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Introduction
Airway inflammation plays a central role in the
pathogenesis of a number of lung diseases, including asthma,
chronic bronchitis, bronchiectasis, and chronic obstructive
pulmonary disease (COPD). Terpenes are widely used in the
treatment of upper and lower airway diseases, such as chronic
sinusitis and bronchitis[1,2].
As the active agent and main component of eucalyptus
oil, 1,8-cineol shows particular anti-inflammation properties.
Juergens et al have reported that the monoterpene
1.8-cineol revealed a steroid-like suppression of arachidonic acid
metabolism, TNF-α, and interleukin (IL)-1β production in
human blood monocytes in
vitro[1]. Furthermore, a non-controlled study showed significant inhibition of
LTB4 and IL-1β in stimulated monocytes
ex vivo after additional therapy with 200 mg 1,8-cineol tid administered in enteric-coated
capsules[3]. They revealed that long-term systemic therapy with
1,8-cineol had a significant steroid-saving effect in
steroid-dependent asthma[2]. 1,8-Cineol significantly inhibited
cytokine production in human unselected lymphocytes of
TNF-α, IL-1β, IL-4, IL-5, and in lipopolysaccharide
(LPS)-stimulated monocytes of TNF-α, IL-1β, IL-6, and
IL-8[4]. However, studies on the mode of the anti-inflammation
properties of 1,8-cineol (eucalyptol) are still infrequent.
To elucidate the anti-inflammation mechanisms of
1,8-cineol, in the present study, we investigated the effects
of 1,8-cineol on the subcellular localization of early growth
response factor-1 (Egr-1), the expression of Egr-1,
and
NF-κB in the human monocyte THP-1 cell line.
Materials and methods
Drugs and reagents 1,8-cineol was obtained from the
National Institute for the Control of Pharmaceutical and
Biological Products (Beijing, China). LPS (from
Escherichia coli 026:B6),
N-α-tosyl-L-lysine chloromethyl ketone (TLCK),
phorbol 12-myristate 13-acetate (PMA), protease
inhibitors, and propidium iodide (PI) were purchased from Sigma (St
Louis, MO, USA). 2'-Amino-3'-methoxyflavone (PD-98059)
was obtained from Calbiochem (La Jolla, CA, USA). Rabbit
polyclonal anti-Egr-1 and anti-NF-κB/p65 antibodies (Ab)
were obtained from Santa Cruz Biotechnology (Santa Cruz,
CA, USA). Horseradish peroxidase (HRP)-conjugated goat
anti-rabbit IgG, fluorescein isothiocyanate
(FITC)-conjugated goat anti-rabbit IgG were obtained from Jackson
Immuno Research (West Grove, PA, USA). The gel shift
assay system was from Bio-Rad (Hercules, CA, USA).
Cell culture and treatments The human monocyte cell
line THP-1 was obtained from American Type Culture
Collection (ATCC, No TIB-202, Manassas, VA , USA), with
distinct monocytic markers and the potential character of
macrophage[5]. The cells were cultured and treated in
RPMI-1640 medium (Invitrogen, Grand Island, NY, USA),
supplemented with 10% heat-inactivated fetal bovine serum,
glutamine 2 mmol/L, 100 kU/L benzylpenicillin, and 100
kU/L streptomycin at 37 °C under 5%
CO2. The cells were incubated with 1,8-cineol at a concentration of 1 mg/L, 10 mg/L,
and 100 mg/L respectively, and emulsified with Tween
80/phosphate-buffered saline (PBS), or 50
µmol/L TLCK, or 25 µmol/L PD98059 for 30 min prior to 1 mg/L LPS (dissolved in
PBS) treatment for 30 min. The vehicle control was
incubated with 0.001% Tween 80/PBS at the same volume for 30
min, and incubated with PBS for 30 min. Then the
subcellular localization and the expression of Egr-1 and
the expression of NF-κB were examined.
Immunofluorescent staining Immunofluorescent
staining was the same as the protocol of Yoo et
al[6]. The cell monolayers, which adhered to coverslips induced by 20
µg/L PMA, were washed twice with cold PBS, then fixed with
freshly prepared 3% paraformaldehyde for 15 min and
permeabilized with 0.5% Triton X-100 for 15 min. After 1 h of
blocking with 10% normal goat serum/PBS, the cells were
incubated with the primary antibody against Egr-1 at a
dilution of 1:50 in PBS for 2 h at 37 °C under a humidified
atmosphere. The coverslips were washed 5 times with PBS
and then incubated with FITC-conjugated IgG (1:100)
diluted in PBS for 1 h at 37 °C. In order to identify the nuclei,
the FITC-labeled samples were counterstained with 25 mg/L
PI for 2 min.
To acquire dual-color images, the cells were examined by
a 510 confocal laser scan microscope (Carl Zeiss,
Oberko-chen, Germany), which was equipped with a Zeiss inverted
research biological microscope and a 100×oil immersion
objective (NA 1.30). The samples labeled with both FITC
and PI were excited at 488 nm, and the fluorescence
emissions were captured through a 510_550 nm (530 nm in center)
and 590_ 620 nm (605 nm in center) band pass with spectral
grating, respectively.
Preparation of nuclear protein and whole cell protein
extracts The nuclear protein[7] and whole cell protein
extracts[8] were prepared as described with some
modifica-tions. For the nuclear protein extract, after the established
time of culture, the cells were collected and washed twice
with cold PBS, lysed in 400 µL cold buffer A
[10 mmol/L HEPES (pH 7.9), 10 mmol/L KCl, 0.1 mmol/L EDTA, and
EGTA,1 mmol/L phenylmethanesulphonylfluoride (PMSF),
and dithiothreitol (DTT), 1 mg/L aprotinin, leupeptin, and
pepstatin A]. After 15 min, 0.1% NP-40 was added to the
homogenates and the tubes were vigorously shaken for 1
min. Then the homogenates were centrifuged at 14 000 r/min
at 4 °C for 5 min. The supernatant fluid (cytoplasmic extracts)
was removed. The nuclear pellets were washed once with
cold buffer A, then suspended in 50 µL cold buffer B [20
mmol/L HEPES (pH 7.9), 420 mmol/L NaCl, 0.1 mmol/L
EDTA, and EGTA, 1 mmol/L PMSF, and DTT, and 1 mg/L
aprotinin, leupeptin, and pepstatin A], and vigorously shaken
at maximum speed at 4 °C for 30 min. The solution was
clarified by centrifugation at 14 000 r/min for 5 min, and the
supernatant fluid (nuclear extract) was stored in aliquots at
-70 °C. For the whole cell protein extract, the sample cells
were lysed in ice-cold buffer C [50 mmol/L Tris-HCl (pH 7.2),
150 mmol/L NaCl, 1% sodium deoxycholate, 1% Triton
X-100, 0.1% SDS, 10 mmol/L NaF, 160 µmol/L
Na3VO4, and
1 mmol/L PMSF]. The suspension was cooled at 4 °C and
sonicated for 2×10 s (40 W) with a probe sonicator.
Following centrifugation at 14 000 r/min at 4 °C for 15 min, the
supernatant fluid (whole cell protein extract) was stored in aliquots
at -70 °C. The protein concentration was determined by the
Folin method.
Western blot analysis The nuclear protein and whole
cell protein extracts (30 µg each lane) were separated by 8%
SDS-PAGE and transferred to polyvinylidene difluoride
membranes using a Mini Trans-Blot module (Bio-Rad,
Hercules, CA, USA). The membranes were blocked in 5% non-fat dried
milk/Tris-buffered saline (TBS)/0.05% Tween 20 (blocking
buffer). Then the membranes were incubated with the
primary antibody in blocking buffer and washed 5 times with
TBS/0.05% Tween 20 before incubation with a secondary
HRP-conjugated antibody in blocking buffer (1 h, room
temperature). After successive washes, the membranes were
developed with an enhanced chemiluminescence kit (ECL,
Santa Cruz). Anti-Egr-1 Ab and anti-NF-κB/p65 Ab,
HRP-conjugated IgG were applied at a dilution of 1:2000. A
semiquantitative analysis of immunoreactivity was measured
by Lab Works image acquisition and analysis software (UVP
GDS 8000, Upland, CA, USA), and the results were expressed
as OD (optical density) value.
Statistical analysis The data are presented as mean±SEM
and compared with ANOVA and least significant difference
test using the SPSS statistical program(Edition 10.0, SPSS
Inc, Chicago, IL, USA). The level of the statistical
significance was set at P<0.05.
Results
Effect of 1,8-cineol on the subcellular localization of
Egr-1 in THP-1 cells The dual-color images of the
FITC-labeled Egr-1 and PI-labeled nuclei in each group were
detected by indirect immunofluorescence and confocal
microscopy. The samples were immunocytochemically
labeled with FITC for the Egr-1 protein in green, followed by
incubation with the nuclear stain PI in red (Figure 1). Normal
THP-1 cells were labeled in the absence of the primary
antibody against Egr-1 to identify autofluorescence and
nonspecific labeling. A faint or invisible signal of the FITC label
(green), but only the PI label (red) was observed in these cell
sheets (Figure 1A). A strong nuclear and perinuclear
localization staining of Egr-1 appeared after stimulation with LPS
at a concentration of 1 mg/L for 30 min in the THP-1 cells
(Figure 1C) compared with the unstimulated cells (Figure
1B). The nuclear and perinuclear localization staining of
Egr-1 caused by LPS was reduced by pretreatment with 100
mg/L 1,8-cineol and PD98059 (Figure 1D, 1E).
Effect of 1,8-cineol (eucalyptol) on the expression of
Egr-1 in the nuclei or the whole cell The expression of Egr-1 in the
nuclei and whole cell protein increased markedly after LPS
stimulation (1 mg/L, 30 min). The increase induced by LPS
was canceled by 1,8-cineol pretreatment in a
dose-dependent manner. PD98059 dramatically inhibited LPS-induced
Egr-1 expression, but TLCK did not block the induction of
Egr-1 in LPS-stimulated THP-1 cells (Figures 2, 3).
The concentration of 1,8-cineol, TLCK, and PD-98059 was
determined from our previous work showing significant
effects in THP-1 cells without toxicity. The cell viability,
evaluated by MTT assay, did not change in the THP-1 cells
at all doses used (data not show).
Effect of 1,8-cineol on the expression of
NF-κB in the nuclei The NF-κB/p65 protein level in the nuclei of
LPS-stimulated THP-1cells markedly increased at 30
min. There was no change on the expression of
NF-κB/p65 in the nuclei after 1,8-cineol pretreatment. However, TLCK dramatically
inhibited LPS induction of NF-κB/p65 expression in the
nuclei (Figure 4).
Discussion
Egr-1 is a transcription factor that plays a regulatory role
in the expression of many important genes for inflammation.
The induction of Egr-1 has been demonstrated in cells
exposed to various stimuli, including phorbol ester, ionizing
radiation, inflammation, oxidative agents, and mechanical
stretch/relaxation[9,10]. Its target genes include cytokine,
chemokines, cell adhesion molecules, and
immunoreceptors[9_12].
The human monocyte cell line THP-1 is often used as a
model for tissue macrophages. Macrophages are key
inflammatory cells that have been documented to play a
critical role in various airway
disorders[13]. Upon stimulation with molecules such as LPS, these cells secrete an array of
pro-inflammatory cytokines and oxidants, including
TNF-α, IL-1β, and macrophage inflammatory
protein-2[14]. These pro-inflammatory cytokines genes are regulated by various
transcription factors, including Egr-1 and
NF-κB[15_17]. Therefore, modulation of
Egr-1 or NF-κB may provide a direct way of inhibiting inflammatory
mediators[18].
In this study, we investigated the effects of 1,8-cineol on
the subcellular localization of Egr-1, the expression of Egr-1
in the nuclei and whole cell, and the expression of
NF-κB in the nuclei of human THP-1 cells. Our studies showed that in
a dose-dependent fashion, 1,8-cineol inhibited the Egr-1
synthesis and nuclear localization induced by LPS in THP-1 cells,
suggesting that 1,8-cineol can inhibit the LPS-mediated
Egr-1 nuclear internalization. Those results indicate that 1,8-
cineol might suppress the expression of many genes
important for inflammation by inhibiting Egr-1 synthesis and nuclear
localization, which represents one of the anti-inflammatory
mechanisms of 1,8-cineol.
In addition, PD98059, which is a selective MAP kinase
kinase (MEK) inhibitor[19], dramatically inhibited LPS-induced
Egr-1 expression. However, TLCK, a serine protease
inhibitor[20], showed no effect on Egr-1 expression. The finding is
in accordance with what Mackman et al demonstrated
previously, that is that TLCK did not block the induction of
Egr-1 in LPS-stimulated monocytic
cells[21]. The result suggests that the inhibitory
effect of 1,8-cineol on the Egr-1
expression in LPS-induced THP-1 cells primarily was due to
the MEKextracellular signal-regulated kinase (ERK)1/2
pathway and not by serine protease phosphorylation pathway.
In contrast, we found that 1,8-cineol did not significantly
affect the LPS-induced NF-κB expression in nuclei. As we
know, Egr-1 is different from NF-κB in the signal pathway of
activation. The nuclear translocation of NF-κB depends on
the IκB rapid degrading through a
phosphorylation-dependent and ubiquitination-dependent
mechanism[22]. However, mitogenic stimulation of the Egr-1 gene has been shown to
be mediated in many cell types through the
RasRaf-1-MEK-ERK1/2 signal transduction
pathway[19,23,24]. Thus, the anti-inflammation mechanisms of 1,8-cineol is more likely related
to Egr-1 rather than NF-κB.
In conclusion, these observations suggest that 1,8-
cineol might block the effect of Egr-1 by inhibiting the
synthesis of Egr-1 and preventing Egr-1
nuclear internalization, which might at least represent one of the anti-inflammatory
mechanisms of 1,8-cineol. This provides evidence for the
role of 1,8-cineol in controlling airway inflammation.
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