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Structure Characterization of a Novel Polysaccharide from Dictyophora indusiata and its Macrophage Immunomodulatory Activities Wenzhen Liao, Zhen Luo, Dan Liu, Zhengxiang Ning, Jiguo Yang, and Jiaoyan Ren J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf504677r • Publication Date (Web): 19 Dec 2014 Downloaded from http://pubs.acs.org on December 23, 2014
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Journal of Agricultural and Food Chemistry
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Structure Characterization of a Novel Polysaccharide from
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Dictyophora indusiata and its Macrophage Immunomodulatory
3
Activities
4
Wenzhen Liao†, Zhen Luo‡, Dan Liu†, Zhengxiang Ning†, Jiguo Yang†*, Jiaoyan Ren†*
5 6
(1 College of Light Industry and Food Sciences, South China University of
7
Technology, Guangzhou 510640, China. 2 R&D Center, Infinitus (China) Co,.LTD.
8
Guangzhou 510665, China)
9 10 11 12 13 14 15 16 17 18
Co-corresponding authors:
19
Jiguo Yang, Department of Food Science and Technology, South China
20
University of Technology, Wushan Road 381, Guangzhou, Guangdong, China.
21
Tel: (+86)20-87112594; Fax: (+86)20-38897117; E-mail:
[email protected] 22
Jiaoyan Ren, Department of Food Science and Technology, South China
23
University of Technology, Wushan Road 381, Guangzhou, Guangdong, China.
24
Tel: (+86)20-87112594; Fax: (+86)20-38897117; E-mail:
[email protected] 25 1
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Abstract
2
A novel polysaccharide, here named DP1, was isolated from the fruiting body of
3
Dictyophora indusiata using water extraction method. Structure characterization
4
revealed that DP1 had an average molecular weight of 1,132 kDa and was consisted of
5
glucose (56.2%), galactose (14.1%) and mannose (29.7%). The main linkage type of
6
DP1 were proved to be (1→3)-linked α-L-Man, (1→2, 6)-linked α-D-Glc,
7
(1→6)-linked β-D-Glc, (1→6)-linked β-D-Gal and (1→6)-linked β-D-Man by
8
periodate oxidation−Smith degradation and NMR analysis. Immuno-stimulating assay
9
indicated that DP1 could significantly promote macrophage NO, TNF-α and IL-6
10
secretion in murine RAW 264.7 cells involving complement receptor 3 (CR3). The
11
immune activities of DP1 was quite stability under thermal processing (100 °C,
12
121 °C and 145 °C). Besides, DP1 retained stably after acidic/alkline treatment ( pH
13
4.0-10.0), which enable it to be an ideal complementary medicine or functional food
14
for therapeutics of hypoimmunity and immunodeficiency diseases.
15 16
Key words polysaccharide, Dictyophora indusiata, structure characterization, immuno-stimulating, complement receptor 3.
17 18 19
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INTRODUCTION
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Dictyophora indusiata, a member of the Phallaceae Corda family, is one of the
3
most popular edible mushrooms worldwidely consumed. Due to its attractive
4
appearance, distinctive flavour and high nutritive value, Dictyophora indusiata is also
5
regarded as “queen of the mushrooms” 1. Dictyophora indusiata can also be used as
6
biopharmaceutical material with various therapeutic properties. In recent decades,
7
many active compounds (e.g polysaccharides, flavonoids or proteins) from
8
Dictyophora indusiata have been reported to possess immunoregulation, antioxidant,
9
anti-tyrosinase, anti-inflammatory and neuroprotective activities
2-4
. Thereamong, the
10
polysaccharides of Dictyophora indusiata received much more attention because of
11
their multiple beneficial potency and relatively low toxicity
12
polysaccharides with various bioactivities have been purified from the fruiting body
13
of Dictyophora indusiata, but rare information is available concerning their chemical
14
structure and immunomodulatory activities.
15
5, 6
. To date, a couple of
Macrophage is one kind of the phagocyte which plays pivotal roles in the innate 7, 8
16
immune responses
When the host is invaded by pathogenic organisms,
17
macrophages can serve as antigen-presenting cells (APC) that present antigen to T
18
lymphocytes and thus induce the adaptive immune response
19
macrophages also play important roles in embryogenesis, tumorigenesis, cutaneous
20
wound healing and hematopoiesis
21
cell models to evaluate the immunemodulatory properties of bioactive compounds.
22
Some botanical polysaccharides have been found to enhance the viability of
23
macrophage against pathogenic microorganisms and tumorigenesis by increasing the
24
secretion of nitric oxide (NO) and the production of cytokines including tumor
25
necrosis factor (TNF-a), interleukin (IL-1, IL-2, IL-6, IL-8, IL-10 and IL-12) 16 -18 and
10-15
9
. Furthermore,
. Thus, macrophages are usually used as ideal
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it has been thought that polysaccharides can bind to specific membrane receptors of
2
macrophage and activate the immune response through distinct signal transduction
3
pathways
4
macrophages by polysaccharides are still not clear. For this sake, it is meaningful to
5
explore the structural characterizations, biological functions and molecular
6
mechanisms of the polysaccharides from Dictyophora indusiata.
19, 20
. However, the exact mechanisms involved in the activation of
7
In the present study, a new polysaccharide, here called Dp1, with a purity of >
8
97.5%, was separated from Dictyophora indusiata. The primary chemical structure
9
and conformation of Dp1 were characterized. Also, its immunomodulatory activities
10
and membrane receptors were examined using murine RAW264.7 macrophages
11
model. The thermal stability of the Dp1 bioactivity against heat treatment was
12
investigated and its stability against acidic/alkali treatment at different pH conditions
13
was studied as well. In addition, the possible signal transduction pathways involved in
14
macrophage activation by Dp1 were discussed and the results from this study might
15
supply useful information for better understanding the chemical structure and
16
immunomodulatory activities of botanical polysaccharides.
17
MATREIALS AND METHODS
18
Materials and Reagents
19
Dictyophora indusiata was collected from Zhijin, Guizhou Province, China. The
20
murine macrophage cell line RAW264.7 was obtained from American Type Culture
21
Collection (ATCC, Rockville, MD, USA). Dulbecco’s modified Eagle’s medium
22
(DMEM), Fetal bovine serum (FBS), phosphate-buffered saline (PBS, pH 7.4),
23
penicillin and streptomycin were purchased from Gibco Life Technologies (Grand
24
Island, NY, USA). Anti-TLR2 antibody, anti-CR3 antibody and anti-TLR4 antibody
25
were got from Abcam Inc. (Cambridge, MA). Laminarin and lipopolysaccharide (LPS) 4
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were
obtained
from
Sigma
Company
(St.
Louis,
MO,
USA).
DEAE
2
(diethylaminoethyl)-52 cellulose and Sephadex G-200 were purchased from GE
3
Healthcare Life Science (Piscataway, NJ, USA). Nitricoxide (NO)-detecting kit was
4
purchased from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China).
5
Mouse IL-6 ELISA kit and Mouse TNF-α ELISA kit were acquired from
6
Neobioscience Technology Co., Ltd. (Shenzhen, China). Standards of dextrans, uronic
7
acid, phycite, glycerol, glycol reference and monosaccharide references (e.g glucose,
8
galactose, xylose, mannose and rhamnose) were purchased from Sigma Company (St.
9
Louis, MO, USA). The ultrapure water was obtained from a Milli-Q water
10
purification system of Millipore (Millipore, Bedford, MA, USA). All the other
11
chemical reagents used in this study were of analytical grade.
12
Extraction and Purification of Polysaccharides from the Fruiting Body of
13
Dictyophora indusiata
14
The fruiting body of Dictyophora indusiata was dried in a hot air-drying oven at
15
40 °C and crushed into powder using a tissue triturator. The powder of fruiting body
16
was extracted with boiling water at a ratio of 1: 30 (w/w) for 2 h and the obtained
17
extract was centrifuged at 4000 × g for 15min. The supernatant was then collected and
18
concentrated at 60 °C. After that, the concentrated supernatant was deproteinated
19
according to the Sevag method
20
deproteination process was repeated 10 times. The resulting solution was mixed with
21
three volumes of absolute ethanol and kept overnight at 4 °C. The supernatant and
22
precipitate were then separated by centrifugation at 5000 × g for 15 min. Finally, the
23
precipitate was dried using vacuum freeze drying and collected as crude
24
polysaccharides.
21
. To remove the protein as much as possible, this
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One hundred miligrams of crude polysaccharides were dissolved in 10 mL of
2
ultrapure water and loaded onto a DEAE-52 anion-exchange chromatography column
3
(2.5 cm × 60 cm). The elution was conducted using ultrapure water first and then by a
4
series of NaCl solution in the sequence of 0.05, 0.1, 0.2, 0.3 and 0.5 M, respectively,
5
at a flow rate of 0.5 mL/min. The eluent fractions were collected and analyzed by the
6
phenol−sulfuric acid method
7
DP2 (eluted by 0.05 M NaCl) were obtained, dialysed at 4°C for 48h and lyophilized.
8
In this study, we mainly focused the investigation of DP1 and the other fraction will
9
be studied later.
22
. Two fractions, DP1 (eluted by ultrapure water) and
10
After that, DP1 fraction was further purified by Sephadex G-200 column
11
chromatography. Twenty milligrams of DP1 was redissolved in 4 mL of ultrapure
12
water and applied to a Sephadex G-200 column (2.5 cm × 60 cm). The sample was
13
eluted with ultrapure water at a speed of 0.5 mL/min, then the eluent was collected
14
and detected using the phenol−sulfuric acid method for the determimation of 97.5%
15
content.
16
Structure Characterization of DP1
17
Molecular Weight Determination of DP1
18
The weight-average molecular weight distribution of DP1 was analyzed by
19
high-performance gel permeation chromatography (HPGPC, Waters, Milford, MA,
20
USA) on a Waters HPLC instrument with a TSK-GEL G-5000 PWXL column (300
21
mm × 7.8 mm i.d., 10 µm) and a TSK-GEL G-3000PWXL column (300 mm × 7.8
22
mm i.d., 6 µm, Tosoh Co., Ltd., Tokyo, Japan), using 0.01 mol/L KH2PO4 as the
23
mobile phase at a flow rate of 0.6 mL/min.
24
Infrared Spectrum Analysis
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The infrared spectra of DP1 was analyzed according to the KBr-disk method
23
2
using a Fourier transform infrared (FT-IR) spectrophotometer (Bruker, Ettlingen,
3
Germany) in the range of 400–4000 cm-1.
4
Chemical Composition of DP1
5
Twenty miligrams of DP1 was hydrolyzed by 4 mL trifluoroacetic acid (TFA) at
6
105 °C for 6 h. Excess of TFA was removed by rotary evaporation. Then the residue
7
was redissolved in methanol and dried by rotary evaporation. Finally, the residue was
8
dissolved in ultrapure water and analyzed by ion exchange chromatography (Dionex
9
ICS-3000) equipped with an integrated amperometric detector and Carbopac PA20
10
column (2 × 250 mm). The elution was conducted at a rate of 0.6 mL/min at column
11
temperature 20 °C. Gradient elution was carried out according to the following
12
process: 0-2 min, 100% 200 mmol/L NaOH; 2.1 - 20 min, 10% 20 mmol/L NaOH and
13
90% ultrapure water; 20.1-40 min, 100% 200 mmol/L NaOH. The glucose, galactose,
14
xylose, mannose and rhamnose standards were derivatised as previously reported.
15
Acetyl inositol was used as the interior reference.
16
Periodate Oxidation-Smith Degradation
17
4
The periodate oxidation-Smith degradation method was taken out according to 24
18
the report of Chi
with some modification. Twenty-five milligrams of DP1 sample
19
was dissolved in 12.5 mL of ultrapure water and incubated with 12.5 mL of NaIO4 (30
20
mmol/L) in the dark at room temperature. During the incubation period, 0.1 mL of the
21
reaction liquid was taken out from the reaction system at different time intervals (0, 6,
22
12, 24, 36, 48 and 60 h), diluted into 100 mL with ultrapure water, and measured at
23
223 nm by a UV-Vis spectrophotometer (Model UV-18000, Shimadzu, Japan) until
24
the absorbance value became invariable. The process of periodate oxidation was
25
stopped by the addition of 2 mL glycol. Then 2 mL of periodate product was titrated 7
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by 0.01 mol/L NaOH to quantify the production of formic acid. The rest of periodate
2
product was dialyzed in distilled water for 72 h and concentrated by rotary
3
evaporation. Then 70 mg of NaBH4 was added to the residuary solution and kept in
4
the dark for 24 h. The solution was adjusted to pH 5.0 with 0.1 mol/L acetic acid and
5
dialyzed in distilled water for another 72 h. The solution was then concentrated and
6
freeze-dried. Ten milligrams of the residues were hydrolyzed by 4 mL of 2 mol/L TFA
7
at 105 °C for 6 h. The lysate was acetylized with 1 mL of pyridine and 10 mg of
8
hydroxylamine hydrochloride at 90 °C for 30 min. Then 1 mL of acetic anhydride was
9
added to the reaction system and continuously heating at 90 °C for another 30 min.
10
The production of acetate derivative was analyzed by a gas chromatograph system
11
(Aglient, 6890N, Agilent Technologies Inc., Santa Clara, California, USA) with a
12
DB-1701 capillary column (30 m × 0.25 mm × 0.25 µm, J&W Scientific, Fulsom, CA
13
95630, USA) and a flame ionization detector. The linearly heating program is from
14
80 °C to 220 °C at a speed of 2 °C/min, from 220 °C to 250 °C at a speed of 5 °C/min
15
and kept at 250 °C for 5 min. The temperature of detector was set at 300 °C. The
16
phycite, glycerol, glycol, rhamnose, arabinose, xylose, galactose, mannose and
17
glucose were used as standards.
18
Nuclear Magnetic Resonance (NMR)
19
Thirty-five milligrams of DP1 was dissolved in 0.5 mL of D2O in a nuclear
20
magnetic resonance tube and then analyzed by a Bruker 600 MHz Nuclear Magnetic
21
Resonance Apparatus (Bruker Corp., Fallanden, Switzerland) 25.
22
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Thermogravimetric (TG) Analysis
3
The thermostability of DP1 was analyzed using a thermogravimetric analyzer
4
(Pyris Diamond TG-DTA, Perkin Elmer Co., Waltham, MA, USA). Five milligrams
5
of DP1 sample was placed in the Al2O3 pan and heated from 60 °C to 600 °C with a
6
heating rate of 10 °C/min. The analysis was conducted under a pure nitrogen gas
7
atmosphere at a flow rate of 30 mL/min.
8
Immunomodulatory Activity and Stability Assay
9
Cell Culture and Immunomodulatory Activity Test
10
RAW264.7 cells were cultured with DMEM plus with 10% of fetal bovine serum
11
(FBS), 100 µg/mL of streptomycin and 100 U/mL of penicillin, and incubated at
12
37 °C with 5% CO2 humidified atmosphere in a carbondioxide cell incubator. Cells
13
were adjusted to a concentration of 1 × 106 cells/mL in the exponential phase, loaded
14
onto the 96-well microplate (100 µL/well) and continuously incubated for 24 h. Then
15
the culture medium was changed to new medium with DP1 samples and incubated for
16
another 24 h. Antibodies (anti-TLR2, anti-CR3 and anti-TLR4, at a concentration of 5
17
µg/mL) and laminarin (500 µg/mL, the inhibitor of dectin-1) were added to cells to
18
investigate the membrane receptors of DP1 on the RAW264.7 cells. The cells were
19
pretreated with antibodies and laminarin for 2 h prior to stimulation with different
20
concentration of DP1 (125, 250, 500, and 1000 µg/mL) and incubated for 24 h. LPS
21
(50 µg/mL) was used as the positive control. After that, the supernatants of cells were
22
collected and the levels of NO, TNF-α and IL-6 were measured using NO-detecting
23
kit, Mouse TNF-α ELISA Kit and Mouse IL-6 ELISA Kit, respectively, according to
24
the manufacturer’s instructions.
25
Immunomodulatory Stability Assay
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Thermal Treatment. DP1 was dissolved in PBS (pH 7.4) to make final
2
concentration of 500 µg/mL and subjected to different heat treatment (100 °C × 30
3
min, 121 °C × 30 min and 145 °C × 30 min, respectively) and then cooled to room
4
temperature. After that, DP1 was sterilized by 0.22 µm microporous filtering film and
5
then incubated with RAW 264.7 cells for 24 h. The changes of NO, TNF-α and IL-6
6
levels were detected as described above.
7
Acidic/alkaline Treatment. DP1 (500 µg/mL) was dissolved in different
8
phosphate buffer to make final pH 2.0, 4.0, 6.0, 7.4, 8.0 and 10.0, respectively, and
9
then kept over night at room temperature. DP1 with different pH value was sterilized
10
by 0.22 µm microporous filtering film and then incubated with RAW 264.7 cells for
11
24 h. The changes of NO, TNF-α and IL-6 levels were detected as described above.
12
Combined Thermal Treatment and Acidic/alkaline Treatment. DP1 (500
13
µg/mL)
was dissolved in different phosphate buffer at pH 2.0, 4.0, 6.0, 7.4, 8.0 and
14
10.0, respectively. Then the solution were heated at 121 °C for 30 min. All samples
15
were sterilized by 0.22 µm microporous filtering film and then incubated with RAW
16
264.7 cells for 24 h. The changes of NO, TNF-α and IL-6 levels were detected as
17
described above.
18
Statistical Analysis
19
Data were presented as mean ± SD for at least three replicates and analyzed by
20
SPSS 16.0 (SPSS Inc., Chicago, IL, USA). Significance was determined at P values
21
below 0.05 by analysis of variance. The difference between three or more groups was
22
analyzed by one-way ANOVA multiple comparisons.
23
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RESULTS AND DISCUSSION
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Extraction and Purification of Polysaccharides from Dictyophora Indusiata
3
Crude polysaccharides were isolated from the fruiting body of Dictyophora
4
indusiata with a yield of 6.36%. The extracts were first purified by DEAE-52
5
anion-exchange chromatography column (Figure. 1A). Two independent peaks, DP1
6
(eluted by ultrapure water) and DP2 (eluted by 0.05 mol/L of NaCl) were obtained. In
7
the present study, we mainly focused on DP1, and the property of DP2 will be studied
8
in our future work. The DP1 fraction was further purified by Sephadex G-200 column.
9
As shown in Figure. 1B, a single peak of DP1 was observed. Finally, DP1 fraction
10
with a purity of > 97.5% was obtained after dialysis and lyophilization.
11
Characterization of DP1
12
Molecular Weight of DP1
13
The
weight-average
molecular
weight
of
DP1
was
measured
by
26
14
high-performance gel permeation chromatography (HPGPC)
15
1C, a single peak was observed on the chromatogram and the molecular weight of
16
DP1 was determined to be 1,132 kDa. This result showed a great difference from the
17
previous research by Hua et al.
18
polyacchrides from Dictyophora indusiata were 2100.99 and 18.16 kDa. The
19
difference of the extraction method, the habitat and the strain of Dictyophora
20
indusiata may lead to the differences of molecular weights.
21
Monosaccharide Composition Assay
22
The
monosaccharide
1
. As shown in Figure.
which indicated that the molecular weight of two
composition
of
DP1
was
analysed
using
ion
23
chromatography. As shown in Figure. 1D, three monosaccharides including galactose,
24
glucose and mannose were identified according to the elution time of relative
25
monosaccharide standards. The molar percentages of galactose, glucose and mannose 11
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were 14.1%, 56.2% and 29.7%, respectively. The results were different from that of
2
Hua et al. (2012) who found that water-soluble polysaccharides from Dictyophora
3
indusiata were composed of xylose, galactose, glucose and mannose with the molar
4
percentages of 22.6%, 18.6%, 13.9% and 44.9% 1.
5
Periodate Oxidation-Smith Degradation Analysis
6
The results for the periodate oxidation analysis of DP1 showed that 1.27 mol of
7
periodate was consumed by every molar of sugar residues and 0.63 mol of acid was
8
produced, indicating the existence of (1→) or (1→6)- linked glycosidic bonds. The
9
periodate-oxidized DP1 sample was further analyzed by Smith degradation. Glycerol,
10
erythritol, galactose, glucose and mannose were observed after smith degradation
11
(Figure. 1E). The presence of glycerol indicating the existence of (1→2) or (1→6)-
12
linked glycosidic bonds, while erythritol corresponding to the presence of
13
(1→4)-linked glycosidic bonds. The presence of monosaccharides revealed the
14
appearance of (1→3)-linked glycosidic bonds.
15
Infrared Spectrum Analysis
16
The IR spectra of DP1 is shown in Figure. 2A. The stretching intense
17
characteristic peaks at 3335.69 cm-1 indicated the hydroxyl stretching vibration of the
18
polysaccharide chains. The peak around 2926.33 cm-1 was attributed to the C–H
19
stretching vibration 27. The band at 1648.01 cm-1 may be due to the bound water. The
20
absorption peaks between 1430 and 1200 cm-1 indicated the presence of the O-H
21
deformation vibrations and C-O stretching vibrations, respectively
22
1074.32 and 1040.25 cm-1 suggested that the skeletal modes of DP1 were pyranose
23
rings. The absorption at 866 cm-1 indicated the β- pyranoside linkage in DP1. A
24
characteristic peak at 796.81 cm-1 represented the presence of mannose.
25
NMR Analysis of DP1 12
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. The bands at
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The spectra of 1H NMR and 13C NMR of DP1 are shown in Figure. 2B and 2C.
2
The signals of DP1 in 1H NMR and 13C NMR spectra were attributed on the basis of
3
monosaccharide, glycosidic linkages and chemical shifts 29. The resonances signals of
4
13
5
L-mannose, L-arabinose, D-galactose, D-glucose and L-rhamnose
6
95.902 ppm suggested the presence of C-1 of (1→3)-linked α-L-Man unit and the
7
peak at 100.723 ppm indicated the presence of C-1 of (1→3)-linked α-D-Gal unit.
8
Another peak at 104.978 ppm proved the presence of C-1 of (1→2,6)-linked α-D-Glc
9
unit and the peak at 69.469 ppm corresponded to C-6 of (1→6)-linked β-D-Glc unit.
10
Besides, the peat at 68.742 ppm suggested the presence of C-6 of (1→6)-linked
11
β-D-Gal unit and the peak at 66.569 ppm corresponded to the C-6 of (1→6)-linked
12
β-D- Man unit. The peaks in the region of 78.287 ppm to 81.436 ppm (78.287,
13
78.593,78.921,and 81.436 ppm) were attributed to the presence of (1→2), (1→3)
14
or (1→4)-linked glycosidic bonds. The configuration of the glycosidic linkage of DP1
15
was analyzed by 1H NMR spectra. The signals of 1H NMR in the region of
16
5.400-5.843 ppm (5.483, 5.517, 5.569, 5.623, 5.673, 5.678, 5.715 and 5.843 ppm)
17
indicated the presence of α-form furanose. The peaks at 5.000-5.400 ppm (5.005,
18
5.117, 5.131, 5.206, 5.217, 5.244, 5.257, 5.296 and 5.348 ppm) were attributed to the
19
presence of α-form pyranose.
C NMR between 95.0 ppm to 110.0 ppm belonged to the anomeric carbon atoms of
13
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Immunomodulatory Activities of DP1
2
Effects of DP1 on Macrophage NO, TNF-α and IL-6 Production
3
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Modulation of the innate immune system can enhance the host’s resistant ability 30
4
to exogenous pathogens threats
. It is thought that the stimulation of the
5
immuno-system might mainly through the activation of macrophage and complement
6
system 31,32. Macrophage activation by immunomodulators is considered to be via the
7
induction of NO expression and enhanced production of cytokines (TNF-a, IL-1, IL-6,
8
IL-12 and IL-10, etc.)
9
IL-6 production were analyzed in this study. Untreated RAW 264.7 cells secreted very
10
little NO, whereas, the addition of DP1 in the medium significantly increased NO
11
secretion in a dose-dependent manner (125−1000 µg/mL) (Figure. 3A). The
12
concentration of NO reached to 11.72 µmol/mL after treatment by 500 µg/mL of DP1,
13
this is higher than a polysaccharide from Lentinula edodes reported by Xu et al. 19. In
14
Xu’s study, the concentration of NO was lower than 4 µmol/mL after treatment by 500
15
µg/mL of Lentinula edodes polysaccharide. Meanwhile, DP1 had similar
16
dose-dependent manner effects on macrophage TNF-α and IL-6 production (Figure.
17
3B and 3C). The macrophage IL-6 levels (eg. 1134 pg/mL of IL-6 at 250 ug/mL of
18
DP1) treated by DP1 is higher than that of some polysaccharide fractions isolated
19
from Opuntia polyacantha 14 (lower than 1000 pg/mL of IL-6 at 250 ug/mL of some
20
Opuntia polyacantha fractions). Whereas, the TNF-α levels (eg. 588 pg/mL of TNF-α
21
at 250ug/mL of DP1) treated by DP1 is lower than that of a polysaccharide from
22
Ganoderma atrum ( over 1400 pg/mL of TNF-α at 160 mg/mL Ganoderma atrum
23
polysaccharide).34
24
immunomodulatory activities by upregulation of NO, TNF-α and IL-6 secretion in
25
RAW 264.7 cells.
33
The
. Thus, the effects of DP1 on macrophage NO, TNF-α and
results
suggested
that
DP1
14
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Page 15 of 34
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Roles of CR3, TLR2, TLR4, and Dectin-1 on DP1-induced Macrophage
3
Immunomodulation
4
It has been proposed that macrophage activation is mediated primarily through
5
the recognition of pathogen-associated molecular patterns (PAMPs) by pattern
6
recognition receptors (PRRs) on the surface of macrophage cells. Stimulation of PRRs
7
leads to the initiation of intracellular signaling pathways and eventually induces
8
transcriptional activation and expression of cytokines. Recent evidence suggests that
9
macrophages might bind polysaccharides and mediate immunomodulating effects
10
mainly through complement receptor 3 (CR3), Toll-like receptors (TLR2 and TLR4)
11
or dectin-135. In the present study, the roles of CR3, TLR2, TLR4 and dectin-1 on
12
DP1-induced
13
including anti-CR3, anti-TLR2, anti-TLR4 and the inhibitor of dectin-1, laminarin,
14
were preincubated with RAW264.7 cells for 2 h and then incubated with DP1 for
15
another 24 h. Laminarin is a β-glucan which can specifically bind to dectin-1 and
16
block the dectin-1-mediated immune responses
17
IL-6 production were determined. As shown in Figure. 4A, 4B and 4C, after treatment
18
of anti-CR3 and DP1, the levels of NO, TNF-α and IL-6 were all visibly decreased
19
comparing to the group treated by DP1 only (p < 0.05). However, no decrease was
20
found in the groups treated by anti-TLR2, anti-TLR4. As for laminarin, it was even
21
enhanced due to its own immuno-stimulating activity. These results indicated that
22
CR3 was one of the receptors of DP1 on RAW264.7. Previous studies have proved
23
that CR3 possessed sugar specificity and the polysaccharides containing
24
N-acetyl-d-glucosamine or mannose in addition to glucose are most likely recognized
25
by CR3 34. Therefore, the mannose and glucose composition of DP1 may contribute to
macrophage
immunomodulation
were
investigated.
Antibodies
36
. The macrophage NO, TNF-α and
15
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Journal of Agricultural and Food Chemistry
1
the specific binding of DP1 to CR3 on macrophage. Besides, there might be some
2
other receptors of DP1 on the membrane of macrophage, since the NO level
3
(Figure.4A), TNF-α level (Figure.4B) and IL-6 level (Figure.4C) in treated group by
4
anti-CR3 and DP1 were still higher than those in the group absence of DP1 treatment.
5
CR3 has been proved to be involved in the activation of the phosphoinositide-3-kinase
6
(PI3K), phosphorylation of the downstream serine/threonine kinase Akt and
7
mammalian target of rapamycin (mTOR), activation of the mitogen-activated protein
8
kinase (MAPK), and nuclear factor-κB (NF-κB) signal transduction pathways.
9
Activation of these signaling pathways ultimately leads to gene transcription and
10
production of pro-inflammatory cytokines 16. Therefore, we can infer that the possible
11
molecular mechanism of DP1-induced macrophage immunomodulation was mainly
12
through activation of the PI3K/Akt/MAPK/NF-κB signaling transduction pathways
13
(Figure. 4D), and these pathways will be characterized in our future study.
14
Effects of Thermal Treatment and Acidic/Alkali Treatment on DP1-induced
15
Macrophage Immunomodulation
16
It is well established that severe environmental conditions such as high
17
tempreature, high pressure and extreme pH have great influence on the advanced
18
structure of macromoleculars
19
and acidic/alkali treatments on the immunomodulation activity of DP1 were
20
investigated. It was found that with the increase of temperatures, DP1 exhibited a
21
substantial decrease effects on macrophage NO, TNF-α and IL-6 production, as
22
shown in Figure.5A, 5B and 5C, respectively. It may be due to that high temperature
23
(over 100 °C) can cause the break down of non-covalent bonds such as ionic bonds,
24
hydrogen bonds and hydrophobic effects of DP1, and thus as compared with TNF-α
25
and IL-6, the NO level was more sensitive to heat treatment and the 145 °C
37
. In this study, the effects of different heat treatment
16
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1
processing for 30 min destroyed 77.6 % of its native activity. At this point, the
2
immue-sting activities of TNF-α and IL-6 were 24.9% and 57.0% of its native
3
activities, respectively. Although being sensivity to heat treatment, DP1 was still able
4
to upgrade the macrophage NO, TNF-α and IL-6 levels after treatment at 145 °C. The
5
reason for these results may mainly because that thermal treatment just destroyed the
6
higher structure (eg. triple helical) of DP1. Recent evidence confirmed that the
7
helix-coil transition of polysaccharide was induced by heat treatment at about
8
130-145 °C due to the breakage of hydrogen bonds 19. The primary structure may not
9
be damaged under the selected temperatures, although higher structure may likely
10
contributed much to the biological activity of DP1, the primary structure of DP1 also
11
maintained a considerable part of its immue-sting activity. The thermostability of DP1
12
was further analyzed using thermogravimetric analysis (TGA). As shown in Figure
13
5D, neglecting the small weight loss at around 50 °C attributed to the vaporization of
14
retained moisture, the decomposition peak of DP1 was found at 329 °C. This
15
degradation temperature of DP1 was much higher than that of the polysaccharides
16
from haloalkalophilic Bacillus sp. I-450 (290°C) as reported by C. Ganesh Kumar. etc
17
38
18
extreme heat treatments.
. These results further confirmed that DP1exhibited good thermostability under
19
Previous studies have also reported that the non-covalent bonds can also be
20
destroyed in the strong acidic or alkline conditions 39. Thus, the effects of pH changes
21
on DP1 was investigated as well in the present study. The DP1 samples were
22
pretreated by phosphate buffers with various pH (form 2.0 to 10.0) for 12 h. As shown
23
in Figure. 6A, 6B and 6C, it was found that the cells without DP1 protection were
24
very sensitive to different pH treatments, while the addition of DP1 remarkably
25
enhanced the macrophage NO, TNF-α and IL-6 secretion. 17
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Especially for IL-6, the
Journal of Agricultural and Food Chemistry
1
protection effect of DP1 was more obvious. It turns out that even at pH 4.0, DP1
2
could still protect the cells to recover 50% of their original NO, TNF-α and IL-6
3
concentration. It demonstrated that the structure of DP1 remained stable in a broad
4
spectrum of pH levels. At alkline conditions (pH 10.0), DP1 showed less protection
5
on the NO level of the cells, but it still contributed to the TNF-α and IL-6 secretion of
6
macrophage.
7
The stability of DP1 against both heat treatment and acidic/alkline treatment
8
were further explored. DP1 samples were dissolved in phosphate buffers with
9
different pH values (form 2.0 to 10.0) and then heated at 121 °C for 30 min. The
10
results demonstrated that DP1 still retained its immunomodulatory activities to
11
enhance macrophage NO (Figure. 6D), TNF-α (Figure. 6E), and IL-6 (Figure. 6F)
12
secretion in RAW 264.7 cells. In this way, DP1 was proved to retain stable
13
immunomodulatory activities even undergoing combined heat treatment and
14
acidic/alkline treatment. It was very interesting that at pH 10.0, the results showed
15
that the contribution of DP1 to the macrophage NO level was even enhanced after
16
combined treatments (Figure. 6D) than single alkline treatment (Figure. 6A) and the
17
reason was not clear yet. With the above results, we suggested the potential of DP1 as
18
a novel immunomodulatory agent.
19
In this study, a new polysaccharide DP1 of 1132 kDa was purified from
20
Dictyophora indusiata. DP1 was consisted of (1→3)-linked α-L-Man, (1→2,
21
6)-linked α-D-Glc, (1→6)-linked β-D-Glc, (1→6)-linked β-D-Gal and (1→6)-linked
22
β-D-Man. DP1 possessed significant immunomodulation activities by upregulating
23
macrophage NO, TNF-α and IL-6 secretion. One of the membrane receptors of DP1
24
on RAW264.7 was confirmed to be CR3. The PI3K/Akt/MAPK/NF-κB signaling
25
pathways were thought to be involved in the immunomodulation function of DP1. 18
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DP1 retained stably across broad spectrum of pH and thermal treatment. Thus, DP1
2
demonstrated
3
hypoimmunity and immunodeficiency population.
4
ACKNOWLEDGEMENTS
the
potential
applications
as
complementary
medicine
for
5
This study was supported by the National Key Technology R&D Program of
6
China in the 12th five-year period (No. 2012BAD33B11), the Fundamental Research
7
Funds for the Central Universities (No. 2014ZZ0063 & 2013ZZ0061) and Guangdong
8
Natural Science Funds for Distinguished Young Scholars (No.S2013050013954).
9
19
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(40) Martínez-Felipe, A.; Cook, A. G.; Wallage, M. J.; Imrie, C. T., Hydrogen
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5 6
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FIGURE LEGENDS
2
Figure 1. The chromatography of the polysaccharides from Dictyophora indusiata
3
(DP1) by (A) DEAE (diethylaminoethyl)-52 cellulose (DEAE-52), (B) Sephadex
4
G-200, (C) high-performance gel permeation chromatography (HPGPC), (D) Ion
5
exchange chromatography of monosaccharides mixture (D1) and DP1 sample (D2),
6
(E) Gas chromatography of monosaccharides mixture (E1) and DP1 sample (E2).
7
Figure 2. (A) Fourier transform infrared (FT-IR) spectrum, (B)
8
and (C) 1H NMR spectrum of the polysaccharides from Dictyophora indusiata (DP1).
9
Figure
3.
Effects
of
different
concentrations
of
13
C NMR spectrum
Dictyophora
indusiata
10
polysaccharides (DP1) on macrophage (A) Nitric oxide (NO), (B) Tumor necrosis
11
factor (TNF-α), and (C) Interleukin (IL-6) secretion in RAW 264.7 cells. The group
12
without adding DP1 was used as the control and lipopolysaccharide (LPS, 50 µg/mL)
13
was used as the positive control.
14
Figure 4. Roles of CR3, TLR2, TLR4 and Dectin-1 on DP1 (the polysaccharides of
15
Dictyophora indusiata)-induced (A) Nitric oxide (NO), (B) Tumor necrosis factor
16
TNF-α, and (C) Interleukin (IL-6) secretion in RAW 264.7 cells. The cells were
17
incubated with monoclonal antibodies (anti-TLR2, anti-CR3, anti-TLR4) for 2 h and
18
then washed with PBS 3 times before stimulating with DP1. For the dectin-1 group,
19
cells were incubated with laminarin (inhibitor of dectin-1) for 2 h before stimulating
20
with DP1 without washing with PBS. (D) the possible molecular mechanism of
21
DP1-induced macrophage immunomodulation.
22
Figure 5. Effects of different temperature treatment on DP1 (the polysaccharides of
23
Dictyophora indusiata)-induced (A) Nitric oxide (NO), (B) Tumor necrosis factor
24
(TNF-α), and (C) Interleukin (IL-6) secretion in RAW 264.7 cells. DP1 samples were
25
divided into three groups and treated at 100 °C, 121 °C and 145 °C for 30min, 26
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respectively. Then the cells were incubated with heat treatment DP1 samples for 24 h.
2
(D) The thermogravimetry- differential therogravimetry (TG-DTG) curves of DP1.
3
Figure 6. (A), (B) and (C), effects of different pH treatment on DP1 (the
4
polysaccharides of Dictyophora indusiata)-Induced (A) Nitric oxide (NO), (B) Tumor
5
necrosis factor (TNF-α), and (C) Interleukin (IL-6) secretion in RAW 264.7 cells.
6
Cells were incubated with DP1 at different pH of phosphate buffers for 24 h. The cells
7
of control group were incubated at different pH of phosphate buffers (without DP1)
8
for 24 h. (D), (E) and (F), co-effects of heat (121°C) and different pH treatment on
9
DP1-induced (D) Nitric oxide (NO), (E) Tumor necrosis factor (TNF-α), and (F)
10
Interleukin (IL-6) secretion in RAW 264.7 cells. Cells were incubated with heat
11
treatment (121°C) DP1 at different pH of phosphate buffers for 24 h, versus the
12
control groups (without DP1).
13
27
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2.0
B
1.8
Concentration of NaCl (mol/L)
1.6
0.4
1.4 1.2
DP2
1.0
0.3
0.8
0.2
0.6 0.4
0.1
0.2
C
1.0 0.8
0.6 0.4 0.2 0.0
0.0
0.0
0
10
20
300
60
D2
N o rm .
40 35 30
40
E2
25
20
20
40 35
glucose
20
mannos e
45
m in 1 5
25
30
E1 galactose
40
D1
10
xylos e
N orm .
Time (min)
5
16
arabinose
14
Mannose
60
12
Xylose
80
10
Glucose
100
Galactose
Rhamnose
8
rhamnose
0
6
phy cite
0 120
60
45
Mannose
80
50
E
Glucose
100
Galactose
The peak height (nC)
D
40
mannos e
Tube number
120
30
Tube number
glucose
250
phycit e
200
gly col
150
glycerol
100
gly col
50
glyc erol
0
The peak height (nC)
Absorbance (490nm)
1.2
0.5
DP1
Absorbance (490nm)
A
Page 28 of 34
30
20
25
0 6
8
10
12
14
20
16
0
Time (min)
Figure 1 28
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mi n
15
20
25
30
Page 29 of 34
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DP1
A
50 40
20 4000
3500
3000
2500
2000
629.53 572.39
890.28 796.81
1547.68
30
1500
1202.53 1074.32 1040.25
60
1374.28
2926.33
1727.93 1648.01
70
1425.46 1319.07 1253.63
80
3335.69
Transmittance (%)
90
2131.46
2348.73
100
1000
Wavenumber (cm-1)
B
C
Figure 2 29
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500
Journal of Agricultural and Food Chemistry
35
A Concentration of NO (µmol/L)
a 30
25
20
b 15
bc c
10
d 5
e 0
control
125
250
500
1000
LPS
µg/mL 1000
a
Concentration of TNF-a (pg/mL)
B 800
600
d
cd
125
250
bc
b
500
1000
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200
0
control
LPS
µg/mL
C Concentration of IL-6 (pg/mL)
2000
a b
1500
cd bc
d 1000
500
e 0
control
125
250
500
1000
µg/mL
Figure 3
30
ACS Paragon Plus Environment
LPS
Page 30 of 34
Page 31 of 34
Journal of Agricultural and Food Chemistry 25
A Concentration of NO (µmol/L)
a 20
15
e
c
ce
e
d 10
b
5
f
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-
LP S
DP1 (500 µg/mL)
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a
c e
1000
Concentration of IL-6 (pg/mL)
e
e d
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600
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ef
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bg
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Figure 4 31
ACS Paragon Plus Environment
S
in m
in
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in ar m in
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4
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DP1 (500 µg/mL)
0
LP
Concentration of TNF-a (pg/mL)
+
1000
B
400
+
Journal of Agricultural and Food Chemistry
A
a
1200
B
40
Page 32 of 34
a
30
d
d
25 20
c 15 10
1000
Concentration of TNF-a (pg/mL)
b
5
e
c 600
b 400
200
b 0
untreated
100?
121?
145?
control
2000
a e
untreated
LPS
100?
121?
145?
control
LPS 2
110
D
100
e
DTG
0
90
1500
-2
80
W eight (% )
Concentration of IL-6 (pg/mL)
d
800
0
C
e
d 1000
c
70
-4
60 -6
50 40
500
-8
30 20
b
10
0
untreated
100?
121?
145?
control
-10
TG
LPS
-12
0
Figure 5
0
100
32
ACS Paragon Plus Environment
200
300
400
Temperature (°C)
500
600
D erivative weight (% m in -1 )
Concentration of NO (µmol/L)
35
A
Journal of Agricultural and Food Chemistry
DP1 control
25
900
B
800
20
c 15
d e
10
A
f 5
bf
C
C BD
Concentration of TNF-a (pg/mL)
Concentration of NO (µmol/L)
a
DP1 control
B
a
700 600
b b
B
AB e 300
A AB
B
400
C
200
0
0
pH2.0
pH4.0
pH6.0
pH10.0
pH8.0
c
1000
100
E
pH6.0
pH4.0
pH8.0
pH10.0
600
d
d
400
200
PBS
pH4.0
pH2.0
a
Concentration of NO (µmol/L)
cd bd
15
b
10
e 5
A
C D
D
CD
800
a
700 600
b b AB
B
e 300
A AB
B
400
C
200 100
B
0
0
pH2.0
pH4.0
pH6.0
pH8.0
500µg/mL
pH10.0
PBS
pH4.0
pH6.0
pH10.0
PBS
pH8.0
pH10.0
PBS
500µg/mL
c
800
b
b 600
d 400
200
0
pH2.0
pH8.0
A
ac
1000
c
500
A
a
DP1 control
1200
a
Concentration of IL-6 (pg/mL)
c
20
Concentration of TNF-a (pg/mL)
D
F
DP1 control
pH6.0
500µg/mL
900
E
A
A
A
A
500µg/mL
DP1 control
b
b 800
0
pH2.0
PBS
a
1200
c
500
DP1 control
1400
C
a
Concentration of IL-6 (pg/mL)
Page 33 of 34
D
pH2.0
C
pH4.0
BC
pH6.0 500µg/mL
Figure 6
33
ACS Paragon Plus Environment
AB
pH8.0
B
pH10.0
A
PBS
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