Turkish Journal of Biology
Turk J Biol
(2015) 39: 657-665
© TÜBİTAK
doi:10.3906/biy-1405-50
http://journals.tubitak.gov.tr/biology/
Research Article
Machilus thunbergii extract inhibits inflammatory response in
lipopolysaccharide-induced RAW264.7 murine macrophages via
suppression of NF-κB and p38 MAPK activation
1,
2,
2
2
3,4
4,5,
Shu-Ju WU *, Wen-Bin LEN *, Chien-Yi HUANG , Chian-Jiun LIOU , Wen-Chung HUANG , Chwan-Fwu LIN **
Department of Nutrition and Health Sciences, Chang Gung University of Science and Technology, Kwei-Shan, Tao-Yuan, Taiwan,
R.O.C.
2
Department of Nursing, Chang Gung University of Science and Technology, Kwei-Shan, Tao-Yuan, Taiwan, R.O.C.
3
Graduate Institute of Health Industry Technology, Chang Gung University of Science and Technology, Kwei-Shan, Tao-Yuan, Taiwan,
R.O.C.
4
Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Kwei-Shan, Tao-Yuan, Taiwan,
R.O.C.
5
Department of Cosmetic Science, Chang Gung University of Science and Technology, Kwei-Shan, Tao-Yuan, Taiwan, 333, R.O.C.
1
Received: 20.05.2014
Accepted/Published Online: 22.09.2014
Printed: 30.09.2015
Abstract: Machilus thunbergii Sieb. & Zucc. (Lauraceae) is a medicinal plant used to treat edema and pain in China and Taiwan. The
anti-inflammatory effects of M. thunbergii were not clear. In this study, we evaluated the anti-inflammatory effects of M. thunbergii
extract in LPS-stimulated RAW 264.7 cells. The cells were pretreated with various doses (12.5 µg/mL–100 µg/mL) of M. thunbergii
extract and activated with LPS. We found that 25 µg/mL M. thunbergii extract inhibited nitric oxide (NO), prostaglandin E2 (PGE2),
inducible nitric oxide synthetase (iNOS), and cyclooxygenase-2 (COX-2) expression, as well as levels of proinflammatory cytokines
including interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α). Furthermore, M. thunbergii extract also suppressed translocation
of nuclear transcription factor kappa-B (NF-κB) subunit p65 proteins into the nucleus and induced a dose-dependent inhibition of the
phosphorylation of p38 mitogen-activated protein kinase (MAPK). M. thunbergii extract also increased HO-1 and Nrf2 production
in a concentration-dependent manner. Taken together, these results suggest that M. thunbergii extract has an anti-inflammatory effect
because it reduces levels of proinflammatory cytokines and mediators via the suppression of NF-κB and p38 MAPK activation in RAW
264.7 cells.
Key words: Cyclooxygenase-2, inducible nitric oxide synthase, Machilus thunbergii, NF-κB, p38 MAPK
1. Introduction
As a part of innate immunity, macrophages recognize
the lipopolysaccharide (LPS) of gram-negative bacteria
and become activated, inducing signal transduction and
expression of pro-inflammatory mediators (Peri et al.,
2010). An appropriate inflammatory response enhances
the host immune response to microbial invasion; however,
an excessive inflammatory response can cause severe
tissue injury (Wolff, 2011). Activated macrophages induce
an inflammatory response through a series of complex
processes (Kobayashi, 2010; Yoon et al., 2010). LPSinduced macrophages secrete proinflammatory cytokines,
including interleukin (IL)-6, tumor necrosis factoralpha (TNF-α), and IL-1β (Guha and Mackman, 2001;
Dinarello, 2002); express inducible nitric oxide synthase
(iNOS) to produce nitric oxide (NO); and synthesize
cyclooxygenase-2 (COX-2) to induce prostaglandin E2
(PGE2) production (MacMicking et al., 1997; Wu, 2003). It
is possible that inhibiting the macrophage-induced release
of these inflammatory substrates could reduce tissue injury
during the inflammatory process.
Nuclear transcription factor kappa-B (NF-κB) is a
transcription factor that regulates the expressions of
many genes (Oeckinghaus et al., 2011). In LPS-induced
macrophages, NF-κB subunit p65 is activated and
translocated into the nucleus where it binds the promoter
of inflammatory-associated genes, leading to transcription
of proinflammatory cytokines and mediators (Ruland,
* Shu-Ju WU and Wen-Bin LEN are equal contributors to the paper.
** Correspondence: [email protected]
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2011). There is also a relationship between the cellular
inflammatory response and mitogen-activated protein
kinase (MAPK) pathways (Coskun et al., 2011), such that
LPS-induced activated macrophages express iNOS and
COX-2.
Machilus thunbergii Sieb. & Zucc. (Lauraceae) grows
mainly in China, Taiwan, Korea, and Japan (Tokuda et al.,
2008). In Chinese medicine, M. thunbergii is used to treat
abdominal distension, anti-inflammatory response, edema,
and pain (Moon and Jung, 2006; Tokuda et al., 2008).
Recent studies found that M. thunbergii suppresses matrix
metalloproteinase-9 expression, following treatment
with ultraviolet irradiation, in fibroblast WS-1 cells and
human keratinocyte cells (HaCaT) (Moon and Chung,
2005; Lee et al., 2009). Meso-dihydroguaiaretic acid was
isolated from M. thunbergii and was found to suppress the
activity of hepatic stellate cells via inhibition of AP-1 (Park
et al., 2005). Obtusilactone B was isolated from Machilus
thunbergii and was found to target the nuclear envelope
for the treatment of cancer (Kim et al., 2013). A recent
study found that butanolides isolated from M. thunbergii
could decrease the NO level in lipopolysaccharide (LPS)activated macrophages (Kim et al., 2003; Ryu et al., 2003).
However, the anti-inflammatory effects of M. thunbergii
have not been studied. Here, we investigate whether M.
thunbergii suppresses the expression of proinflammatory
cytokines, iNOS, and COX-2 and explore whether
this activity is regulated by NF-κB or MAPK signaling
pathways in LPS-activated murine macrophages.
2.3. MTT assay for cell viability
RAW 264.7 cells (105 cells/mL) were cultured in 96-well
plates and treated with different doses of M. thunbergii
extract for 24 h, as previously described (Gholami, 2014).
The supernatant was removed, and cells were washed with
PBS. Then MTT (50 mg/mL; Sigma, St Louis, MO, USA)
was added to each well to determine cytotoxicity. Briefly,
the plate was incubated for 4 h at 37 °C. The wells were
washed with PBS, and isopropanol was added to dissolve
the formazone crystal. Finally, the optical density of each
well was measured at 570 nm using the microplate reader
(Multiskan FC, Thermo, Waltham, MA, USA).
2. Materials and methods
2.6. Determination of PGE2 levels
PGE2 was measured by ELISA (R & D Systems), as
previously described (Liou and Huang, 2011). Briefly,
RAW 264.7 cells (2 × 106 cells/mL) were pretreated with M.
thunbergii for 1 h and then incubated with LPS (1 µg/mL)
for 24 h. Samples were mixed with primary antibody for
1 h at room temperature. Next, PGE2 conjugate solution
was added, and samples were incubated for 2 h at room
temperature. Finally, the plate was washed, and substrate
solution was added to assay the levels of PGE2 by OD450nm
measurements.
2.1. Machilus thunbergii collection and extraction
The aerial parts of Machilus thunbergii Sieb. & Zucc
(Lauraceae) were collected from Tao-Yuan, Taiwan, in
July 2006 and identified by the taxonomist Mr Jun-Chih
Ou, who previously worked with the National Research
Institute of Chinese Medicine. The M. thunbergii samples
(25 kg) were extracted with MeOH (140 L) at 50 °C for
72 h. The MeOH extract was evaporated under vacuum to
1178 g. The yield was 4.7%.
2.2. Cell line and culture medium
The murine macrophage cell line RAW 264.7 was
purchased from the Bioresource Collection and Research
Center (BCRC, Taiwan). RAW 264.7 cells were cultured
in Dulbecco’s modified Eagle’s medium (Invitrogen-Gibco,
Paisley, Scotland) supplemented with 10% fetal bovine
serum (Biological Industries, Haemek, Israel) and 100 U/
mL each of penicillin and streptomycin. The cells were
incubated in an atmosphere of 5% CO2 at 37 °C and were
subcultured twice every week.
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2.4. Determination of NO production
Nitrite levels were measured to determine NO production
in culture supernatants of RAW 264.7 cells. Briefly, RAW
264.7 cells (2 × 106 cells/mL) were pretreated with M.
thunbergii for 1 h then incubated with LPS (1 µg/mL) for
24 h. Next 50 μL of supernatant were mixed with 50 μL of
Griess reagent (Sigma) for 15 min at room temperature.
The optical density of each well was measured at 570 nm
using the microplate reader (Multiskan FC, Thermo).
2.5. Measurement of proinflammatory cytokine levels
RAW 264.7 cells (2 × 106 cells/mL) were pretreated with
different doses of M. thunbergii for 1 h and then LPS (1
μg/mL) was added for 8 h. Cytokines were measured by
ELISA, as previously described (Liou et al., 2010; Nasir
et al., 2014). The culture supernatants were measured
using ELISA kits specific to IL-6 and TNF-α (R & D
Systems, Minneapolis, MN, USA), and there were three
independent experiments in each group (n = 9).
2.7. Preparation of total and nuclear protein
For total protein, macrophage cells (5 × 106) were
pretreated with different concentrations of M. thunbergii
(12.5, 25, 50, or 100 µg/mL) for 1 h in 10-cm culture dishes
(Corning, NY, USA). Then 1 µg/mL LPS was added for
24 h. Subsequently, the cells were lysed with protein lysis
buffer (50 mM Tris-HCl, pH 8; 150 mM NaCl; 0.5% NP40;
0.1% SDS, 1 mM EDTA) containing a protein inhibitor
cocktail (Sigma). To detect protein phosphorylation, cells
(5 × 106) were treated with M. thunbergii (12.5 µg/mL–100
WU et al. / Turk J Biol
µg/mL) and cultured for 24 h at 37 °C in 10-cm culture
dishes. Then cells were cultured with 1 µg/mL LPS for
30 min to detect phosphorylated-p38, phosphorylatedERK 1/2, and phosphorylated-JNK. In addition, nuclear
proteins were extracted with NE-PER nuclear and
cytoplasmic extraction reagent kits (Pierce, Rockford, IL,
USA) to detect NF-κB subunit p65. Protein concentrations
were determined with the BCA protein assay kit (Pierce).
2.8. Western blot analysis
Protein samples (10 µg) were separated on 10% SDS
polyacrylamide gels and transferred onto PVDF membranes
(Millipore, Billerica, MA, USA), as previously described
(Wang et al., 2014). The membranes were incubated with
primary antibodies, including COX-2, HO-1, iNOS, p65,
PCNA, (Santa Cruz, CA, USA), phospho-ERK 1/2, ERK,
phospho-JNK, JNK, phospho-p38, p38, Nrf2 (Millipore),
and β-actin (Sigma), overnight at 4 °C. The membranes
were washed and incubated with secondary antibodies for
1 h. Finally, the membranes were incubated with solution
from the ECL detection kit (Millipore) for 3 min and
exposed with the BioSpectrum 600 (UVP, Upland, CA,
USA).
2.9. Statistical analysis
Statistical analyses were performed using one-way
ANOVA followed by the post-hoc Dunnett’s test. All values
were expressed as mean ± SD of at least three independent
experiments, and a P value less than 0.05 was considered
statistically significant.
3. Results
3.1. Cytotoxicity of M. thunbergii in RAW264.7
macrophages
Prior to other experiments, the cytotoxicity of M.
thunbergii was determined by the MTT assay (Figure
1a). Cell viability was not significantly altered by doses
of M. thunbergii up to 100 µg/mL. Therefore, cells in all
experiments were treated with concentrations of M.
thunbergii from 12.5 to 100 µg/mL.
3.2. M. thunbergii inhibited the levels of proinflammatory
cytokines in LPS-stimulated RAW264.7 macrophages
To assess the effects of M. thunbergii on proinflammatory
cytokine production by LPS-stimulated RAW264.7
macrophages, culture supernatants were harvested
and measured by ELISA (Figure 2). Treatment with M.
thunbergii dose-dependently inhibited the levels of IL-6 and
TNF-α in RAW264.7 cells. M. thunbergii concentrations
≥50 µg/mL inhibited IL-6, while concentrations ≥25 µg/
mL suppressed TNF-α secretion. Obviously, the extract
of M. thunbergii suppressed levels of proinflammatory
cytokines in LPS-activated macrophages.
3.3. Effect of M. thunbergii on LPS-induced NO and
PGE2 production
To investigate the effects of M. thunbergii on NO and PGE2
production, RAW264.7 cells were pretreated with various
doses of M. thunbergii (12.5, 25, 50, or 100 µg/mL) and
exposed to LPS (1 µg/mL) for 24 h; then the levels of nitrite,
the stable metabolite of NO, and PGE2 were measured.
Figure 1. Effects of M. thunbergii (MT) on cell viability (a). RAW264.7 cells were
pretreated with the indicated doses of M. thunbergii for 1 h, and then the cells were
stimulated with LPS (1 µg/mL). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01;
compared to the LPS-treated group.
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Figure 2. Effects of M. thunbergii (MT) production of IL-6 (a) and TNF-α (b). RAW264.7 cells were
pretreated with the indicated doses of M. thunbergii for 1 h, and then the cells were stimulated with LPS
(1 µg/mL). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01; compared to the LPS-treated group.
LPS-treated cells exhibited increased NO production
(approximately 13-fold) compared with unstimulated cells.
However, pretreating cells with M. thunbergii significantly
reduced NO levels in a dose-dependent manner (Figure
3a). M. thunbergii treatment also significantly and dosedependently decreased PGE2 secretion in RAW264.7
macrophages (Figure 3b).
3.4. Effects of M. thunbergii on LPS-induced iNOS and
COX-2 protein levels
Next, we determined whether the inhibitory effects of
M. thunbergii on the levels of NO and PGE2 were related
to suppression of iNOS and COX-2 expression (Figures
3c and 3d). The protein levels of iNOS and COX-2 were
significantly increased by LPS, and pretreatment with
M. thunbergii significantly suppressed iNOS and COX2 protein expression in a dose-dependent manner. Thus,
inhibition of NO and PGE2 production by M. thunbergii
likely resulted from inhibition of iNOS and COX-2,
respectively.
3.5. M. thunbergii affected NF-κB translocation into the
nucleus in LPS-stimulated RAW264.7 macrophages
We investigated whether M. thunbergii could suppress
NF-κB (active subunit p65) translocation from cytosol
to nucleus in LPS-stimulated RAW264.7 cells (Figure 4).
In unstimulated cells, NF-κB was constant in the cytosol
and hardly translocated into the nucleus. However, the
nuclear translocation of NF-κB subunit p65 increased
in LPS-stimulated RAW264.7 cells. Interestingly,
pretreatment with various doses of M. thunbergii (25, 50,
or 100 µg/mL) significantly suppressed NF-κB p65 nuclear
translocation, compared with the LPS group. Hence, M.
thunbergii displayed a substantial influence on NF-κB p65
translocation into the nucleus.
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3.6. M. thunbergii affected activation of MAPK pathways
in LPS-stimulated RAW264.7 macrophages
To determine whether MAPK signaling pathways were
suppressed by M. thunbergii, RAW264.7 cells were treated
with various doses of M. thunbergii for 24 h and then
were stimulated with LPS for 30 min. Evaluation of the
phosphorylation of MAPK-signaling molecules, including
ERK1/2, JNK, and p38, by western blot revealed that M.
thunbergii did not suppress phosphorylation of ERK1/2
and JNK compared with the LPS-stimulated macrophages
(Figure 5). However, LPS-stimulated macrophages that
were pretreated with M. thunbergii exhibited significant and
dose-dependent decreases in the phosphorylation levels of
p38. These results indicated that only phosphorylation of
p38 MAPK is effectively blocked by M. thunbergii in LPSactivated macrophages.
3.7. Effect of M. thunbergii on LPS-induced HO-1 and
Nrf2 expression in RAW264.7 cells
Heme oxygenase (HO)-1 has an antioxidant role and antiinflammatory function in the inflammatory process (An
et al., 2012). We found that M. thunbergii could modulate
HO-1 and Nrf2 expression compared with normal
macrophages (Figure 6).
4. Discussion
In response to LPS released by gram-negative bacteria,
activated macrophages release proinflammatory cytokines
and mediators that cause acute or chronic inflammation
(Schett, 2008). LPS combines with LPS binding protein to
bind CD14, a macrophage receptor, inducing activation of
Toll-like receptor-4 (TLR4) (Kawai and Akira, 2007; Peri et
al., 2010). This complex induces an intracellular signaling
cascade, including activation of myeloid differentiation
factor 88 (MyD88) and TNF receptor-activated factor 6
(TRAF6) (Zanoni and Granucci, 2010), which activates the
IκB kinase (IKK) complex and MAPK signaling pathways.
WU et al. / Turk J Biol
Figure 3. Effect of M. thunbergii (MT) on the LPS-induced production of nitrite (a) and PGE2 (b).
Protein levels of iNOS (c) and COX-2 (d) were detected by western blot. β-Actin expression was used
as an internal control. The densitometry values of three independent experiments were analyzed and
expressed as mean ± SD. * P < 0.05, ** P < 0.01; compared to the LPS-treated group.
The activated IKK complex gives rise to phosphorylation
of IκB and NF-κB (subunits p50 and p65) (Schmid and
Birbach, 2008). Finally, activated p65 can translocate from
cytosol to nucleus, where it binds the promoter to activate
transcription of proinflammatory-associated genes. The
MAPK signaling pathway also modulates activation of
NF-kB (Huang et al., 2010). Therefore, suppression of the
translocation of NF-κB p65 or of MAPK activation may be
useful for modifying the production of proinflammatory
cytokines and mediators in LPS-activated macrophages.
We found that M. thunbergii extract suppressed the
levels of IL-6, TNF-α, NO, and PGE2 in LPS-activated
RAW 264.7 murine macrophages. In addition, production
of iNOS and COX-2 proteins was inhibited, decreasing
production of proinflammatory mediators via suppression
of activated NF-κB and the p38 phosphorylation pathway.
Therefore, M. thunbergii extract has good ability to inhibit
inflammatory response.
Activated macrophages induce iNOS proteins to
produce NO (MacMicking et al., 1997). The extra NO
helps activated macrophages to kill microorganisms;
therefore, iNOS expression is closely related to protection
against pathogen invasion. However, overproduction
of NO can cause severe tissue or cell injury in acute
and chronic diseases (Kobayashi, 2010). Additionally,
COX-2 expression is linked to production of PGE2, a
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Figure 4. Inhibitory effect of M. thunbergii on nuclear translocation of NF-κB in RAW 264.7 cells. Cells were pretreated with different
doses of M. thunbergii (12.5–100 µg/mL) for 1 h, and then LPS (1 µg/mL) was added for 1 h. PCNA in the nucleus and β-actin in cytosol
were used as internal controls. The densitometry values of three independent experiments were analyzed and expressed as mean ± SD.
* P < 0.05, ** P < 0.01; compared to the LPS-treated group.
Figure 5. Effect of M. thunbergii on LPS-induced phosphorylation of MAPK. The cells were stimulated with or without LPS (1 µg/
mL) for 30 min. Protein samples were analyzed by western blot with phospho-specific antibodies. The total MAPK levels were used as
internal controls. The densitometry values of three independent experiments were analyzed and expressed as mean ± SD. * P < 0.05, **
P < 0.01; compared to the LPS-treated group.
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Figure 6. Western blot of HO-1 and Nrf2 protein expression in RAW 264.7 cells. First, 106 cells/mL
were pretreated with the indicated concentration of M. thunbergii for 1 h, and then they were stimulated
with LPS for 24 h. β-Actin and PCNA expressions were used as internal controls.
highly inflammatory substance that can accumulate and
cause swelling, increased pain (Jiao et al., 2009), and
tissue or cell damage. In the present study, M. thunbergii
significantly decreased overproduction of PGE2 and NO
in LPS-activated murine macrophages. We suggest that
M. thunbergii extract suppressed production of these
proinflammatory molecules via suppression of iNOS and
COX-2 expression.
Proinflammatory cytokines IL-6 and TNF-α are
secreted by activated macrophages (Zanoni and Granucci,
2010). IL-6 induces the synthesis of C-response proteins
to enhance the inflammatory response (Valledor et al.,
2010). Low levels of TNF-α can injure tissue and enhance
fibroblast growth, and high levels in conjunction with
sepsis can injure both inflammatory and noninflammatory
tissue (Yan and Hansson, 2007). M. thunbergii suppressed
the levels of IL-6 and TNF-α, which would decrease the
inflammatory response to injured tissues or cells.
Production of proinflammatory cytokines and
mediators is closely related to the transcription factor NF-κB
(Ruland, 2011), which plays a critical role in inflammation,
immune disease, apoptosis, and tumorigenesis (Brasier,
2006). Research has shown that suppression of NF-κB
activation decreases the ability of NF-κB to bind promoters
of inflammatory-associated genes and thus decreases
levels of proinflammatory cytokines and mediators (BenNeriah and Karin, 2011; Ruland, 2011). We found that M.
thunbergii significantly suppressed p65 translocation and
decreased expression of IL-6, TNF-α, iNOS, and COX2, thereby inhibiting p65 translocation in LPS-activated
macrophage. HO-1, an antioxidant enzyme, could protect
against cell injury in inflammatory response and oxidative
stress (An et al., 2012). HO-1 could convert heme to iron,
biliverdin, and CO.
Hence, HO-1 is recognized as a key factor in the
cellular defense system. Nrf2 is a transcription factor
translocated into the nucleus for HO-1 expression. Recent
studies found that HO-1 could decrease the production
of inflammatory mediators in activated macrophages. In
the present study, M. thunbergii promoted the nuclear
translocation of Nrf2 and increased HO-1 expression. We
thought that M. thunbergii enhanced HO-1 for antioxidant
effect and decreased iNOS and COX-2 expressions for
anti-inflammatory response.
A previous study found that major MAPKs,
including ERK 1/2, JNK, and p38, were important for
regulating the expression of proinflammatory cytokines
or mediators in the signaling pathways (Cargnello and
Roux, 2011). However, M. thunbergii only significantly
inhibited phosphorylation of p38 and did not decrease
the phosphorylation of ERK 1/2 or JNK. Phosphorylation
of p38 can enhance and stabilize the required NF-κB
transcription factor-related gene (Cargnello and Roux,
2011; Cuadrado and Nebreda, 2010), as well as TNF-α
mRNA, enhancing production of TNF-α (Cargnello
and Roux, 2011). We suggest that M. thunbergii not only
suppressed p65 translocation into the nucleus, but also
decreased phosphorylation of p38 in order to destroy the
stability of TNF-α mRNA and decrease the level of TNF-α.
In conclusion, our data suggest that M. thunbergii
has anti-inflammatory effects that include inhibiting the
production of proinflammatory cytokines and mediators,
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WU et al. / Turk J Biol
as well as suppressing the iNOS/NO and COX-2/PGE2
pathways by blocking the p38 MAPK and NF-κB pathways.
In the future, we will try to isolate specific compounds
from M. thunbergii to clarify the details of the antiinflammatory mechanism and the effects of M. thunbergii.
Acknowledgments
This study was supported in part by grants from the
Chang Gung Memorial Hospital (CMRPF3A0012 and
CMRPF180031) and the Chang Gung University of Science
and Technology (EZRPF3D0071 and EZRPF3D0081).
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