Extract
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Introduction
Numerous studies have demonstrated that certain components present in dietary mushrooms have been responsible for
the modulation of cellular and physiological changes in the host. It is for this reason that mushrooms are often used as cancer
therapeutic agents[1_4]. Hericium
erinaceum, a well-known traditional edible mushroom, contains valuable constituents
including polysaccharides, lectins, proteins, lipids, hericenone, erinacol, erinacine, and
terpenoids[5_7]. Recently these components, including water-soluble polysaccharides of
H erinaceum, were isolated from its
fruiting
bodies and induced intriguing biological activities such as cytotoxicity, synthesis of a nerve growth factor, and antimicrobial
function[6,8_11]. However, not much attention has been given to the elucidation of other immunological activities and therefore,
limited data and references are available.
Interleukin (IL)-1 is a pluripotent and proinflammatory cytokine that orchestrates inflammatory and host-defense responses.
Biologically active IL-1b is a 17.5-kDa protein resulting from cleavage of an inactive 31_34 kDa
pro-IL-1b[12,13]. IL-1b augments T-cell responses to
mitogens[14,15], indirectly activates B
cells[16,17], increases expression of vascular adhesion
molecules[18], and induces other proinflammatory cytokines and
chemokines[19,20]. IL-1 is produced mainly by monocytes and macrophages
when stimulated with various antigenic stimulants, including viruses or bacterial components such as lipopolysaccharide
(LPS)[21_23]. Numerous studies have demonstrated that nuclear factor-kappa B
(NF-kB), activator protein 1 (AP-1), nuclear factor interleukin-6 (NF-IL6), and cAMP response element (CRE)/activating transcription factor (ATF) regulate IL-1
transcription in macrophages upon
stimulations[24_27].
Since IL-1 is a proinflammatory cytokine, agents that induce the activity of IL-1 have recently gained particular
therapeutic and clinical
interest[28_31]. In the present study, in order to characterize the immunological properties of
H erinaceum, we prepared water extract
from H erinaceum (WEHE) and investigated the inductive effect of WEHE on
IL-1b expression in vitro.
Materials and methods
Reagents and chemicals LPS (from Salmonella
typhosa) was obtained from Sigma-Aldrich (St Louis, Missouri, USA)
and b-D-(1,3)-(1,6)-glucan from VPGmbH (Hergestellt,Germany). All reagents for RT-PCR were purchased from Promega
(Madison, Wisconsin, USA), with the exception of
recombinant Taq DNA polymerase (rTaq) and dNTP, which were
purchased from Takara Bio Inc (Otsu, Shiga, Japan). Rabbit anti-mouse
IL-1b antibody and anti-actin antibody were purchased
from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA) and Sigma-Aldrich, respectively. The mouse macrophage-like cell
line RAW 264.7 (TIB-71) was purchased from the American Type Culture Collection (Manassas, Virginia, USA).
WEHE extraction, isolation and chemical analysis
Dried mushrooms, H erinaceum, were obtained from the Korean
Mushroom Corporation (Pochon, Korea). One hundred grams of the mushroom was washed several times with distilled
water, soaked in 1.5 L of pyrogen-free water for 2 h, and then boiled for 2 h. Solid particles and aggregates were removed by
centrifugation at 3000×g for 30 min and the supernatants were lyophilized. Finally, 26.35 g of the lyophilized water extract was
obtained and used in this experiment. The general chemical composition of WEHE was analyzed in triplicate according to the
methods of the Association of Official Analytical
Chemists[32]. Analyses were conducted for moisture (AOAC method
930.15), crude protein (AOAC method 984.13), acid detergent fiber (AOAC method 962.09), ash (AOAC method 942.05) and ether
extract (AOAC method 920.39). For the high-performance thin-layer chromatography (HPTLC) analysis, Interlucan 500
[b-D-(1,3)-(1,6)-glucan from Pharmode] and WEHE were dissolved in HPLC-grade methanol and applied to the pre-washed silica
gel 60 F254 HPTLC plates (size 10×10 cm; thickness of the silica gel 0.2 mm; Merck, Darmstadt, Germany) with an automated
applicator (Linomat IV, CAMAG, Merck KGaA, Germany). The samples were then separated (migration distance 75 mm)
using HPLC-grade chloroform/methanol/water/formic acid (48:48:2:2). The migrated components were visualized at 254 nm
using Reprostar 3 with a digital camera (CAMAG, Germany).
Culture of RAW 264.7 cells RAW 264.7 cells were cultured with Dulbecco¡¯s modified Eagle¡¯s medium (DMEM, Cellgro
Mediatech, Herndon, Virginia, USA) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, Utah,
USA), 1×105 unit/L penicillin, and 100 mg/L streptomycin at 37
oC in a 5% CO2 humidified incubator.
Animals Specific pathogen-free 6-week-old C3H/HeJ mice were purchased from Charles River Japan Inc (Hino Breeding
Center, Yokohama, Japan). On arrival, the randomized mice were transferred to cages (5 mice per cage) containing a saw dust
bedding and quarantined for 1 week. The mice were given food (Purina Certified Lab Chow) and
water ad libitum. Their thymocytes were isolated and used for IL-1
bioassay when their body weight reached 17 to 20 g. The temperature of the animal
care facility was kept at 21_24 oC and 40%_60% relative humidity with a 12 h light/dark cycle. All animal studies were
conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the US
National Institutes of Health.
IL-1 bioassay An IL-1 bioassay to determine functional
IL-1 levels in media was performed as previously
described[33]. Briefly, RAW 264.7 cells were plated at
5×105 cells/mL in
24-well culture plates and stimulated with 0, 0.1, 1, or 10
mg/L WEHE for 48 h. Indomethacin was added to prevent
prostaglandin synthesis in the IL-1 assay. The culture media were harvested and assayed for the activity of IL-1 to induce proliferation
of thymocytes isolated from the 6-week-old C3H/HeJ mice. The thymocytes
(1.5×106) were cultured for 68 h with 10-fold
dilutions of the IL-1-containing supernatants in the presence of phytohemagglutinin and then incubated with
[3H]-thymidine for 4 h. The cultures were harvested onto glass filter paper and the
[3H]-thymidine uptake was measured by scintillation
counting.
RT-PCR RAW 264.7 cells
(5×105 cells/mL, 10 mL) were treated with 0, 1, 5, or 10 mg/L of WEHE or 500 µg/L of LPS as a
positive control for 3 h. The total RNA was isolated from each group of the cells using TRIzol reagent (InVitrogen, Carlsbad,
California, USA) and equal amounts of RNA were reverse transcribed into cDNA with random hexamers (Promega Corporation,
Madison, Wisconsin, USA). For PCR, the amplifications were performed in a total volume of 30 µL containing 0.5 units of
rTaq and 10 pmol of primers specific
to murine IL-1b (5¡¯- AAGCTCTCACCTCAATGGA-3¡¯ and
5¡¯-TGCTTGAGAGGTGCTGATGT-3¡¯) and b-actin (5¡¯-GTG GGGCGCCCCAGGCACCA-3¡¯ and 5¡¯-CTCCTTAATGTCA CGCACGATTTC-3¡¯). The amplifications
were performed for 25 cycles for b-actin and for 30 cycles for
IL-1b. Equal volumes of RT-PCR products were separated on an
agarose gel (1%) and visualized by ethidium bromide staining with a gel documentation system (Gel Doc 2000, Life Science
Research, Hercules, CA, USA). Relative expression of the
IL-1b to the b-actin control was quantitated using a densitometer
with Multi gauge software (Fujiphoto Film Co Ltd, Tokyo, Japan).
Transient transfection of RAW 264.7 cells and CAT
assay RAW 264.7 cells were transiently transfected with an
IL-1b reporter gene construct pIL-1(870 bp)-CAT, which expresses CAT reporter genes solely regulated by the activity of the
IL-1b promoter[33], using the DEAE-Dextran method as previously
described[34]. The cells were then adjusted to
5×106 cells per 10 mL of the media, placed onto 100 mm
plates, and incubated in a 5% CO2 humidified incubator at 37
oC for 24 h. The transfectants were treated with 2, 5 or 10 mg/L of WEHE. Eighteen hours later, the cells were washed with ice-cold PBS, resuspended in
0.25 mmol/L Tris (pH 7.8) and subjected to 3 cycles of freezing and thawing.
The lysates were centrifuged (12
000×g for 10 min at 4 oC) and the supernatant was assayed for CAT activity by the TLC
method[35]. The amount of radioactivity was determined
by an image analyzer (Phosphor Imager, Molecular Dynamics, Sunnyvale, CA, USA) for quantitative analysis.
Western blotting RAW 264.7 cells
(5×105 cells/mL, 10 mL) were placed onto a 100 mm tissue culture dish in DMEM
supplemented with 10% FBS and antibiotics
(1×105 unit/L penicillin and 100 mg/L streptomycin). Cells were treated with 0, 1,
5 or 10 mg/L of WEHE for 24 h. At the end of incubation, cells were washed once with PBS and lysed with RIPA buffer
(Upstate Biotechnology, Lake Placid, NY, USA) as recommended by the manufacturer. Twenty micrograms of the whole-cell
lysate were separated by 10% SDS-polyacrylamide gel electrophoresis and electro-transferred to a PVDF membrane (Millipore,
Bedford, MA, USA). The membrane was incubated with a blocking buffer (5% BSA/1X TBS/0.1% Tween-20) at room
temperature for 1 h and then was kept on ice overnight with the same buffer containing rabbit polyclonal antibodies against
IL-1b or actin. After washing 3 times with TBS-T (1×TBS/0.1% Tween-20), the membrane was incubated with HRP-conjugated
anti-rabbit IgG in the blocking buffer at room temperature for 1 h. Then, after washing 3 times with TBS-T, the immunoreactive
bands were detected with enhanced chemiluminescence reagents (Amersham Biosciences, Piscataway, NJ, USA).
Electrophoretic mobility shift assay (EMSA)
The cells (5×106 cells/mL) were treated with 1, 5, or 10 mg/L of WEHE or 500
µg/L of LPS as a positive control for 90 min. Nuclear extracts were prepared as previously
described[34]. Briefly, cells were lysed with hypotonic buffer (10 mmol/L Hepes,
1.5 mmol/L MgCl2, pH 7.5) and the nuclei were pelleted
by centrifugation at 3000×g for 5 min. Nuclear lysis was
performed using a hypertonic buffer (30 mmol/L Hepes, 1.5
mmol/L MgCl2, 450 mmol/L KCl, 0.3 mmol/L EDTA, 10% glycerol, 1 mmol/L dithiothreitol (DTT), 1 mmol/L phenylmethyl-sulfonyl fluoride (PMSF), and 1
mg/L each of aprotinin and leupeptin). Following lysis, the samples were centrifuged at 14
500×g for 20 min, and the supernatant was retained for use in the DNA binding assay. Double-stranded deoxyoligo nucleotides containing each consensus
recognition site (in italics) of the NF-kB, AP-1, NF-IL6, and CRE/ATF are as follow:
NF-kB; 5¡¯-GATCTCAGAGGGGACTTTCCGAG AGA-3¡¯, AP-1;
5¡¯-GATCTGCATGAGTCAGACACA-3¡¯, NF-IL6;
5¡¯-GATCTACATGTTGTGCAACTTGCCTA-3¡¯, and CRE/ATF;
5¡¯-CTCGAGAGAGATTGCCTGACGTCAGAGAGCTA
GAGATCT-3¡¯. The oligonucleotides were synthesized and end-labeled with
[g-32P]-dATP. Nuclear extracts (5 µg) were incubated with 1
mg poly (dI-dC) and the 32P-labeled DNA probe in the binding buffer (100 mmol/L KCl, 30 mmol/L Hepes,
1.5 mmol/L MgCl2, 0.3 mmol/L EDTA, 10% glycerol, 1
mmol/L DTT, 1 mmol/L PMSF, and 1 mg/L of each aprotinin and leupeptin) for 20 min at room
temperature. Protein/DNA binding complexes were separated from free probe using a 4.8%
polyacrylamide gel in 0.5×TBE (44.5 mmol/L Tris, 44.5
mmol/L boric acid, and 1 mmol/L EDTA). Following electrophoresis, the gel was dried and subjected to
autoradiography.
Statistical analysis The mean±SD was determined for each treatment group in a given experiment. The treatment groups
were compared to appropriate controls to find significant differences using a Dunnett¡¯s
two-tailed t-test.
Results
Basic chemical composition of WEHE The general
chemical composition of WEHE showed a relatively higher
concentration of crude protein (44.82%) and carbohydrate (27.63%) than other components (Figure 1A). Further analysis of
carbohydrate using HPTLC showed that b-glucan was one of the major components of WEHE (Figure 1B).
WEHE treatment augments IL-1 production in RAW
264.7 cells The effect of WEHE on IL-1 induction was examined in
RAW 264.7 cells using bioassay. A significant
(P<0.05) increase in the activity of IL-1 was observed when the cells were
stimulated with 1 or 10 mg/L of WEHE (Figure 2A). The
IL-1b protein expression was examined using Western blotting since
the bioassay may have been interferred with other similar or closely related molecules although we added appropriate
inhibitors. As shown in Figure 2B, the level of
IL-1b in the macrophage cells was increased in proportion to the concentration
of WEHE. Meanwhile, no change in the actin expression was observed, indicating specific induction of IL-1 in macrophages
by WEHE. Cell viabilities in any of the WEHE treatment groups were not affected, in which live cells always exceeded 90%
as determined by trypan blue staining (data not shown).
WEHE-mediated augmentation of IL-1b production was due to up-regulation of its
transcription Next we examined the steady-state levels of mRNA encoding
IL-1b in RAW 264.7 cells treated with WEHE. As shown in Figure 3A, the cells maintained in normal conditions
expressed undetectable levels of IL-1b mRNA, whereas the level of expression dramatically increased when the cells were
stimulated with 500 µg/L of LPS, which served as a positive control. It is evident that WEHE induced a dose-dependent
increase of IL-1b mRNA. Since the enhancement of
IL-1b transcripts could be due to either increased stability of
IL-1b mRNA or up-regulation of IL-1b transcription, we performed an
in vitro transfection assay with a reporter gene, pIL-1(870bp)-CAT,
where the expression of CAT is regulated by IL-1b promoter. As shown in Figure 3B, the activity of
IL-1b promoter increased in cells treated with WEHE compared to the negative control.
Positive regulation of transcription factors,
NF-kB, AP-1, and NF-IL6 by WEHE We have further investigated
the mechanism of WEHE for the transcriptional activation of the
IL-1b gene. DNA-binding activity of transcription factors,
NF-kB, NF-IL6, AP-1, and CRE/ATF, all of which have been known to exist on the promoter sequence of
IL-1b and contribute to its transcriptional regulation, are examined using EMSA. As shown in Figure 4, the DNA-binding ability of
NF-kB increased in the cells upon exposure to WEHE in a dose-dependent manner. It was further evident that the DNA-binding activities of
NF-IL6 and AP-1 also increased, although the effects were less dramatic than
NF-kB. However, no change was observed in the DNA binding activity of CRE/ATF to its cognate DNA recognition sequences (Figure 4).
Discussion
There is increasing interest in the use of mushrooms and mushroom extracts, not only as dietary supplements, but also as
therapeutic supplements based on theories and
findings, with a paucity of data, which indicate that they modulate immune
function. In the present study, WEHE induced the secretion of
IL-1b and its mRNA expression in murine macrophages.
Although some other possibilities cannot be excluded, our study indicates that WEHE induction of
IL-1b expression in murine macrophage might be primarily due to the up-regulation of
IL-1b mRNA as determined by the in vitro CAT reporter
gene assay. This is further supported by the fact that WEHE induced activation of responsible transcription factors such as
NF-kB, AP-1 and NF-IL6, leading to an increase in
IL-1b transcription.
IL-1 represents a potent inflammatory cytokine with numerous biological activities that regulate host defense and immune
responses[12,18]. In addition to the role of IL-1 as an important immunoregulator, it also appears to be involved in anticancer
activity. IL-1 has a suppressive effect on the proliferation of human prostate
cancer[36], ovarian
cancer[37], and breast
cancer[38]. Conversely, a strong carcinogen,
2-acetylaminofluorene inhibits IL-1b
expression[25]. Together with these
aforementioned reports, our findings on the inductive effect of WEHE on the expression of IL-1 could give an important insight into
the explanation of a possible mechanism by which polysaccharides isolated from
H erinaceum produce antitumor
activities[11].
We further demonstrated that WEHE stimulating
IL-1b expression in murine macrophages was due to the up-regulation of
transcription factors, especially NF-kB, where the changes of
NF-kB were superior to other transcription factors. It is well
established that the activation of NF-kB is strictly regulated by the binding of its inhibitor,
IkBa[39]. Phosphorylation and subsequent proteasomal degradation of
IkBa disrupts the IkBa-NF-kB complex, thus allowing for the translocation of
activated NF-kB into the nucleus[39_42]. In the nucleus,
NF-kB binds onto its consensus sequence on the promoter regions
of various genes, including IL-1b, TNF-a, IL-6, and
iNOS[40,43]. Therefore, it is very likely that the expression of not only
IL-1b, but also other proinflammatory mediators, could be affected by WEHE through a similar mechanism to those observed in
this study.
In conclusion, the present study suggests that WEHE has a stimulatory effect on IL-1 production in macrophages via
activation of transcription factors such as NF-kB. This study could be valuable in that WEHE might have therapeutic
potential, although further examination is necessary in terms of efficacy in animal studies and safety, including adverse
effects.
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