang
induced by the binding of LPS or TNF-α in mammary
epithelial cells [8–10]. MAPKs are a family of serine/
threonine protein kinases, which include members such as
extracellular signal receptor-activated kinase (ERK) 1/2,
p38, and c-Jun N-terminal kinase (JNK). It is widely
accepted that mitogen-activated protein kinase (MAPK)
and the nuclear factor-κB (NF-κB) pathways play vital
roles in inflammation, both of which can induce the
expressions of various inflammatory mediators and pro-
inflammatory cytokines [11–16].
Thymol (2-isopropyl-5-methylphenol) is a natural
monoterpene phenol primarily found in thyme, oregano,
and tangerine peel (Fig. 1). It has been credited with a
series of pharmacological properties, including antimicro-
bial, anti-inflammatory, and antioxidant effects [17–21].
However, there are very limited literatures about the anti-
inflammatory property of thymol on mastitis. The aim of
this study was to verify the potential anti-inflammatory
effect of thymol on LPS-stimulated inflammatory response
in mouse mammary epithelial cells (mMECs).
MATERIALS AND METHODS
Animals
BALB/c mice (10–15-day gravid), weighing approx-
imately 18–22 g, were purchased from Norman Bethune
University of Medical Science of Jilin University. The
mice were housed in special room with 24±1 °C and 40–
80 % humidity. All the animals received food and water
adequately. All the animal studies were conducted
according to the experimental practices and standards
approved by the Animal Welfare and Research Ethics
Committee at Jilin University (approval ID 20111106–2),
and all efforts were made to minimize suffering.
Fig. 1. Chemical structure of thymol.
Chemicals and Reagents
Thymol (purity >99.9 %) was purchased from the
National Institute for the Control of Pharmaceutical and
Biological Products (Beijing, China). The thymol stock
solution was prepared with dimethyl sulfoxide (DMSO),
and the DMSO concentration in the solution did not exceed
0.1 %. LPS (Escherichia coli 055:B5) and 3-(4,5-dimeth-
ylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT)
were purchased from Sigma Chemical Co. (St. Louis,
MO, USA). Dulbecco's modified Eagle's medium (DMEM
F12/1:1), fetal calf serum (FCS), and trypsin/EDTA were
purchased from Hyclone (Logan, UT, USA). Collagenase I
and collagenase II were purchased from Invitrogen Corp.
(Carlsbad, CA, USA). Epidermal growth factor (EGF),
transferrin, and T3 were purchased from PeproTech;
mouse TNF-α and IL-6 enzyme-linked immunosorbent
assay (ELISA) kits were purchased from Biolegend (San
Diego, CA, USA). Mouse Specific iNOS antibody, COX-2
antibody, mouse monoclonal phospho-specific p46–p54
JNK antibodies, mouse monoclonal phospho-specific p42–
p44 ERK antibodies, mouse monoclonal phospho-specific
p38 antibodies, mouse mAb phospho-NF-κB, mouse mAb
NF-κB, mouse mAb phospho-IκBα, and rabbit mAb IκBα
were purchased from Cell Signaling Technology, Inc.
(Beverly, MA, USA). β-actin and horseradish peroxidase-
conjugated goat anti-rabbit and goat anti-mouse antibodies
were provided by Tianjin Sungene Biotech Co., Ltd.
(Tianjin, China). All other chemicals were of reagent grade
and were endotoxin free.
Mouse Mammary Epithelial Cell Isolation
and Culture
Primary cultured mMECs were prepared as previously
described by Smalley [22]. Briefly, the mammary tissues
were removed aseptically from 15-day gravid BALB/c mice
and minced into pasty fine tissues. The minced tissues were
digested by collagenase I/II/trypsin mixture (Invitrogen) and
shaken at 37 °C. After filtration to remove undissociated
tissue and debris, the cells were collected by centrifugation
at 250×g for 5 min three times. The cell pellets were
resuspended in DMEM/F12 containing 10 % FCS and
incubated for 1 h at 37 °C, and then the supernatant was
collected. This step was repeated three times until the
fibroblasts were removed. After the last incubate, the cells
were resuspended in DMEM/F12 containing 10 % FCS,
0.5 % transferrin, 0.1 % T3, and 0.5 % EGF, and were
cultured at 37 °C with 5 % CO2. The medium was changed
Thymol Inhibits LPS-Stimulated Inflammatory Response
every day. In all the experiments, the cells were allowed to
acclimate for 24 h before any treatments.
MTT Assay for Cell Viability
The effect of thymol on cell viability was evaluated
using the MTT assay. The mouse mammary epithelial
cells were exposed to thymol at a concentration of 200–
0 μg/mL), followed by stimulation with 1 μg/mL of LPS
for 24 h in a 96-well microplate. Subsequently, 20 μL
MTT (5 mg/mL) was added to each well. The resulting
solution was incubated at 37 °C until blue deposits were
visible. Cell-free supernatants were then removed and
resolved with 150 μL DMSO in each well. The optical
density was measured at 570 nm on a microplate reader.
Cytokine Assay
5
The mMECs were plated onto 24-well plates (4×10
cells/well) and pretreated with or without various
concentrations of thymol (10, 20, 40 μg/mL) for 1 h and
then were stimulated with LPS (1 μg/mL) for 12 h. The
supernatant was collected to measure the levels of TNF-
α, IL-1β, and IL-6. All the operations were followed from
the instruction of the ELISA kits.
Western Blot Analysis
The primary cultured mMECs were treated with
various concentrations of thymol for 1 h and stimulated
with 1 μg/mL of LPS for 24 h (for iNOS and COX-2) or
1 h (for NF-κB and MAPKs). Total cellular proteins were
extracted using mammalian protein extraction reagent
(Pierce, Rockford, IL, USA) supplemented with a 1:100
dilution of phenylmethylsulfonyl fluoride (Sigma). The
concentrations of the protein samples were determined
using BCA assay kits. For western blot analysis, 20 μg of
total protein per lane was resolved by SDS-PAGE and
blotted onto PVDF membrane. Mouse-specific iNOS
antibody, COX-2 antibody, rabbit anti-phospho-p38,
anti-phospho-JNK, anti-phospho-ERK, anti-phospho-
IκB-3, and mouse anti-phospho-IκB-α monoclonal anti-
body (Cell Signaling Technology) were used for protein
detection, and the corresponding antibodies were used
for the detection of unphosphorylated molecules. The
β-actin protein served as an internal control. The proteins
were visualized using secondary HRP-conjugated goat
anti-rabbit and goat anti-mouse antibodies (Jackson
ImmunoResearch Laboratories, West Grove, PA, USA)
and Pierce Supersignal West Pico Chemiluminescent
Substrate. The images were captured using a MicroChemi
4.2 system (DNR Bio Imaging Systems, Jerusalem, Israel).
The intensities of the bands were measured with ImageJ
software.
Statistical Analysis
All values were expressed as the mean ± SEM.
Differences between the mean values of normally
distributed data were assessed with a one-way ANOVA
(Dunnett's t test) and two-tailed Student's t test. P values
of 0.05 or less were considered statistically significant.
RESULTS
Effect of Thymol on mMEC Viability
MTT is a monotetrazolium salt; the reduction of
which is one of the most frequently employed methods for
the detection of cell cytotoxicity or viability following
exposure to toxic substances. This water-soluble salt is
converted to an insoluble purple formazan by succinate
dehydrogenase within the mitochondria of the living cells,
and the purple formazan is in direct proportion to the
healthy cell numbers. In this study, the potential cytotox-
icity of thymol was evaluated using the MTT assay after
incubating the cells for 24 h in the absence or presence of
LPS, and the result showed that cell viability was not
affected by thymol at the concentrations used (10, 20, and
40 μg/mL) (Fig. 2). Thus, the effects of thymol on mMECs
were not attributable to cytotoxic effect.
Effect of Thymol on Cytokine Release
in the Supernatants of LPS-stimulated mMECs
LPS-induced inflammatory response is characterized
by the release of pro-inflammatory cytokines such as
TNF-α, IL-1β, and IL-6 [6, 7]. These cytokines play
crucial roles during inflammation. TNF-α, along with
IL-1β and IL-6, is recognized as an important early
inflammatory mediator, and the over-expression of this
cytokine can lead to severe pro-inflammatory reactions. To
determine whether thymol affects the expression of
inflammatory cytokines in LPS-stimulated mMECs, the
expression of TNF-α, IL-1β, and IL-6 was detected using
ELISA. As shown in Fig. 3, the levels of TNF-α and IL-6
significantly increased after LPS challenge compared with
those of the control group (P<0.01), while the pretreatment
with thymol (20 and 40 μg/mL) reduced the production of
 Liang, Li, Fu, Cao, Song, Wang, Wang, Guo, Zhou, Li, Yang, and Zhang

Fig. 2. Effect of thymol on the cell viability of mMECs. The cells were cultured with different concentrations of thymol (10, 20, and 40 μg/mL) in the
absence or presence of 1 μg/mL LPS for 24 h. The cell viability was determined using MTT assay. The values are presented as mean ± SEM (n=8).

TNF-α and IL-6 in a dose-dependent manner, and the not observed in our experiment, and the data were not
levels of cytokines were marked lower than those in the shown. The results showed that thymol attenuated the
LPS group (P<0.01). However, the level of IL-1β was inflammatory response via suppressing the pro-inflamma-

Fig. 3. Effects of thymol on the expression of TNF-α (a) and IL-6 (b) in LPS-stimulated mMECs. The cells were treated with 1 μg/mL LPS in the
absence or presence of thymol (10, 20, and 40 μg/mL) for 12 h. Levels of TNF-α (a) and IL-6 (b) in culture supernatants were measured using ELISA.
The data are representative of three independent experiments, and the values are presented as mean ± SEM. #P<0.01 significant difference from control;
*P<0.05 and **P<0.01 significant difference from LPS only.
Thymol Inhibits LPS-Stimulated Inflammatory Response

tory cytokine production in primary cultured LPS-stimu-
lated mMECs.
Effect of Thymol on iNOS and COX-2 Protein
Expression in LPS-stimulated mMECs
Both iNOS and COX-2 are important enzymes
involved in mediating the inflammation process. Up-
regulation of iNOS and COX-2 occurs in many patho-
logical conditions, such as mastitis. Therefore, the anti-
inflammatory activity of thymol can be demonstrated by
its effect on iNOS and COX-2 expression in LPS-induced
mMECs. As shown in Fig. 4, western blot analysis
showed that the expression of iNOS and COX-2 were
strongly increased by LPS, while the application of
thymol (10, 20, and 40 μg/mL) 1 h prior to LPS treatment
resulted in a dramatic reduction in the levels of iNOS and
COX-2 proteins in a dose-dependent manner in mMECs
(P<0.01 or P<0.05).
Effect of Thymol on the Activation of NF-κB
and MAPK Signaling Pathways
NF-κB activation is critical in regulating gene
expression in LPS-induced inflammatory responses [23].
Since COX-2 and iNOS are frequently regulated through
the activation of NF-κB and MAPK signaling pathways in
diverse mechanisms, it is of importance to investigate the
potential inhibitory activity of thymol on the activation of
NF-κB and MAPKs in mMECs. The levels of p-NF-κB
p65 and p-IκBα in the NF-κB signaling pathway and the
levels of p-p38, p-ERK, and p-JNK in the MAPK signaling
pathway were analyzed using western blot (Figs. 5 and 6).
The results showed that LPS treatment significantly
activated the NF-κB and MAPK signaling pathways. In
contrast, pretreatment with thymol (10, 20, and 40 μg/mL)
inhibited the up-regulation of phosphorylation of NF-κB
p65, p-IκBα, p38, ERK, and JNK as compared with the
LPS group. These results of our immunoblot analyses
suggest that the inhibition of TNF-α, IL-6, iNOS, and
COX-2 expression by thymol might block the LPS-
induced NF-κB and MAPK activation.
DISCUSSION
Mastitis is typically defined as the inflammation of
the mammary gland which is usually due to bacterial
infection in mammals [24]. Mammary epithelial cells
directly contact with the invading pathogens and involved
in initiating innate immune response during the process of

Fig. 4. Effects of thymol on inducible nitric oxide synthase and COX-2 protein expression in LPS-stimulated mMECs. The mMECs were treated with
different concentrations (10, 20, and 40 μg/mL) of thymol for 1 h and stimulated with 1 μg/mL of LPS for 24 h. Total cellular protein (20 μg) was
resolved by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and visualized by western blot analysis. Quantification of iNOS and COX-
2 protein expression was normalized to β-actin using a densitometer. Data are representative of three independent experiments, and the values are
presented as mean ± SEM. #P<0.01 significant difference from control; *P<0.05 and **P<0.01 significant difference from LPS only.
 Liang, Li, Fu, Cao, Song, Wang, Wang, Guo, Zhou, Li, Yang, and Zhang

Fig. 5. Effects of thymol on phosphorylation of IκBα and NF-κB p65 in LPS-stimulated mMECs. The results illustrated that thymol could inhibit the
phosphorylation of IκBα and NF-κB p65. mMECs were treated with different concentrations (10, 20, and 40 μg/mL) of thymol for 1 h and stimulated
with 1 μg/mL of LPS for 1 h. Total cellular protein (20 μg) was resolved by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and
visualized by western blot analysis. Quantification of p-IκBα and p-p65 protein expression was normalized to β-actin using a densitometer. Data are
representative of three independent experiments, and the values are presented as mean ± SEM. #P<0.01 significant difference from control; *P<0.05 and
**P<0.01 significant difference from LPS only.

mammary inflammation [2]. Thymol is a p-cymene-
derived compound primarily found in thyme, oregano,
and tangerine peel, which has been used in traditional
Chinese medicine for its multiple biological properties. In
the present study, we demonstrated that thymol exerted
beneficial effects in LPS-induced mouse primary cultured
epithelial cells, which was associated with the attenuation
of inflammatory responses.
LPS, which is capable of eliciting cellular activation
of alveolar macrophages and epithelial cells, and trigger-
ing the release of pro-inflammatory cytokines such as
TNF-α, IL-1β, and IL-6 [2, 25], is known as the major
virulence factor of clinical mastitis. These pro-inflamma-
tory cytokines are typical examples of multifunctional
mediators involved in the regulation of the immune
response and inflammation. TNF-α and IL-1β are two
key components of the innate immune response to
infection [7]. Likewise, IL-6 is a pleiotropic cytokine
with strong influences on inflammatory responses and a
major effector of the acute phase reaction [2]. They are
produced by a variety of activated immune cells including
T cells, B cells, macrophages, and some of epithelial cells
[26]. The over-expression of these cytokines can lead to
severe pro-inflammatory reactions. Thus, anti-TNF-α
therapeutic strategies may play a role in many inflamma-
tory diseases, such as mastitis. In this experiment, we
found that the concentrations of TNF-α and IL-6 were
markedly increased in the culture 12 h after LPS
stimulation, and pretreatment with thymol inhibited the
production of TNF-α and IL-6 in a dose-dependent
manner. Interestingly, the IL-1β level was not observed
under our experimental conditions. A possible explana-
tion of this result may be the lower concentration of LPS
used and the difference in LPS challenging time in
mMECs compared to those reported in an in vitro study
[27]. Our results indicated that thymol attenuated the pro-
inflammatory cytokine TNF-α and IL-1β release to exert
positive effects against LPS-challenged mMECs.
COX-2 and iNOS have been implicated as important
enzymes that mediate inflammatory process [28]. COX-2
and iNOS are not usually expressed in quiescent cells; they
are transcriptionally induced in response to bacterial
endotoxins and pro-inflammatory cytokines in macrophages
and various other cell types. A variety of studies have shown
that iNOS and COX-2 are up-regulated by pro-inflamma-
tory cytokines and further aggravate the inflammatory
immune response during infection [29–31]. The pleiotropic
inflammatory mediators NO and PGE2, which are generated
Thymol Inhibits LPS-Stimulated Inflammatory Response

Fig. 6. Effects of thymol on the phosphorylation of extracellular p38, ERK, and JNK in LPS-stimulated mMECs. The results illustrated that thymol
could inhibit the phosphorylation of p38, ERK, and JNK. mMECs were treated with different concentrations (10, 20, and 40 μg/mL) of thymol for 1 h and
stimulated with 1 μg/mL of LPS for 1 h. Total cellular protein (20 μg) was resolved by SDS-PAGE, transferred to a polyvinylidene difluoride membrane,
and visualized by western blot analysis. Quantification of p-p38, p-ERK, and p-JNK protein expression was normalized to β-actin using a densitometer.
Data are representative of three independent experiments, and the values are presented as mean ± SEM. #P<0.01 significant difference from control;
*P<0.05 and **P<0.01 significant difference from LPS only.

by iNOS and COX-2, are involved in the pathogenesis of
inflammation and infection. Thymol has been shown to
inhibit the LPS-stimulated NO generation in mouse
macrophage-like RAW 264.7 cells [32]. Marsik et al.
concluded that thymol exhibited inhibitory effect on COX-
2 in vitro [33]. In this study, we examined the effects of
thymol on iNOS and COX-2 expression in LPS-stimulated
mMECs. In accordance with the previously described
studies, we found that thymol (10, 20, and 40 μg/mL)
suppressed the LPS-induced iNOS and COX-2 protein
expression in mMECs in a dose-dependent manner, which
indicated that the beneficial effect of thymol on inflamma-
tory responses may be partially attributed to the inhibition of
iNOS and COX-2 expression in LPS-challenged mMECs.
The NF-κB signaling pathway has been considered
to play a pivotal role in inflammatory phenotypic changes
in various pathophysiological conditions [34, 35]. NF-κB
is one of the most ubiquitous and important transcription
factors that regulate the expression of genes involved in
controlling inflammatory responses, such as the expres-
sion of iNOS, COX-2, and other inflammatory-related
cytokines, including TNF-α, IL-1β, and IL-6 [36, 37].
Under normal conditions, NF-κB is present in its inactive
cytoplasmic form bound to the repressor of NF-κB
 Liang, Li, Fu, Cao, Song, Wang, Wang, Guo, Zhou, Li, Yang, and Zhang
(IκBs). However, in response to certain stimuli, such as
LPS, the degradation and phosphorylation of IκBα will
increase, which results in the release of free NF-κB p65
and translocation from the cytoplasm to the nucleus,
leading to the transcription of specific target genes, such
as inflammatory-related cytokines, chemokines, adhesion
molecules, and other immune effector molecules [11, 12,
38, 39]. The activation of JNK, p38, and ERK1/2 MAPK
proteins is known to be involved in the regulation of
iNOS and COX-2 expression [28]. MAPK signalling
cascades have also been shown to be involved in the
activation of NF-κB following LPS stimulation [23]. Our
laboratory has recently reported that both the NF-κB and
MAPK pathways were activated in LPS-induced mastitis
[16, 40–42]. In the present study, we examined the effects
of thymol on the phosphorylation of IκBα and NF-κB
p65 in the NF-κB signaling pathway, and the phosphor-
ylation of p38, ERK, and JNK in the MAPK signaling
pathway. We found that pretreatment with thymol signif-
icantly suppressed the phosphorylation of NF-κB p65,
IκBα, p38, ERK, and JNK. The results demonstrated that
thymol plays a central anti-inflammatory role in the
regulation of inflammatory responses, possibly through
inhibiting the NF-κB and MAPK signaling pathways.
Therefore, the targeted inhibition of the induction and
activity of NF-κB, COX-2, and/or iNOS has been
identified as a new therapeutic method in inflammatory
diseases. The results in our study suggested that thymol
could promote the anti-inflammatory effect against LPS-
induced disfunction. Yet, these results are very primary,
and more work is needed to identify the exact target of
thymol in mammary epithelial cells.
In summary, our study demonstrated that the effect of
thymol on anti-inflammation not only comes from the
expression of inflammatory cytokines but also from the
iNOS and COX-2 pathway in LPS-stimulated mMECs. The
promising anti-inflammatory effect of thymol may involve in
the inhibition of NF-κB and MAPK signaling pathways. Our
results provide useful and additional mechanistic explana-
tions for the anti-inflammatory effects of thymol. Thereby,
thymol may be a potential therapeutic agent against mastitis.
ACKNOWLEDGMENTS
This work was supported by grants from the National
Natural Science Foundation of China (no. 31272622 and no.
3120195) and the Research Fund for the Doctoral Program
of Higher Education of China (no. 20110061130010 and
20120061120098).
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