Extract
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Introduction
Antrodia camphorata is a medicinal mushroom in
Taiwan well-known as a folk medicine for the treatment of
intoxication caused by alcohol or drugs, and is also used in
the treatment of diarrhea, abdominal pain, hypertension, skin
itching, and liver cancer[1]. It specifically parasitizes on the
inner cavity of the wood of the endemic species
Cinnamomum kanehirai and grows extremely slowly in nature. Therefore,
the submerged liquid culture using the parasitic hyphae of
A camphorata for the production of mycelia has being
become one of the most important methods. The biological
functions of A camphorata have been studied in various
bioassays. In the research of aqueous extracts from
A camphorata mycelia, the suppression of oxidative
hemolysis and lipid/protein peroxidation in erythrocytes has been
demonstrated[2]. The inhibition ability of its fermented
filtrate on H2O2-induced lipid peroxidation and its extracts on
the CCl4-induced rat liver damage was also
reported[3,4]. The extracts also showed a concentration-dependent inhibition
of N-formyl-methionyl-leucyl-phenylalanine or phorbol
12-myristate 13-acetate-induced reactive oxygen species
production in peripheral human neutrophils or mononuclear
cells[5]. Moreover, the fermented culture broth of
A camphorata has been found to have inhibitory capabilities
on the production of lipopolysaccharide (LPS)-induced
tumor necrosis factor (TNF)-α, interleukin (IL)-1β, inducible
nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2
expression[6]. Water extracts of submerged culture ofA camphorata have being shown to have antitumor
activity[7_10]. Polysaccharides extracted from fruiting bodies or
cultured mycelia of A camphorata exhibit antihepatitis B
virus activity[11]. As the polysaccharide components of
mushroom species have been established as the most promising
pharmacologically-active anti-tumor
portion[12,13], the partially-purified polysaccharide component from
A camphorata mycelia was also found to have antitumor effects on both the
in vitro and in vivo
model[14]. Fungous LPS from A
camphorata was found to inhibit bacterial LPS-induced intercellular
adhesion molecule-1 expression and the subsequent monocyte
adhesion process in vascular endothelial
cells[15]. However, there is no report about the anti-inflammatory activity of the
A camphorata polysaccharide fraction.
Cells of the monocytic/macrophage lineage play an
important role in response to inflammation and infection, as
the main players performing innate immune response and
contributors rendering adaptive immune response.
Bacterial LPS, a component of the gram-negative bacteria cell wall,
activates macrophages to secrete pro-inflammatory
cyto-kines, such as IL-6, TNF-α, and secondary mediators such
as leukotrienes, prostaglandins (PG) and nitric oxide (NO).
The iNOS is responsible for high output formation of NO by
macrophages against invading
microorganisms[16] or tumor
cells[17]. Although large production of NO by iNOS may
promote host defensive potency, it also contributes to
septic and hemorrhagic shock, rheumatoid arthritis and chronic
infections[18]. The COX-2 is an essential enzyme in the
production of inflammatory PG, and is inducible in activated
macrophages, fibroblasts, and several other cell types.
In vivo, the expression of COX-2 is observed in chronic
inflammatory conditions such as
arthritis[19] and human colon cancer
tissue[20]. In vitro, COX-2 expression was induced in
response to stimuli such as LPS and growth
factors[21,22]. Therefore, the screening of chemopreventive products from
natural resources with the functions focused on inhibiting
NO and PGE2 production would be an effective and direct
method.
The anti-inflammatory effects of the polysaccharide
fractions from A camphorata have never been studied. The
objective of this study was to investigate the effects of the
polysaccharides from the sequential extractions of
A camphorata mycelia on LPS-induced inflammation-related
gene expression by using mouse macrophages.
Materials and methods
Materials LPS (Escherichia coli 055:B5) and
anti-β-actin antibodies were purchased from Sigma-Aldrich (St Louis,
MO, USA). SuperSignal West Pico Chemiluminescent
Substrate was obtained from Pierce (Rockford, IL, USA).
Antibodies against COX-2, Sp1 and TLR4 were purchased from
Santa Cruz (Santa Cruz, CA, USA). The antibody against
NF-κB p65 was from Abcam (Cambridge, UK). The
antibody against iNOS/NOS II was from Upstate (Lake Placid, NY,
USA). PRO-PREPTM Protein Extraction Solution was from
iNtRON Biotechnology (Kyungki-Do, Korea). Bio-Rad
protein assay was purchased from Bio-Rad (Hercules, CA, USA).
TRIzol reagent and SuperScript II were from Invitrogen
(Carlsbad, CA, USA).
Cell culture RAW 264.7 cells were purchased from
Bioresource Collection and Research Center (Hsinchu,
Taiwan) and cultured at 37 ºC in a 5%
CO2 atmosphere in Dulbecco's modified Eagle's medium (DMEM; GIBCO,
Carlsbad, CA, USA) supplemented with 10% fetal bovine
serum FBS (Biowest, Rue de la Caille, Nuaille, France). The
endotoxin concentration of FBS was below 0.15 ng/mL, which
was certificated by Biowest.
Pretreatment of the mycelial powder Prior to the
extraction of the polysaccharides, a supercritical fluid carbon
dioxide (SC-CO2, 99.5% in purity) extraction was performed to
remove some inherently existing oil soluble substances in
order to minimize the interference to the polysaccharides,
according to a previous report[23]. Briefly, 100 g of the
lyophilized mycelial powder was weighed and added with 5%
n-hexane to serve as a modifier. The extraction was carried
out at 60 °C and 5000 psi in a supercritical gluid extraction
apparatus (ISCO SFXTM 2-10, Isco, Lincoln, NE, USA)
attached with a modified extraction vessel. In the beginning, a
dynamic continuous extraction was adopted at a flow rate of
1 mL SC-CO2/min for 1 h, and was then followed by a static
extraction for an additional l h. The oil soluble extracts were
collected in 95% ethanol. The residue
(ACR1) remaining in the extraction vessel was used in the subsequent
experi-mentations.
Preparation of AC-1 and AC-2 The method of Ker
et al[23] was followed for the preparation of the polysaccharide
fraction AC-1 and AC-2. For the AC-1 fraction,
ACR1 (100 g) was extracted with a reflux 3 times with 2000 mL double distilled
water (DDW) at 90 °C and was constantly stirred at 400 r/min
for 2 h. The extracts were filtered with aspiration after
cooling; the residue (ACR2) was kept for further
experimenta-tion. One mol/L HCl was added to the filtrate to adjust the
pH to 4.0, and then a 2 fold volume of ethanol (95%) was
added to precipitate the water-soluble polysaccharides, which
were collected and further purified in 400 mL of hot water
(100 °C). Finally, the water-soluble polysaccharides were
precipitated with the addition of a 3 fold volume of ethanol
(95%), and then collected and lyophilized (AC-1). For the
AC-2 fraction, the residue ACR2 was added with 1000 mL of
2% NaOH and extracted 3 times, and constantly stirred at
400 r/min and refluxed. The extracts were filtered with
aspiration after cooling; the residue was stored for the other
experimentation. The filtrate was collected and adjusted to
pH 4.0 with 12 mol/L H2SO4, and left to stand overnight. The
sediment was collected, dialyzed and lyophilized to recover
the isoelectric precipitate (AC-2). The yields of the
polysaccharide of A camphorata were 2.92%
(w/w) in the AC-1 fraction, and 10.38%
(w/w) in the AC-2 fraction. The average
molecular masses of AC-1 and AC-2 were 508 kDa and 394
kDa by gel permeation chromatography analysis.
Cell viability assay RAW 264.7 cells were plated in a 6
well plate at 2.8×106 cells per well and allowed to adhere to
the plate overnight; then the culture medium were refreshed
by new medium containing AC-1 or AC-2. After the
introduction of AC-1 or AC-2 for 1 h, the cells were stimulated
with LPS (500 μg/L). The cell numbers were counted after 18
h of exposure to LPS. Cell viability was examined using trypan
blue exclusion and counted using hemocytometer and phase
contrast microscopy.
Nitrite determination RAW 264.7 cells were cultured in
a 24 well plate at a density of
5×105, 1 d before LPS treatment. The cells were treated with AC-1 or AC-2 1 h before the
introduction of 500 μg/L LPS. After LPS treatment for 18 h,
the extracellular medium containing nitrite ion
(NO2_) was used as an indication of NO production, and the amount of
NO2_ in the culture medium was determined according to the
colorimetric method by using Griess reagent. The isolated
supernatants were incubated with an equal volume of Griess
reagent and incubated at room temperature for 10 min.
Absorbance at 540 nm was then read and compared with known
standard solutions of NaNO2.
Protein extraction RAW 264.7 cells were precultured in
a 3.5 cm dish for cell lysate extraction 1 d before LPS treatment.
The cells were treated with each polysaccharide 1 h prior to
the introduction of 500 μg/L LPS. The total cell lysates were
prepared by lysing the cells in buffer containing
PRO-PREPTM Protein Extraction Solution containing 10 mmol/L
NaF and 1 mmol/L orthovanadate at 4 ºC for 15 min and
centrifuged at 7500×g at 4 ºC for 30 s. The supernatants
containing the protein extracts were stored at -80 ºC for
stabilization.
Nuclear extract preparation The cells from the 10 cm
dishes were washed twice with phosphate-buffered saline
PBS and scraped in 1 mL of PBS. The cells were collected by
centrifuging at 7500×g for 30 s, resuspended in 0.4 mL of
buffer A [10 mmol/L
N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid HEPES, pH 7.9, 10 mmol/L KCl, 1.5
mmol/L MgCl2, and 0.5 mmol/L EDTA] at 4 ºC for 10 min. Then nuclei
were pelleted by centrifugation at
7500×g for 30 s. The pellets were resuspended in 0.1 mL of buffer C [20 mmol/L HEPES,
pH 7.9, 420 mmol/L NaCl, 1.5 mmol/L
MgCl2, 0.2 mmol/L EDTA, and 25% glycerol
(v/v)] at 4 ºC for 20 min. The suspension
was centrifuged by centrifugation at
7500×g for 2 min. The supernatants were collected and stored at -80 ºC until use.
Both buffers A and C contained the following protease
inhibitors: 0.5 mmol/L phenylmethylsulfonyl, 1 mmol/L
orthovanadate, 2 μg/mL pepstatin A, and 2 μg/mL leupeptin.
Western blotting Protein concentration was determined
by using the Bio-Rad protein assay reagent. The extracted
protein (30 μg) was separated in 8% SDS-PAGE and
transferred to a polyvinylidene fluoride PVDF membrane. After
blotting, the membrane was incubated with specific primary
antibodies overnight at 4 ºC, then further incubated for 1 h
with a horseradish peroxidase HRP-conjugated secondary
antibody and eventually incubated with SuperSignal West
Pico Chemiluminescent Substrate for 2 min. The bounded
antibodies were detected by Kodak Digital
ScienceTM (Image Station 4000 MM, New Haven, CT, USA).
RT-PCR RAW 264.7 cells were cultured in a 3.5 cm dish
the day before LPS treatment. The cells were treated with
each inhibitor 1 h prior to the introduction of 500
μg/L LPS. The RNA was extracted with TRIzol reagent and detected by
RT-PCR technique. RT was performed on 2 μg of total RNA
by random primers (9 mers) and SuperScript II, then 1/20
volume of reaction mixture was pooled, followed by PCR
with mouse COX-2 specific primers (5'-CAGCAAATCCTTGCTGTTCC-3' and
5'-TGGGCAAAGAATGCAAACATC-3'), mouse iNOS specific primers
(5'-GTCAACTGCAAGA-GAACGGAGAAC-3' and
5'-GAGCTCCTCCAGAGGGTAGGCT-3'), mouse IL-6 specific primers
(5'-AGTAAGTTCCT-CTCTGCAAGAGACT-3' and
5'-CACTAGGTTTGCCGAGTAGATCTC-3'), mouse IL-10 specific primers
(5'-CGTCGGATCCGCCATGCCTGGCTCACCACTGCT-3' and
5'-CGTCTCTAGATTAGCTTTTCATTTTGATCA-3'), or β-actin specific
primers (5'-CCTAAGGCCAACCGTGAAAA-3' and 5'-TCTTCATGGTGCTAGGAGCCA-3'). Cycle numbers of PCR were
25 cycles for each primer. The RT-PCR products were
separated on 1% agarose gel and analyzed.
Protein chip assay RayBio Mouse Cytokine Antibody
Array I was purchased from RayBiotech (Norcross, GA,
USA). It was employed to assay cell culture conditioned
medium and used according to the manufacturer's
instruc-tions. The confluent cells were replaced from medium
containing 10% FBS to serum-free medium; in the meantime, AC
preparations were added into the cells. LPS was added 1 h
after AC treatment. The conditioned medium was collected
after LPS treatment for 9 h. Twenty-two different cytokines
were evaluated: granulocyte-colony stimulating factor
(GCSF), granulocyte-macrophage colony stimulating factor
(GM-CSF), IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10,
IL-12p40/p70, IL-12p70, IL-13, IL-17, interferon-γ, monocyte
chemoat-tractant protein-1 (MCP-1), monocyte chemoattractant
protein-5 (MCP-5), regulated upon activation, normal T-cell
expressed, and presumably secreted (RANTES), stem cell
factor(SCF), sTNFR1, TNF-α, thrombopoietin, and vascular
endothelial growth factor. The bounded cytokines were
detected by biotin-labeled antibodies and horseradish
peroxidase-labeled streptavidin, and were detected by
SuperSignal West Pico Chemiluminescent Substrate for 2 min.
The bounded antibodies were analyzed by Kodak Digital
ScienceTM (Image Station 4000MM).
Statistical analysis Differences among the data of the
LPS-treated control and further treatments with each
polysaccharide fractions were analyzed by Student's
t-test. Statistical probability was expressed as
P<0.05.
Results
Effects of AC-1 and AC-2 on the viability of RAW 264.7
cells Before testing the effects of AC-1 and AC-2 on
LPS-induced nitrite and COX-2 expression, we tested the effects
of AC-1 and AC-2 on cell viability. After 18 h treatment of
AC-1 or AC-2 and LPS in mouse macrophage RAW 264.7
cells, cell numbers were counted individually. The results
presented in Figure 1 demonstrate that cell numbers were
not affected by AC-1 at 100 mg/L or AC-2 at 50 mg/L. A
minute decrease was found at 200 mg/L of AC-1 mg/L or 100
mg/L, and 200 mg/L of AC-2 (Figure 1). After statistical
analysis, we suggested that there was no significant
cytotoxicity under 200 mg/L of AC-1 or AC-2. Further study on
the inflammation-related gene expression affected by AC-1
and AC-2 under the concentration of 200 mg/L was conducted.
Effects of AC-1 and AC-2 on LPS-induced NO
production NO synthesized by iNOS has been implicated as a
mediator of inflammation. The inhibitory effects of AC-1 and
AC-2 on LPS-induced NO production were investigated. As
shown in Figure 2, NO production was greatly increased
after LPS treatment for 18 h. Only AC-2, but not AC-1,
showed a dose-dependent reduction of NO production
between 50 and 200 mg/L. This suggests that the AC-2 fraction
has the ability to inhibit LPS-induced NO production.
Effects of AC-1 and AC-2 on LPS-induced expression of
the iNOS protein and mRNA In general, iNOS is not present
in the resting cells, but is induced by various stimuli.
Increased expression of iNOS has been associated with
inflammatory disorders. Because LPS-induced NO
production could be reduced by AC-2, we further tested the
effects of AC-1 and AC-2 on the LPS-induced iNOS protein and
mRNA expression. As shown in Figure 3, AC-2, but not
AC-1, dose-dependently inhibited the LPS-induced iNOS protein
(Figure 3A) and mRNA (Figure 3B) expression in RAW
264.7 cells. This suggests that the inhibitory effects of AC-2 on
LPS-induced NO production is due to the suppression of
LPS-induced iNOS gene expression.
Effects of AC-1 and AC-2 on LPS-induced expression of
COX-2 protein and mRNA Arachidonic acid is released from
the cell membrane catalyzed by phospholipase
A2, converted into PGH2 by cyclooxygenase, and further metabolized to
PGE2. The effects of AC-1 and AC-2 on the expression of the
COX-2 protein and mRNA were investigated. As shown in
Figure 4, 200 mg/L AC-1 or 200 mg/L AC-2 did not suppress
either the LPS-induced COX-2 protein (Figure 4A) or mRNA
(Figure 4B) expression. Furthermore, we detected the
PGE2 concentration in the culture medium. We also found that
that 200 mg/L AC-1 or 200 mg/L AC-2 did not inhibit
LPS-induced PEG2 production (data not shown). These data
suggest that AC-1 and AC-2 had no inhibitory effect on
LPS-induced COX-2 gene expression and
PGE2 production.
Effects of AC-1 and AC-2 on LPS-induced cytokine
expression In order to widely observe the effects of AC-1 and
AC-2 on LPS-induced cytokine expression, the mouse
cytokine antibody array (Figure 5A) was applied. When the
cells were incubated with AC-1 or AC-2 for 10 h, neither
AC-1 nor AC-2 changed the cytokine expression pattern, compared
to the control cells (Figure 5Bi, iii, v). After LPS treatment for
9 h, the protein expression of GCSF, GM-CSF, IL-6, IL-10,
MCP-1, MCP-5, RANTES, sTNFRI, and TNF-α all increased
(Figure 5Bi, ii). Each LPS-increased dot was quantified and
the result is shown in Figure 5C. It indicated that AC-2, but
not AC-1, inhibited LPS-induced protein expression of IL-6,
IL-10, MCP-5 and RANTES, and neither AC-1 nor AC-2
inhibited LPS-induced GCSF, GM-CSF, MCP-1, sTNFRI, and
TNF-α protein secretion in mouse macrophages.
Effects of AC-2 on LPS-induced IL-6 and IL-10 mRNA
expression We further identified whether AC-2 inhibited
LPS-induced mRNA expression of IL-6 and IL-10. The mRNA
expression of IL-6 and IL-10 were monitored by
RT-PCR. AC-2 dose-dependently inhibited LPS-induced IL-6 mRNA
expression (Figure 6A); it also inhibited IL-10 mRNA
expression (Figure 6B). The inhibitory effect on LPS-induced IL-6
and IL-10 gene expression by AC-2 is important in
the regulation of IL-6 and IL-10 genes in mouse macrophages.
Effects of AC-2 on TLR4 and LPS-induced
NF-κB translocation Because AC-2 has the ability to repress some
specific gene activation by LPS, we further identified the effects
of AC-2 on the upstream linkage of LPS signaling. TLR4 is a
membrane receptor for the recognition of LPS. After binding
to LPS, it will activate the NF-κB pathway to induce the
expression of many inflammatory
genes[24]. Next, we analyzed whether TLR4 and
NF-κB were affected by LPS and AC-2. As shown in Figure 7A, the protein expression of the TLR4
receptor was unchanged after LPS and AC-2 treatment.
NF-κB p65 translocation is one of the important signal pathways
activated by LPS; therefore, the effect of AC-2 on
LPS-induced NF-κB p65 translocation was monitored. It showed
that LPS increased the nuclear translocation of p65, and
AC-2 inhibited LPS-induced p65 translocation (Figure 7B).
Discussion
A camphorata (Polyporaceae, Aphyllophorales), the
cause of brown heart rot of Cinnamomum kanahirai
Hay in Taiwan, is a new basidiomycete and a scarce traditional
medicine that has attracted great attention due to its antioxidant
and antitumor effects in vivo and in
vitro. It was identified in 1990 as a traditional Chinese medicine in Taiwan. The present
study was undertaken to elucidate the pharmacological and
molecular effects of the partially purified polysaccharide
fraction from A camphorate on LPS-induced inflammatory
mediators in macrophages. The results indicate that AC-2 is an
effective inhibitor of LPS-induced inflammatory mediators
such as IL-6, IL-10, MCP-5, RANTES, and NO. This
indicates that the polysaccharide fraction of A
camphorate appears to have some potential bioactive compounds in
inhibiting LPS-induced inflammatory gene expression,
including IL-6, IL-10, iNOS, MPC-5, and RANTES.
IL-6 was originally identified as a B-cell differentiation
factor, but it is now known to play a central role in host
defense because it is released in response to infection, burns,
trauma and tumor, and its functions range from key roles in
acute phase protein induction to B- and T-cell growth and
differentiation[25]. IL-6 can induce a variety of acute-phase
proteins, such as fibrinogen, serum amyloid A, and
the C-reactive protein in human
hepatocytes[26]. IL-6-deficient mice also show a severely defective inflammatory
acute-phase response after tissue damage or
infection[27]. An unregulated, high-level production of IL-6 could generate
an undesired inflammatory state, a circumstance that can
cause various diseases. Several reports indicate that IL-6 is
implicated in the pathogenesis of a number of human
disorders, including rheumatoid arthritis and inflammatory
bowel disease[28,29]. AC-2, a partially purified
polysaccharide fraction from A camphorate could inhibit LPS-induced
IL-6 gene expression, which could provide a therapeutic clue
in IL-6-related diseases.
IL-10 was discovered as a cytokine synthesis inhibiting
factor, and its principal function is the activity inhibition of
Th1 cells[30]. IL-10 has multiple biological functions on
different cell types. In macrophages, IL-10 inhibits
ligand-induced activation and the production of pro-inflammatory
cytokines from macrophages[31,32]. IL-10 inhibits the
proliferation as well as cytokine synthesis of
CD4+ T-cells[33]. Other immunosuppressive effects have also been reported on
eosinophils[34],
neutrophils[35], and dendritic
cells[36]. In contrast to these immunosuppressive effects, IL-10 has been shown
to have the abilities of immunostimulation on
cytotoxic T-cells[37], and is a growth costimulator for thymocytes and
mast cells[38]. In this study, AC-2 also inhibited LPS-induced
IL-10 gene expression.
Besides IL-6 and IL-10, AC-2 also reduced LPS-induced
iNOS gene expression and NO release in RAW 264.7 cells.
The expression inhibition of the iNOS protein responsible
for NO inhibition by A camphorata had been
reported[6]. Here we suggested for the first time that
A camphorata reduced LPS-induced NO production by inhibiting iNOS
mRNA expression. Although nanomolar concentrations of
NO play an important physiological role as a defense
molecule in the immune system[39], overproduction of NO,
predominantly via the upregulation of iNOS in macrophages,
contributes to numerous pathological processes, including
inflammation[40] and
atherosclerosis[41]. The mechanism responsible for NO inhibition of several plant extracts had been
reported, including direct scavenging of
NO[42], suppression of iNOS
activity[43], or the reduction on iNOS gene
expression[44]. In the present data, the AC-2 fraction of
A camphorata expressed anti-inflammatory effects via iNOS
gene inhibition. This inhibition may in part be through
inhibiting LPS-activated NF-κB translocation (Figure 7B). This
data suggests that the AC-2 polysaccharide fraction of
A camphorate could be an attractive candidate for adjunctive
therapy in gram-negative bacterial infections.
AC-2 also has the ability to repress the secretion of
MCP-5 and RANTES. MCP-5 is a potent monocyte active chemokine
that is involved in allergic
inflammation[45]; RANTES is also an important chemoattractant for many immune
cells[46]. Some molecules inhibit LPS-activated macrophages by interfering
LPS binding to cell surface[47,48]. In this study, it is not likely
that AC-2-inhibited cytokine expression is due to interferring
LPS binding to its receptor TLR4 because the protein
expression of TLR4 does not change after AC-2 treatment
(Figure 7A). LPS-induced COX-2, GCSF, GM-CSF, MCP-1,
sTNFRI, and TNF-α were not reduced simultaneously.
Although the inhibition of COX-2 protein expression by
A camphorata had been
reported[6], there was no such bioactive
constituent in this polysaccharide fraction of A
camphorata. It also suggests that the bioactive compounds for inhibiting
COX-2 and iNOS existed in different components of
A camphorata. However, the characterization of candidate
compounds that mediate the inhibition of LPS-induced NO,
IL-6, IL-10, MCP-5, and RANTES secretion requires further
study.
In conclusion, the present study shows that the
fractionated polysaccharides AC-2 of A camphorata
inhibits the production of IL-6, IL-10, MPC-5, RANTES, and NO in
LPS-stimulated mouse macrophages. This inhibition of
IL-6, IL-10, and iNOS was mediated by transcriptional
downregula-tion of IL-6, IL-10, and iNOS genes. Because IL-6, IL-10,
MCP-5, RANTES, and NO are thought to be associated with
acute and chronic inflammation diseases, the inhibitory
effects of A camphorata on LPS-induced IL-6, IL-10 MCP-5,
RANTES, and NO might provide a partial anti-inflammation
function in LPS-induced inflammatory conditions.
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