Structure and immunomodulatory activity of microparticulate

Jul 25, 2019 - In this study, an immunologically active novel microparticulate mushroom β-glucan (PRA-1p) was prepared using an alkali-soluble glucan...
2 downloads 0 Views 929KB Size
Subscriber access provided by BUFFALO STATE

Functional Structure/Activity Relationships

Structure and immunomodulatory activity of microparticulate mushroom sclerotial #-glucan prepared from Polyporus rhinocerus Chaoran Liu, and Peter Chi Keung Cheung J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03206 • Publication Date (Web): 25 Jul 2019 Downloaded from pubs.acs.org on July 27, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36

Journal of Agricultural and Food Chemistry

Structure and immunomodulatory activity of microparticulate

1

mushroom sclerotial

2

prepared from Polyporus rhinocerus

3 4

Chaoran Liu 1,2, Peter C.K. Cheung *, 2

5 6

1

Shenzhen Institute of Standards and Technology, Shenzhen, China

7

2

Food and Nutritional Sciences, School of Life Sciences, The Chinese University of

8

Hong Kong, Shatin, New Territories, Hong Kong SAR, China

9

Running title: Structure and immunomodulatory activity of microparticulate

10

mushroom )*

11 12 13 14 15 16 17 18 19 20 21 22 1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

23

ABSTRACT: In this study, an immunologically active novel microparticulate

24

mushroom )*

25

emulsification and cross-linking method. PRA-1 was a hyper-branched + /0, + /1,*

26

)*2*

27

rhinocerus. PRA-1 had a rod-like conformation, while PRA-1p exhibited monodisperse

28

and homogeneous spherical conformation with a diameter ranging from 0.3-2.0 7' in

29

water. PRA-1p significantly induced NO and ROS production as well as the

30

morphological changes of murine macrophages (RAW 264.7 cells) and up-regulated

31

their phagocytic activity. Furthermore, PRA-1p treatment markedly enhanced the

32

secretion of cytokines including CTACK, G-CSF, MCP-1, = * > MIP-2, RANTES,

33

sTNFRI and TIMP-1. Activation of RAW 264.7 cells triggered by PRA-1p was

34

associated with activation of iNOS,

35

the novel PRA-1p derived from the mushroom sclerotia of P. rhinocerus has potential

36

application as an immunostimulatory agent.

(PRA-1p) was prepared using an alkali-soluble glucan PRA-1 by

with a degree of branching of 0.89 isolated from the sclerotia of Polyporus

*@A ERK and AKT. This work suggests that

37 38

KEYWORDS: mushroom sclerotia, Polyporus rhinocerus microparticulate -glucan,

39

macrophage activation, polysaccharides

40 41 42 43 44 2

ACS Paragon Plus Environment

Page 2 of 36

Page 3 of 36

Journal of Agricultural and Food Chemistry

45

INTRODUCTION

46

Bioactive polysaccharides derived from edible or medicinal mushrooms have been

47

demonstrated to possess a variety of therapeutic properties including antitumor,

48

antidiabetic

49

polysaccharides, )*

50

(IRMs) or immunomodulators.5, 6 As )*

51

they are classified as pathogen-associated molecules that can be recognized by pattern

52

recognition receptors (PRRs) on the surfaces of innate immune cells mainly including

53

macrophages, monocytes and dendritic cells.7-9

and

Several )*

54

effects.1-4

immunomodulating

Among

these

mushroom

are studied extensively as immune response modifiers are nondigestible in the human body,

from mushrooms have been developed into prescription drug used

55

clinically with documented evidences on inducing host-mediated antitumor immune

56

responses.10-13 These medicinal )*

57

krestin from Trametes versicolor and schizophyllan from Schizophyllum commune.14

58

They all shared a common structure of a (1 / 3)-linked main chain substituted at O-6

59

by a single unit of )*2*

60

branching.10 It has been found that the immunomodulatory effects of

61

are closely correlated to their physical state including molecular weight, water

62

solubility and degree of branching.15 It has been reported that different preparations of

63

)*

64

mechanism of their immunomodulatory action. A comparative study found that

65

although both soluble and particulate )*

66

the particulate )*

(

include lentinan from Lentinus edodes,

with different molecular weight and degree of 0*)*2*

s, including the particulate and the soluble ones, could also influence the

could bind to the Dectin-1 receptor, only

was able to activate it.16 In another research, it was found that 3

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

67

0*)*2*

Page 4 of 36

derived from Candida albicans showed higher stimulating ability in a

68

particulate form than in a solubilized form.15 However, researches that focus on the

69

underlying mechanism on how water-insoluble )*

70

immune cells and changes their morphological properties are rare.

mediates the

activation of

71

Polyporus rhinocerus is a well-known medicinal mushroom belonging to the

72

Polyporacea family in China and Southeast Asia.17 Sclerotium is a compact aggregate

73

of mycelia and is one of the developmental stages in the mushroom life cycle. The

74

sclerotia of P. rhinocerus, which are rich in )*

75

and prevention of various human diseases including gastric ulcer, chronic hepatitis, and

76

cancer in Asia.17 Previous study found that )*

77

weight of the sclerotia of P. rhinocerus.7 The potent bioactivity of alkali-soluble

78

polysaccharide and water-soluble polysaccharide-protein complex (PPC) isolated from

79

the sclerotia of P. rhinocerus have been compared in our previous studies.8 It was found

80

that the water-soluble PPC which has a structure of a heteroglycan and protein was

81

more immunopotent than the alkali-soluble homoglycan +)*

82

vitro, which might be explained by their differences on water solubility and molecular

83

conformation in an aqueous medium.8, 17-19 Alkali-soluble polysaccharides are usually

84

aggregated in an aqueous medium and thus have fewer chances to contact with the

85

immune cell surface, not to mention stimulating the cell responses.

have a long history in treatment

constituted for more than 70% dry

, both in vivo and in

In the present study, we aim at preparing an immunologically active microparticulte

86 87

)*

using an alkali-soluble glucan PRA-1 obtained from the sclerotia of P.

88

rhinocerus by employing the emulsification and cross-linking method. Accordingly, 4

ACS Paragon Plus Environment

Page 5 of 36

Journal of Agricultural and Food Chemistry

89

the primary structure of PRA-1 was elucidated by methylation analysis. The

90

morphological properties of PRA-1 and PRA-1p were examined by Fourier transform

91

infrared spectroscopy (FTIR), transmission electron microscopy (TEM), scanning

92

electron microscopy (SEM) and laser light scattering (LLS) spectroscopy. The

93

immunostimulating action such as nitric oxide (NO) production, reactive oxygen

94

species (ROS) generation, phagocytic activity, cytokine profile and the related

95

molecular mechanisms were further studied. This work provides fundamental

96

information of a novel microparticulate mushroom sclerotial )*

97

rhinocerus which has potential for further biomedical applications.

98

MATERIALS AND METHODS

from P.

99

Chemicals and Antibodies. Analytical grade reagents including ethyl acetate and

100

acetone were purchased from Duksan Pure Chemicals Co. (South Korea). Ethanol was

101

purchased from Merck Millipore (USA). Lipopolysaccharides (LPS), Thiazolyl blue

102

tetrazolium bromide (MTT) and FITC-labeled dextran were purchased from Sigma-

103

Aldrich (USA). The primary anti-mouse monoclonal antibodies and horseradish

104

peroxidase (HRP) conjugated secondary antibody used in Western blot analysis was

105

purchase from Cell Signaling Technology (USA).

106

Preparation of PRA-1. The dried Mushroom sclerotia of P. rhinocerus, a

107

commercial product originated from mainland China, were purchased from Chinese

108

herbal store in Hong Kong. After the removal of the peel, the sclerotia were pulverized

109

into powders to pass through a screen with an aperture of 0.5 mm by using a cyclotech

110

mill (Tecator, $G

H

Sweden). Powder of sclerotia of P. rhinocerus were firstly 5

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

111

defatted in ethyl acetate (1 h, 3 times) and then acetone (1 h, 3 times) at 60 oC before

112

being extracted with boiling water. After removing the hot water-soluble extract by

113

centrifugation (4000 g for 10 min), the water-insoluble residue was extracted with 1M

114

NaOH at room temperature, the supernatant was neutralized to pH 7.0 with 0.5 M acetic

115

acid and the precipitate (alkali-soluble polysaccharide fraction PRA-1) was obtained by

116

centrifugation (4000 g for 10 min) and washed with distilled water to remove salt.

117

Preparation of particulate PRA-1. The particulate PRA-1 (PRA-1p) was prepared

118

using the native PRA-1 by emulsification and cross-linking method firstly described to

119

form chitosan nanoparticles with some modifications.20 In brief, PRA-1 was firstly

120

dissolved in DMSO at a concentration of 30 mg/mL. Then, 500 7! of the PRA-1

121

solution was slowly added into 25 mL liquid paraffin containing 3% span 80 followed

122

by 30 min sonication (Model VC 600 processor, Sonics & Materials Inc., Newton, USA)

123

under a fixed frequency of 20 kHz and a power of 600W to obtain a stable dispersion.

124

Afterwards, 0.5 mL of glutaraldehyde was added as a cross-linking agent to the

125

dispersion to harden the formed droplets, followed by 30 min sonication under a fixed

126

frequency of 20 kHz and a power of 600W. The dispersion was stirred using a magnetic

127

stirrer for 3 h and then it was subjected to centrifugation at 1000 g for 15 min. The

128

precipitate was washed with petroleum ether followed by methanol and finally with

129

acetone for five times. PRA-1p obtained was then lyophilized and stored in the

130

refrigerator at 4 °C.

131

Chemical Composition Analysis. The amount of the total carbohydrates in PRA-1

132

were determined by the phenol-sulfuric acid assay and the amount of the total proteins 6

ACS Paragon Plus Environment

Page 6 of 36

Page 7 of 36

Journal of Agricultural and Food Chemistry

133

were determined by the BCA protein assay as described previously.21, 22 Content of total

134

uronic acids in PRA-1 was analyzed colorimetrically using meta-hydroxydiphenyl-

135

sulfuric acid.23

136

Monosaccharide composition analysis. PRA-1 sample was firstly acid hydrolyzed

137

into monosaccharides followed by reduction and acetylation to yield the sugar

138

derivatives of alditol acetates according to the method described previously.24 The

139

derivatives were separated and analyzed by gas chromatography according to the

140

method described previously.25 The column, the gas chromatograph (GC), the mass

141

spectrometry (MS) and the conditions used were the same as previously reported. 7

142

Linkage Analysis by Methylation. The glycosidic linkages in PRA-1 was analyzed

143

by methylation as previously described with some modifications26 The column, oven

144

temperature, MS detector condition used were the same as previously reported.7 Each

145

partially methylated alditol acetates (PMAA) was identified according to the literature

146

database.27

147

Transmission electron microscopy. PRA1 and PRA-1p were dissolved in distilled

148

water at a concentration of 5 mg/mL and were heated to 80 °C in water bath for 2 h

149

with constant stirring. The samples were prepared using holey carbon film (200 mesh,

150

Beijing Zhongjingkeyi Technology, China), which was supported by a copper grid, for

151

the analysis by transmission electron microscopy (TEM) (H-7650, Hitachi, Japan).

152

After filtration through a 0.22 7' nylon syringe filter, a droplet of the sample was

153

deposited on the specimen, which was finally dried in air at ambient temperature and

154

humidity. Molecular morphology of the prepared samples was performed on TEM at 7

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

155

an accelerating voltage of 80 kV.

156

Scanning electron microscopy. Droplets of PRA1 and PRA-1p samples (5 mg/mL)

157

were deposited on the silicon wafer and allowed to dry in air. The test samples were

158

sputtered with a gold layer and analyzed by SEM (Hitachi S-3400N) which was

159

performed at an accelerating voltage of 25 kV.

160

Size distribution. The size distribution of PRA-1p in aqueous solution (1 mg/mL)

161

was measured using laser light scattering spectroscopy with a Mastersizer equipment

162

(Malvern Panalytical, Malvern, UK).

163

Infrared spectrophotometric analysis. The infrared spectra of PRA-1 and PRA-1p

164

were recorded using a Fourier Transform Infrared Spectrometer (FTIR, Nicolet 670) in

165

the range of 4000-400 cm-1 using the KBr-disk method. Briefly, the PRA-1 and PRA-

166

1p samples were blended with KBr powder and pressed into transparent pellet for the

167

FTIR measurement.

168

Nitrite determination. RAW 264.7 cells in 6-well plates (5×104 cell/mL) were

169

preincubated for 24 h. After 72-h stimulation with PRA1 (100 7 I'!, or PRA-1p (100

170

7 I'!, or LPS (100 ng/mL), the cell culture medium were collected. Nitrite oxide

171

production in the culture medium was determined by the Greiss reagent system

172

(Promega) according to the manufacturer’s instructions.

173

Phagocytosis of FITC-labeled dextrans. RAW 264.7 cells (5×104 cells/mL) were

174

incubated with PRA1 (100 7 I'!, PRA-1p (100 7 I'!, and LPS (100 ng/mL)

175

individually. After 24 h, cells were collected and the live cell count was performed by

176

trypan blue dye exclusion assay. About 1x105 live cells were incubated in 1 mL medium 8

ACS Paragon Plus Environment

Page 8 of 36

Page 9 of 36

Journal of Agricultural and Food Chemistry

177

containing FITC-labeled dextrans (1mg/mL) at 37 °C for 1 h. After washing with PBS

178

(3 times) to remove the extra FITC-labeled dextrans, cells were analyzed by a CXP 500

179

flow cytometry (Beckman Coulter, Miami, FL).

180

Reactive Oxygen Species (ROS) generation. The intercellular ROS generation in

181

RAW 264.7 cells treated by PRA1 (100 7 I'!, PRA-1p (100 7 I'!, and LPS (100

182

ng/mL) individually was determined by 2’, 7’-Dichlorodihydrofluorescein diacetate

183

(DCFH-DA) assay. DCFH-DA that measures hydrogen peroxide is able to penetrate

184

through the cell membrane and is rapidly deacetylated and finally oxidized in the

185

presence of the intracellular hydrogen peroxide to a highly fluorescent 2’, 7’-

186

Dichlorodihydrofluorescein. Briefly, the treated RAW 264.7 cells were harvested using

187

trypsin and subjected to centrifugation (800 g for 5 min) to form a pellet. The cells

188

(1x105) then were incubated in DMEM medium containing DCFH-DA (10 7=, at

189

37 °C for 30 min. Then the cells were washed for three times using PBS and the

190

fluorescent signal was immediately measured by a fluorescence microscopy with the

191

excitation and emission wavelength set at 488 and 525 nm, respectively (Carl Zeiss

192

PALM inverted microscope).

193

Determination of Cytokine Profile. RAW 264.7 cells (5×104 cells/mL) were

194

incubated with 100 7 I'! PRA-1p for 24h and then the cell culture medium were

195

collected. The RayBio® Mouse Cytokine Antibody Array II (RayBiotech) was used to

196

detect the relative levels of cytokines secreted by RAW 264.7 cells according to the

197

manufacturer’s instructions.

198

Western Blot Analysis. RAW 264.7 cells (105 cells/well) were plated in a 6-well 9

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

199

plate and incubated with PRA-1 (100 7 I'!, or PRA-1p (100 7 I'!, or LPS (100

200

ng/mL) for 24h. The protein in RAW 264.7 cells were extracted following the method

201

described previously with some modifications.7 The protein concentration was

202

determined using BCA reagent (Pierce). An equal amount of denatured proteins were

203

subjected to western blot analysis for iNOS, (*

204

AKT and -actin as described previously.7

*@A

*@A p-ERK, ERK, p-AKT,

205

Statistical Analysis. Results were represented as mean ± standard deviation (SD).

206

Experiments were performed in triplicate unless specified otherwise. Difference

207

between two groups was analyzed by two-tailed Student’s t test and P < 0.05 (*) or P


macrophage inflammatory protein-1>

32.39 ± 0.37

MIP-2

macrophage inflammatory protein-2

30.48 ± 0.08

MCP-1

monocyte chemoattractant protein-1

13.84 ± 0.20

641 642

#

The fold changes of the cytokines/chemokines are relative to the control.

643 644 645 646 647 648 649 650 35

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

ACS Paragon Plus Environment

Page 36 of 36