Immunomodulatory Activity of Ganoderma atrum ... - ACS Publications

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Immunomodulatory activity of Ganoderma atrum polysaccharide on purified T lymphocytes through Ca2+/CaN and MAPK pathway based on RNA-seq Quan-Dan Xiang, Qiang Yu, Hui Wang, Ming-Ming Zhao, Shi-Yu Liu, Shaoping Nie, and Ming-Yong Xie J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 13 Jun 2017 Downloaded from http://pubs.acs.org on June 14, 2017

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 free 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 accessible to all readers and 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.

Journal of Agricultural and Food Chemistry 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 39

Journal of Agricultural and Food Chemistry

Immunomodulatory activity of Ganoderma atrum

1

2

polysaccharide on purified T lymphocytes through Ca2+/CaN

3

and MAPK pathway based on RNA-seq

4 5

Quan-Dan Xiang#, Qiang Yu#, ∗, Hui Wang&, Ming-Ming Zhao#, Shi-Yu Liu#,

6

Shao-Ping Nie#, Ming-Yong Xie#, ∗

7

#

8

Nanchang 330047, China

9

&

10

State Key Laboratory of Food Science and Technology, Nanchang University,

Institute of Life Science & College of Life Sciences, Nanchang University,

Nanchang 330031, China

11 12

∗Corresponding to: Professor Ming-Yong Xie, PhD; Associate professor Qiang Yu,

13

PhD.

14 15

State Key Laboratory of Food Science and Technology, Nanchang University, 235

16

Nanjing East Road, Nanchang 330047, China

17 18

Tel.&Fax: +86 791-83969009 (M. Y. XIE); +86 791-88304452 (Q. YU)

19

E-mail: [email protected] (M. Y. XIE); [email protected] (Q. YU)

1

ACS Paragon Plus Environment


20

ABSTRACT

21

Our previous study has demonstrated that Ganoderma atrum polysaccharide

22

(PSG-1) has immunomodulatory activity on spleen lymphocytes. However, how

23

PSG-1 exerts its effect on purified lymphocytes is still obscure. Thus, this study aimed

24

to investigate the immunomodulatory activity of PSG-1 on purified T lymphocytes,

25

and further elucidate the underlying mechanism based on RNA-seq. Our results

26

showed that PSG-1 promoted T lymphocytes proliferation and increased the

27

production of IL-2, IFN-γ and IL-12. Meanwhile, RNA-seq analysis found 394

28

differentially expressed genes. KEGG pathway analysis identified 20 significant

29

canonical pathways and 7 biological functions. Furthermore, PSG-1 elevated

30

intracellular Ca2+ concentration and calcineurin (CaN) activity, and raised the p-ERK,

31

p-JNK and p-p38 expression levels. T lymphocytes proliferation and the production of

32

IL-2, IFN-γ and IL-12 were decreased by the inhibitors of calcium channel and

33

MAPKs. These results indicated that PSG-1 possesses immunomodulatory activity on

34

purified T lymphocytes, in which Ca2+/CaN and MAPK pathway play essential role.

35 36

KEYWORDS: Ganoderma atrum polysaccharide; purified T lymphocytes; RNA-seq;

37

Ca2+/CaN pathway; MAPK pathway.

2


Page 2 of 39

Page 3 of 39


38

INTRODUCTION

39

T cells are adaptive immunity cells, and play an irreplaceable role in immune

40

response.1 IL-2, IFN-γ and IL-12 are immunomodulatory cytokines and T cells

41

growth factors that participate in T cell proliferation, differentiation, activation and

42

immunomodulation. As early as the 1980s, Mossman2 and Bob Coffman3 had

43

discovered two subsets of T cells and proposed that Th1 cells mainly produce IL-2

44

and IFN-γ, while Th2 cells mainly produce IL-4, IL-5, and IL-13. Subsequently, it

45

was found that IL-12, IFN-γ induce naïve T cell differentiation into Th1 cells, which

46

involve in cellular immunity and clear intracellular pathogens.4, 5 IL-4 favors naïve T

47

cells differentiated into Th2 cells, which involves in humoral immunity.4, 5

48

Ca2+ has been described as a second signal messenger in various cells of the

49

immune system,6 which participates in cell activation,7 and function.8 The continuous

50

elevation of intracellular Ca2+ is essential for the expression of T cell activation

51

genes.9 Ca2+ channel also involves in the expression of many cytokines, once Ca2+

52

channel is impaired, it will lead to a strongly reduced secretion of several cytokines

53

such as IL-2, IL-4 and IFN-γ in T cells.8 Calcineurin (CaN) is a Ca2+-activated

54

serine/threonine phosphatase, which is a critical enzyme in T cells signal

55

transduction.10 It has been demonstrated that Ca2+ linking with CaN results in

56

activating immune response in B and T cells.11

57

Mitogen-activated protein kinases (MAPKs) are signaling regulators that are

58

responsible for T cell survival, differentiation and function, which include three

59

classic subfamilies: the extracellular signal–regulated kinases (ERKs), c-Jun

60

N-terminal kinases (JNKs) and p38 MAPKs in mammalian cells.12, 13 ERKs function

61

in control of cell division, JNKs are pivotal regulators of transcription, and p38

62

MAPKs may be concerned with diseases like asthma and autoimmunity. ERKs, 3



Page 4 of 39

63

known as physiological function kinases, are activated by mitogens and growth

64

factors and are required for regulating cell proliferation and Th2 cell differentiation.14,

65

15

66

production,16 JNK3 results in preventing neuronal apoptosis in response to excitotoxic

67

stress,17 p38 is specifically activated by pathway that results in IL-2 secretion in T

68

cells18 and is necessary for IL-12 and IFN-γ production.19

JNK1 and JNK2 are essential for naïve CD4+ T cell differentiation and cytokine

69

RNA-seq, also called whole transcriptome sequencing,20 is a recently developed

70

technology that uses deep-sequencing technology to obtain gene information of the

71

transcriptome.21 Specifically, RNA-seq is used to analyze the structure of

72

transcriptome, which is able to extract a wide range of gene ontology,22,

73

fusion,24 the polymorphism of coding sequence25 and the function of the non-coding

74

sequences.26 In addition, RNA-seq can also be used for alternative splicing

75

quantitative analysis27 and capturing the dynamic changes of the transcriptome.28

23

gene

76

Ganoderma atrum has been safely used as ingredient of traditional medicine and

77

functional food for thousands of years in oriental countries.29 Recently, a

78

polysaccharide, named PSG-1 with a purity of > 99.8%, was extract from Ganoderma

79

atrum in our laboratory. PSG-1 was a homogeneous protein-bound polysaccharide,

80

which contained 10.1% protein and 17 general amino acids, and the molecular weight

81

was about 1013 kDa.30 Monosaccharide composition analysis showed that PSG-1 was

82

composed of glucose (72.5%), mannose (8.3%), galactose (5.7%) and glucuronic acid

83

(13.5%).31 Based on methylation and GC-MS analysis, the main linkage type of

84

PSG-1 was 1,3-linked-Glcp, T-Glcp, 1,3,6-Glcp, 1,4-Galp , 1,6-Glcp , 1,2-Manp,

85

1,4-GalpA, 1,4-Manp and 1,4,6-Glcp.30 Our recent studies have demonstrated that

86

PSG-1 possesses a variety of biological functions, for example, anti-tumor,32

87

immunomodulatory,33

protection,34

cardiovascular 4


chemoprotective35

and

Page 5 of 39


88

hypoglycemic activities.36 Among them, of note is that PSG-1 has immunomodulatory

89

effect on spleen lymphocytes.37 However, the experimental conclusion was derived

90

from the mix lymphocytes, which could not illustrate the immunomodulatory effect of

91

PSG-1 on purified lymphocytes. Therefore, in the present study, we first tried to

92

purify T lymphocytes, then screen the key signal transduction pathways in purified T

93

lymphocytes stimulated by PSG-1 via RNA-seq, and further investigate the

94

underlying mechanism involved in the immunomodulatory activity of PSG-1 on

95

purified T lymphocytes.

96

MATERIALS AND METHODS

97

Materials and Reagents. Pan T cell isolation kit II was from Miltenyi Biotec

98

(Bergisch Gladbach, Rheinisch-Bergischer Kreis, Germany). Enzyme linked

99

immunosorbent assay (ELISA) kits were from Wuhan Boster Biological Technology

100

Co. (Wuhan, Hubei, China). Cell counting kit-8 was from Dojindo (Kumamoto,

101

Japan). All antibodies used in flow cytometry were purchased from BD Biosciences

102

(San Jose, CA, USA). The calcineurin assay kit was from Genmed Scientifics, Inc.

103

(Shanghai, China). Antibodies against p44/42 MAPK (ERK1/2), SAPK/JNK

104

(JNK1/2),

105

phospho-SAPK/JNK (p-JNK1/2), phospho-p38 (p-p38) were purchased from Cell

106

Signaling Technology (Beverly, MA USA). The related secondary antibodies and

107

β-actin were purchased from Zhong Shan Golden Bridge Biological Technology Co.

108

Ltd (Beijing, China). PD98059, SP600125, SB203580, Fluo-3/AM, RIPA buffer,

109

BCA protein assay kit, and BeyoECL plus ECL chemiluminescence kit were

110

purchased from Beyotime Biotechnology (Nanjing, Jiangsu, China). Verapamil was

111

purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI 1640 medium,

112

hypotonic lysis buffer, and bovine serum albumin were purchased from Solarbio

p38

MAPK,

phospho-p44/42

MAPK

5


(ERK1/2)

(p-ERK1/2),


113

Science and Technology Co. Ltd. (Beijing, China).

114

T lymphocytes purification. Female Balb/c mice, 6-8 weeks of age, 18−20 g of

115

weight, were purchased from Hunan Slac Laboratory Animal Center, Chinese

116

Academy of Sciences (Changsha, Hunan, China) certificate no. SCXK (Xiang)

117

2016-0002]. All mice used in this study were cared for in accordance with the

118

Guidelines for the Care and Use of Laboratory Animals published by the United

119

States National Institutes of Health (NIH Publication 85-23, 1996), and all

120

experimental procedures were approved by the Animal Care Review Committee,

121

Nanchang University.

122

then suspended with RPMI 1640 medium. Single cell suspensions were prepared by

123

filtering the suspension through a sterile sieve mesh. Red blood cells were lysed with

124

a hypotonic lysis buffer. The cells were washed twice with cold phosphate-buffered

125

saline (PBS, pH7.2), and adjusted to the concentration of 5 × 106 cells/mL in RPMI

126

1640 medium with 10% fetal bovine serum, following by incubating in cell culture

127

dishes for 4 h. The suspended cells were the spleen lymphocytes. Then T lymphocytes

128

were isolated by using Pan T cell isolation kit II according to the manufacturer’s

129

instruction. According to the flow cytometry, the purity of T cells was higher than

130

90%. The survival rate was higher than 95% as detected by trypan blue.

131

Cytokine assay. T lymphocytes (density of 1 × 106 cells/mL) were cultured with 160

132

µg/mL PSG-1 and/or 5 µg/mL ConA, without PSG-1 as the control group. The cells

133

were incubated in 6-well culture plates, after 48 h the levels of cytokines in the

134

supernatant were detected by ELISA kits according to the manufacturer’s instructions.

135

Proliferation assay. T lymphocytes (5×105 cells/well) were incubated with medium

136

alone as the negative control. Cells were pretreated with inhibitors for 30 min,

137

including ERK inhibitor PD98059, JNK inhibitor SP600125, p38 inhibitor SB203580,

The spleens were removed aseptically and cut into pieces and

6


Page 6 of 39

Page 7 of 39


138

and calcium channel inhibitor verapamil. Then cells were cultured with 160 µg/mL

139

PSG-1 or 5 µg/mL ConA in 96-well plate. After 48 h, T lymphocyte proliferation was

140

measured by CCK-8 kit. Each well was added with 10 µL of the CCK-8 solution and

141

incubated for 3 h, then the absorbance was measured at 450 nm by microplate reader

142

(Bio-Rad Laboratories, Hercules, CA, USA).

143

Intracellular calcium concentration measurement. 1 × 106 cells/mL of T

144

lymphocytes were incubated in 6-well plate with PSG-1 at the concentration of 160

145

µg/mL. After 48 h the cells were collected and washed twice with PBS, then cultured

146

with the fluorescent probe Fluo-3/AM at 37 °C for 60 min. The cells were washed

147

with PBS, and Ca2+ concentration was analyzed by BD FACSCalibur™ flow

148

cytometry (BD Biosciences, San Jose, CA, USA).

149

Measurement of CaN activity. The cells (1 × 106 cells/well) were cultured in 6-well

150

plate and stimulated with PSG-1 at the concentration of 160 µg/mL for 48 h. CaN

151

activity was measured using the CaN activity assay kit. Briefly, the cells were treated

152

by cell lysis buffer (reagent B) provided in the kit following the manufacturer’s

153

instructions. The optical density at 660 nm was determined for each sample using a

154

microplate reader.

155

RNA extraction and cDNA synthesis. T lymphocytes (1 × 107 cells/mL) were

156

cultured in tissue culture flasks with PSG-1 (160 µg/mL) for 48 h. Cells were

157

harvested, total RNA was extracted using TRIzol® reagent (Life Technologies,

158

Waltham, MA, USA), and reversed transcribed into cDNA using Prime

159

Script RT reagent Kit (TaKaRa Biotechnology, Dalian, Liaoning, China) according to

160

the manufacturer’s protocol.

161

Quantitative real-time PCR. Target gene was determined using the Quant Studio™

162

7 Real-Time PCR System (Life Technologies, Waltham, MA, USA). Relative 7



163

expression of target gene was analyzed by qPCR using SYBR Premix Ex Taq II kit

164

(TaKaRa Biotechnology, Dalian, Liaoning, China). For PCR, samples were heated to

165

95 °C for 5 min, denatured at 95 °C for 15 s, annealed temperature was 60 °C for 15 s,

166

72 °C for 20 s, and cycled 40 times. The primer sequences were listed in Table S1. To

167

determine the relative expression compared to the group without PSG-1 stimulated, Ct

168

values were normalized to β-actin, and relative expression was calculated using the

169

the ∆∆Ct method.

170

RNA-seq analysis. Library preparation and sequencing were performed at Shanghai

171

Personal Biotechnology Co., Ltd. (Shanghai, China). Total RNA sample libraries

172

were extracted using the Illumina TruSeq Stranded mRNA LT sample preparation kit

173

(Illumina Inc., San Diego, CA, USA), according to the standard manufacturer's

174

instructions. rRNA was removed from the total RNA by ribosomal RNA removal kit

175

(Illumina Inc., San Diego, CA, USA), the purified mRNA was denatured and diluted

176

to 0.1 µg to 0.2 µg of total RNA before interrupted producing libraries with an insert

177

size between 200 and 300 bp. cDNA was then synthesized from RNA using Super

178

Script II Reverse Transcriptase (Invitrogen Corp., Carlsbad, California, USA), the

179

first strand of cDNA was synthesized with hexahedron random primers and reverse

180

transcriptase, and the second strand cDNA was synthesized by using the first strand of

181

cDNA as template. In the second strand of cDNA synthesis, dTTP was replaced by

182

dUTP, thus maintain the strandedness of the library. After the library was constructed,

183

the fragmented library was enriched by PCR amplification, and then the library was

184

selected according to the size of the fragment. The size of the library was 300 - 400 bp.

185

Then 3’-adenylation, adaptor ligation and libraries were subjected to 15 cycles of

186

PCR to generate RNA-Seq libraries ready for sequencing. Before sequencing

187

RNA-Seq libraries were qualified by the Agilent 2100 Bioanalyzer with the high 8


Page 8 of 39

Page 9 of 39


188

sensitivity DNA kit (Agilent Technologies, Palo Alto, California, USA).

189

Quantification of libraries for clustering was performed using the KAPA library

190

quantification kit - Illumina/Universal (KAPA Biosystems, Wilmington, MA, USA)

191

and Quant Studio™ 7 Real-Time PCR System (Life Technologies, Waltham, MA,

192

USA). Sequencing was performed using the Next-Generation Sequencing (NGS)

193

technology, based on Illumina NextSeq500 (NextSeq control software v1.2/Real Time

194

Analysis v2.1) platform. The library was diluted and denatured according to the

195

effective concentration of the library and the number of library required and

196

sequencing was carried out to produce single-end 76 bp reads using NextSeq500 High

197

Output reagent kit (Illumina Inc., San Diego, CA, USA). FastQC was used to quality

198

control, quality score Q > 30 of reads were used to downstream analysis. HTSeq

199

0.6.1p2 was used to count the read counts for each gene in the ensembl annotation.

200

Bioinformatics analysis. For differentially expressed genes analysis, we used the

201

DESeq (version 1.18.0) to detected the differential expression (n=3) with the gene

202

symbol annotation (FDR< 0.05, fold change > 2). In addition, principle component

203

analysis (PCA) was carried out on the genes significantly expressed between all

204

different groups. Enrichment analysis of differentially expressed genes was conducted

205

utilizing the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway

206

enrichment analysis. KEGG, which is a database resource for understanding

207

high-level functions and utilities of the biological system from molecular-level

208

information and the significantly and differentially expressed genes with fold changes

209

information included in the KEGG pathway database. We counted the number of

210

differentially expressed genes at different levels of KEGG pathway, and determined

211

the metabolic pathways and signaling pathways.

212

Western blot analysis. The whole cells were lysed using RIPA buffer containing the 9



213

protease inhibitor. The concentration of protein samples were measured with the

214

enhanced BCA protein assay kit. Then protein samples were denatured at 95 °C for 5

215

min, loaded onto SDS-PAGE and transferred to the PVDF membrane (Millipore Co.,

216

Belford, MA, US). The membrane was sealed with 5% bovine serum albumin (BSA)

217

and incubated with primary antibody overnight at 4 °C and followed by incubation

218

with HRP conjugated anti-rabbit antibody. Then the protein was detected by using

219

Molecular Imager ChemiDoc™ XRS Imaging System (Bio-Rad Laboratories,

220

Hercules, CA, USA), band density was normalized to β-actin, and bands were

221

quantified with the image analysis software Quantity One 4.6.2.

222

Statistical analysis. Data are expressed as means ± SD. The statistical analysis was

223

used SPSS 19.0 software. A p-value of P < 0.05 was considered to be statistically

224

significant.

225

RESULTS

226

Effect of PSG-1 on the proliferation and cytokines production of T lymphocytes.

227

To test the effect of PSG-1 on the proliferation of T lymphocytes, cell proliferation

228

was measured by CCK-8 kit. As Figure 1A showed that PSG-1 in combine with

229

ConA, T lymphocytes proliferation was significantly (P < 0.01) increased, indicating

230

that PSG-1 synergized with ConA to promote the proliferation of T lymphocytes.

231

When PSG-1 was used alone to stimulate T lymphocytes, the proliferation was also

232

significantly (P < 0.05) enhanced, demonstrating that PSG-1 could directly induced T

233

lymphocytes proliferation. To investigate the effect of PSG-1 on the level of

234

cytokines, the cells were cultured with PSG-1 (160 µg/mL) and/or ConA (5 µg/mL).

235

Compared with the control group, PSG-1 increased the secretion of IL-2, IFN-γ and

236

IL-12, when combined with ConA, the production of three cytokines were

237

dramatically (P < 0.01) increased (Figure 1B, C and D), but has no effect on the 10


Page 10 of 39

Page 11 of 39


238

production of IL-4 (data not shown).

239

RNA-seq and bioinformatics analysis. In order to give further insights into the

240

underlying molecular mechanisms involve in the immunomodulatory activity of

241

PSG-1 on T lymphocytes, RNA-seq was performed. Differentially expressed genes

242

analysis showed 394 genes were differentially expressed in a significant manner

243

(FDR< 0.05, fold change > 2) in PSG-1 stimulated T lymphocytes, of which the up

244

and down regulated genes were 283 and 111, respectively. The top 50 up- and

245

down-regulated genes were listed in Table S2 and Table S3. Principle component

246

analysis (PCA) on the complete transcriptome of T lymphocytes in the PSG-1

247

treatment group (P group) and control group (C group), demonstrated that two

248

different groups can be distinguished, illustrating that P and C cluster separately

249

(Figure 2A). Moreover, KEGG pathway enrichment analysis identified significant

250

enrichment of differentially expressed genes involved in 152 canonical pathways.

251

Figure 2B showed the top 20 significantly enriched canonical pathways. The analysis

252

also demonstrated significant enrichment of functions associated with diseases, and

253

the top 7 biological functions were showed in Figure 2C.

254

To certify the RNA-seq results, we randomly selected 9 differential expression

255

genes that were up or down regulated in PSG-1 stimulated T lymphocytes and

256

measured gene expression by RT-qPCR. The result showed that the tendency of

257

RT-qPCR and RNA-seq were the same (Figure 2D).

258

PSG-1 stimulated Ca2+/CaN pathway in T lymphocytes. After exposure to PSG-1

259

(160 µg/mL) for 48 h, Ca2+ concentration and CaN activity were measured by flow

260

cytometry and CaN activity assay kit. We found that PSG-1 increased the level of

261

Ca2+ (Figure 3A), and markedly (P < 0.01) enhanced CaN activity (Figure 3B). In

262

addition, canonical pathway analysis demonstrated that calcium pathway (P < 0.01) 11



263

was significantly associated with immunomodulatory activity of PSG-1 in T

264

lymphocytes (Figure 2C). Heatmap (Figure 3C) demonstrated 7 up-regulated genes

265

and 3 down-regulated genes associated with calcium pathway.

266

Effect of calcium channel inhibitor on proliferation and cytokines production

267

induced by PSG-1 in T lymphocytes. As shown in the Figure 4A, when pretreated T

268

lymphocytes with verapamil for 30 min, and stimulated with PSG-1 (160 µg/mL) for

269

another 48 h, the proliferation of T lymphocytes were significantly suppressed. In

270

addition, Figure 4B/C/D showed that in the presence of PSG-1 and ConA, the

271

production of IL-2, IFN-γ and IL-12 were 2801.56, 8029.83 and 2563.66 pg/mL,

272

respectively. When pretreated with verapamil (40 µM), the levels significantly

273

reduced to 337.7, 3831.84 and 634.75 pg/mL, respectively, which were much lower

274

than the group that PSG-1 combined with ConA.

275

PSG-1 activated MAPKs in T lymphocytes. To assess the possible involvement of

276

three MAPK families: ERK, JNK and p38 MAPK in PSG-1 stimulated T lymphocytes

277

immunomodulation, we used western blot analysis to detect the expression of ERK,

278

p-ERK, JNK, p-JNK, p38 and p-p38 in T lymphocytes after 48 h exposure to PSG-1.

279

The data showed that all proteins expression were increased compared with control

280

group (Figure 5A/C/E). When treated with PD98059, SP600125 or SB203580, the

281

ratio of p-ERK/ERK, p-JNK/JNK and p-p38/p-38 were dramatically (P < 0.01)

282

decreased compared with the group that stimulated with PSG-1 alone (Figure 5B/D/F).

283

Moreover, the heatmap (Figure 5G) identified 14 differentially expressed gene

284

involved in MAPK pathway, of which the up- and down-regulated genes were 12 and

285

2, respectively.

286

Effect of MAPKs inhibitors on proliferation and cytokines production induced

287

by PSG-1 in T lymphocytes. To investigate the role of MAPK families in T 12


Page 12 of 39

Page 13 of 39


288

lymphocyte proliferation, we exposed T lymphocytes to the inhibitors of ERK, JNK

289

and p38 MAPK for 30 min, respectively. The inhibitors significantly suppress T

290

lymphocyte proliferation, of which the inhibitor of p38 (SB203580) suppressed T

291

lymphocytes proliferation most (Figure 6A). To determine the role of MAPKs in the

292

regulation of IL-2, IFN-γ and IL-12 secretion in PSG-1-stimulated T lymphocytes, we

293

pretreated T lymphocytes with PD98059, SP600125, and SB203580 for 30 min,

294

followed by exposure to PSG-1 and ConA for 48 h, and found that all the inhibitors

295

markedly reduced the production of IL-2, IFN-γ and IL-12 (Figure 6B/C/D).

296

DISCUSSION

297

Polysaccharides

are

natural

polymer

compounds

bounded

together

by

298

monosaccharide unit and their derivatives.38 They are not only the structure of cells

299

and energy substances,39 but also biological substances with a variety of physiological

300

functions, of which immunomodulation is the most important and fundamental

301

function.40 Spleen is the largest peripheral lymphoid organ, whose significant immune

302

function is based on various immune cells inside, including lymphocytes,

303

macrophages, and dendritic cells.41 T lymphocytes account for about 35% of

304

lymphocytes, and their primary role is to regulate the immune response caused by

305

antigens and eliminate intracellular microbes.42 Zhong et al. showed that Ganoderma

306

polysaccharide B (GL-B) could significantly increase IFN-γ mRNA expression in T

307

lymphocytes.29 In our previous study, we have found that PSG-1 has

308

immunomodulatory effect on spleen lymphocytes, as evidenced by increased the

309

intracellular Ca2+ concentration and CaN activity, activated NFAT activity, and

310

induced IL-2 production.37 Moreover, PSG-1 increased the numbers of CD4+ T

311

lymphocytes and the ratio of CD4+/CD8+, and restored the IL-2, INF-γ, IL-10 levels

312

of spleen lymphocyte in cyclophosphamide-induced mice.35 However, it lacks of 13



Page 14 of 39

313

study about the immunomodulatory effect of PSG-1 on purified T lymphocytes. T

314

lymphocytes play an important role in immune diseases.43 It is possible to fully

315

understand the corresponding links of the immune response through exploration of T

316

lymphocytes function, which has great significance for researching immune response

317

of the body. To demonstrate the effect of PSG-1 on the proliferation of T lymphocytes,

318

we measured the proliferation by CCK-8 kit. Results showed that PSG-1 dramatically

319

stimulated T lymphocytes proliferation. Moreover, in the presence of stimulus such as

320

ConA favors the proliferation of T lymphocytes. As intercellular signaling proteins,

321

cytokines have important functions in the control of homeostasis in organism.37 It is

322

generally

323

immunomodulation.44 Liu et al. have indicated that Ganoderma lucidum

324

polysaccharides encapsulated in liposome significantly promoted splenocytes

325

proliferation, secretion of IL-6, IFN-γ, IL-4, TNF-α, and activation of CD3+CD4+ and

326

CD3+CD8+ T lymphocytes.45 To investigate whether PSG-1 has immunomodulatory

327

effect on T lymphocytes, we measured the levels of cytokines produced by T

328

lymphocytes and found that PSG-1 promoted the expression of IL-2, IFN-γ and IL-12,

329

but there was no effect on IL-4 expression. The results noted above were consistent

330

with our previous findings, indicating that PSG-1 has an immunomodulatory effect on

331

T lymphocytes. However, the specific mechanism was still unclear.

332

known

that

cytokine

environment

plays

an

vital

role

in

RNA-seq has been considered as a highly efficient, widely used, and conventional

333

molecular biology method to obtain transcriptome information.21,

334

RNA-seq was performed to illustrate the effect of PSG-1 on the differentially

335

expressed genes in T lymphocytes, trying to globally screen the signal transduction

336

pathway of PSG-1 stimulated T lymphocytes. RNA-seq analysis identified 283

337

up-regulated genes, 111 down-regulated genes. Furthermore, KEGG pathway analysis 14


46

Therefore,

Page 15 of 39


338

illuminated significant enrichment canonical pathways and biological functions that

339

were significantly enriched with the differentially expressed genes obtained from the

340

RNA-seq analysis. From the results we could find that the top 7 significant

341

enrichment biological functions were associated with diseases. Besides, 10 out of the

342

top 20 significantly enriched canonical pathways were related to immunity, and

343

calcium and MAPK pathway were two significant pathways in PSG-1 stimulated T

344

lymphocytes. Thus, we focused on these two pathways to further investigate the

345

immunomodulatory effect of PSG-1 on T lymphocytes.

346

Ca2+ is necessary for cell proliferation, activation and functions. T lymphocytes

347

depend on Ca2+ signaling to activate developmental and activation procedures.47 CaN

348

is a kind of calmodulin-dependent enzyme and CaN regulation in vivo is through

349

changing intracellular calcium.48 Ca2+ involves in immune response in T cells mainly

350

through Ca2+/CaN/NFAT pathway, the increasing of intracellular calcium, activates

351

calcineurin and triggers the dephosphorylation of NFAT, and then NFAT translocates

352

into the nucleus, and activates a series of signaling events.49 To explore the role of

353

Ca2+/CaN pathway in PSG-1 stimulated T lymphocytes, we detected Ca2+

354

concentration and CaN activity, and further measured the proliferation and the

355

cytokines production with the pretreatment of calcium channel inhibitor. The results

356

showed that PSG-1 dramatically improved the intracellular Ca2+ concentration and

357

CaN activity, and the calcium channel inhibitor suppressed PSG-1 induced T

358

lymphocyte proliferation and production of IL-2, IFN-γ and IL-12. Moreover,

359

RNA-seq and KEGG pathway analysis significantly highlighted the importance of

360

Ca2+ signaling in immunomodulatory activity of PSG-1 on purified T lymphocytes.

361

These results suggested that PSG-1 exerted immunomodulatory effect on T

362

lymphocytes through Ca2+/CaN signaling pathway. 15



363

MAPKs are evolutionarily conserved enzymes that related to many physiological

364

processes, such as cellular processes, immune responses in mammalian species.13

365

Recently, studies have shown that MAPK inhibitors suppressed the levels of TNF-α

366

protein and TNF-α mRNA expression in macrophages of S-180 tumor-bearing mice.50

367

Our results showed that PSG-1 elevated the levels of MAPK expression. When

368

exposure to inhibitors, the phosphorylation of MAPK were decreased to relatively low

369

levels. To explore whether PSG-1 stimulated T lymphocytes proliferation and the

370

cytokines production through MAPK pathway, we took advantage of ERK, JNK and

371

p38 inhibitors and found that the proliferation of T lymphocytes was inhibited to

372

varying degrees, in which SB203580 has a strongest inhibitory effect. Besides that, all

373

the inhibitors markedly reduced the secretion of IL-2, IFN-γ and IL-12. Furthermore,

374

the heatmap demonstrated the differentially expressed genes involved in MAPK

375

pathway activated by PSG-1. These results suggested that MAPK pathway was

376

involved in the immunomodulatory activity of PSG-1 on purified T lymphocytes.

377

In summary, the present study demonstrated that PSG-1 increased the proliferation

378

and the production of IL-2, IL-12 and IFN-γ in purified T lymphocytes, and

379

elucidated the underlying mechanism that Ca2+/CaN and MAPK pathway played an

380

important role in the immunomodulatory activity of PSG-1 on purified T lymphocytes

381

based on RNA-seq. This study revealed the potential of PSG-1 developed as a novel

382

immunomodulatory agent, and provided a new perspective for the research of

383

bioactive components in functional foods. However, all the experiments were done in

384

vitro, the immunomodulatory mechanism of T lymphocytes in vivo is complex, which

385

needs more detailed investigation.

386

ABBREVIATIONS USED

387

PSG-1, Ganoderma atrum polysaccharide; CaN, calcineurin; IL-2, interleukin-2; 16


Page 16 of 39

Page 17 of 39


388

IL-12, interleukin-12; IFN-γ, Interferon-γ; MAPK, mitogen-activated protein kinases;

389

the extracellular signal–regulated kinases, ERKs; c-Jun N-terminal kinases, JNKs;

390

RNA-seq, RNA sequencing, ConA, concanavalin A.

391

ACKNOWLEDGEMENTS

392

This work was financially supported by the National Natural Science Foundation of

393

China (No. 21265011), Research Project of State Key Laboratory of Food Science

394

and Technology (No. SKLF-ZZA-201611), Natural Science Foundation of Jiangxi,

395

China (No. 20161BAB214161), is gratefully acknowledged.

396

SUPPORTING INFORMATION

397

Table S1. Primer sequences used for real-time qPCR.

398

Table S2. The top 50 up-regulated genes RNA-seq profiling in PSG-1 stimulated T

399

lymphocytes.

400

Table S3. The top 50 down-regulated genes RNA-seq profiling in PSG-1 stimulated T

401

lymphocytes.

402

REFERENCES

403

(1) Saito, S.; Nakashima, A.; Shima, T.; Ito, M., Review article: Th1/Th2/Th17 and

404

regulatory T cell paradigm in pregnancy. Am. J. Reprod. Immunol. Microbiol. 2010,

405

63, 601-610.

406

(2) Mosmann, T. R.; Cherwinski, H.; Bond, M. W.; Giedlin, M. A.; Coffman, R. L.,

407

Two types of murine helper T cell clone. I. Definition according to profiles of

408

lymphokine activities and secreted proteins. J. Immunol. 1986, 136, 2348-2357.

409

(3) Coffman, R. L., Origins of the Th1-Th2 model: a personal perspective. Nat.

410

Immunol. 2006, 7, 539-541.

411

(4) O'Garra, A., Cytokines induce the development of functionally heterogeneous T 17



412

helper cell subsets. Immunity 1998, 8, 275-283.

413

(5) Mosmann, T. R.; Coffman, R. L., Th1-cell and Th2-cell - different patterns of

414

lymphokine secretion lead to different functional properties. Annu. Rev. Immunol.

415

1989, 7, 145-173.

416

(6) Carafoli, E., The calcium-signalling saga: tap water and protein crystals. Nat. Rev.

417

Mol. Cell Biol. 2003, 4, 326-332.

418

(7) Zweifach, A.; Lewis, R. S., Mitogen-regulated Ca2+ current of T lymphocytes is

419

activated by depletion of intracellular Ca2+ stores. Proc. Nat. Acad. Sci. 1993, 90,

420

6295-6299.

421

(8) McCarl, C.-A.; Khalil, S.; Ma, J.; Oh-Hora, M.; Yamashita, M.; Roether, J.;

422

Kawasaki, T.; Jairaman, A.; Sasaki, Y.; Prakriya, M., Store-operated Ca2+ entry

423

through ORAI1 is critical for T cell-mediated autoimmunity and allograft rejection. J.

424

Immunol. 2010, 185, 5845-5858.

425

(9) Prakriya, M.; Lewis, R. S., CRAC channels: activation, permeation, and the

426

search for a molecular identity. Cell Calcium 2003, 33, 311-321.

427

(10) Clipstone, N. A.; Crabtree, Identification of calcineurin as a key signalling

428

enzyme in T-lymphocyte activation. Nat. 1992, 357, 695-697.

429

(11) Molkentin, J. D.; Lu, J.-R.; Antos, C. L.; Markham, B.; Richardson, J.; Robbins,

430

J.; Grant, S. R.; Olson, E. N., A calcineurin-dependent transcriptional pathway for

431

cardiac hypertrophy. Cell 1998, 93, 215-228.

432

(12) Zhang, W.; Liu, H. T., MAPK signal pathways in the regulation of cell

433

proliferation in mammalian cells. Cell Res. 2002, 12, 9-18. 18


Page 18 of 39

Page 19 of 39


434

(13) Chen Dong, R. J. D., and Richard A. Flavell, MAP kinases in the immune

435

response. Annu. Rev. Immunol. 2002, 20, 55-72.

436

(14) Junttila, M. R.; Li, S.-P.; Westermarck, J., Phosphatase-mediated crosstalk

437

between MAPK signaling pathways in the regulation of cell survival. The FASEB

438

Journal 2008, 22, 954-965.

439

(15) Zhang, Y.; Dong, C., MAP Kinases in immune responses. Cell. Mol. Immunol.

440

2005, 2, 20-27.

441

(16) Jeffrey, K. L.; Camps, M.; Rommel, C.; Mackay, C. R., Targeting dual-specificity

442

phosphatases: manipulating MAP kinase signalling and immune responses. Nat. Rev.

443

Drug Discov. 2007, 6, 391-403.

444

(17) Yang, D. D.; Conze, D.; Whitmarsh, A. J.; Barrett, T.; Davis, R. J.; Rincón, M.;

445

Flavell, R. A., Differentiation of CD4+ T cells to Th1 cells requires MAP kinase JNK2.

446

Immunity 1998, 9, 575-585.

447

(18) Matsuda, S.; Moriguchi, T.; Koyasu, S.; Nishida, E., T lymphocyte activation

448

signals for interleukin-2 production involve activation of MKK6-p38 and

449

MKK7-SAPK/JNK signaling pathways sensitive to cyclosporin A. J. Biol. Chem.

450

1998, 273, 12378-12382.

451

(19) Zhang, S.; Kaplan, M. H., The p38 mitogen-activated protein kinase is required

452

for IL-12-induced IFN-γ expression. J. Immunol. 2000, 165, 1374-1380.

453

(20) Morin, R. D.; Bainbridge, M.; Fejes, A.; Hirst, M.; Krzywinski, M.; Pugh, T. J.;

454

McDonald, H.; Varhol, R.; Jones, S. J. M.; Marra, M. A., Profiling the HeLa S3

455

transcriptome using randomly primed cDNA and massively parallel short-read 19



456

sequencing. Biotechnol. 2008, 45, 81-94.

457

(21) Wang, Z.; Gerstein, M.; Snyder, M., RNA-Seq: a revolutionary tool for

458

transcriptomics. Nat. Rev. Genet. 2009, 10, 57-63.

459

(22) Young, M. D.; Wakefield, M. J.; Smyth, G. K.; Oshlack, A., Gene ontology

460

analysis for RNA-seq: accounting for selection bias. Genome Biol. 2010, 11, R14.

461

(23) Trapnell, C.; Williams, B. A.; Pertea, G.; Mortazavi, A.; Kwan, G.; van Baren, M.

462

J.; Salzberg, S. L.; Wold, B. J.; Pachter, L., Transcript assembly and quantification by

463

RNA-Seq reveals unannotated transcripts and isoform switching during cell

464

differentiation. Nat. Biotech. 2010, 28, 511-515.

465

(24) Maher, C. A.; Kumar-Sinha, C.; Cao, X.; Kalyana-Sundaram, S.; Han, B.; Jing,

466

X.; Sam, L.; Barrette, T.; Palanisamy, N.; Chinnaiyan, A. M., Transcriptome

467

sequencing to detect gene fusions in cancer. Nat. 2009, 458, 97-101.

468

(25) Yang, S. S.; Tu, Z. J.; Cheung, F.; Xu, W. W.; Lamb, J. F.; Jung, H.-J. G.; Vance,

469

C. P.; Gronwald, J. W., Using RNA-Seq for gene identification, polymorphism

470

detection and transcript profiling in two alfalfa genotypes with divergent cell wall

471

composition in stems. BMC Genomics 2011, 12, 1-19.

472

(26) Sun, L.; Luo, H.; Bu, D.; Zhao, G.; Yu, K.; Zhang, C.; Liu, Y.; Chen, R.; Zhao, Y.,

473

Utilizing sequence intrinsic composition to classify protein-coding and long

474

non-coding transcripts. Nucleic Acids Res. 2013, 41, e166-e166.

475

(27) Shen, S.; Park, J. W.; Huang, J.; Dittmar, K. A.; Lu, Z.-x.; Zhou, Q.; Carstens, R.

476

P.; Xing, Y., MATS: a Bayesian framework for flexible detection of differential

477

alternative splicing from RNA-Seq data. Nucleic Acids Res. 2012, 40, e61-e61. 20


Page 20 of 39

Page 21 of 39


478

(28) Marguerat, S.; Bähler, J., RNA-seq: from technology to biology. Cell. Mol. Life

479

Sci. 2010, 67, 569-579.

480

(29) Lin, Z. B.; Zhang, H. N., Anti-tumor and immunoregulatory activities of

481

Ganoderma lucidum and its possible mechanisms. Acta Pharmacol. Sin. 2004, 25,

482

1387-1395.

483

(30) Yu, Q.; Nie, S.-P.; Wang, J.-Q.; Huang, D.-F.; Li, W.-J.; Xie, M.-Y., Molecular

484

mechanism underlying chemoprotective effects of Ganoderma atrum polysaccharide

485

in cyclophosphamide-induced immunosuppressed mice. J. Funct. Foods 2015, 15,

486

52-60.

487

(31) Zhang, H.; Nie, S.; Cui, S. W.; Xu, M.; Ding, H.; Xie, M., Characterization of a

488

bioactive polysaccharide from Ganoderma atrum: Re-elucidation of the fine structure.

489

Carbohydr. Polym. 2017, 158, 58-67.

490

(32) Zhang, S.; Nie, S.; Huang, D.; Li, W.; Xie, M., Immunomodulatory effect of

491

Ganoderma atrum polysaccharide on CT26 tumor-bearing mice. Food Chem. 2013,

492

136, 1213-1219.

493

(33) Yu, Q.; Nie, S.-P.; Li, W.-J.; Zheng, W.-Y.; Yin, P.-F.; Gong, D.-M.; Xie, M.-Y.,

494

Macrophage immunomodulatory activity of a purified polysaccharide isolated from

495

Ganoderma atrum. Phytother. Res. 2013, 27, 186-191.

496

(34) Li, W.-J.; Nie, S.-P.; Chen, Y.; Xie, M.-Y.; He, M.; Yu, Q.; Yan, Y., Ganoderma

497

atrum polysaccharide protects cardiomyocytes against anoxia/reoxygenation-induced

498

oxidative stress by mitochondrial pathway. J. Cell. Biochem. 2010, 110, 191-200.

499

(35) Yu, Q.; Nie, S.-P.; Wang, J.-Q.; Liu, X.-Z.; Yin, P.-F.; Huang, D.-F.; Li, W.-J.; 21



Page 22 of 39

500

Gong, D.-M.; Xie, M.-Y., Chemoprotective effects of Ganoderma atrum

501

polysaccharide in cyclophosphamide-induced mice. Int. J. Biol. Macromol. 2014, 64,

502

395-401.

503

(36) Zhu, K.; Nie, S.; Li, C.; Lin, S.; Xing, M.; Li, W.; Gong, D.; Xie, M., A newly

504

identified polysaccharide from Ganoderma atrum attenuates hyperglycemia and

505

hyperlipidemia. Int. J. Biol. Macromol. 2013, 57, 142-150.

506

(37) Yu, Q.; Nie, S.-P.; Wang, J.-Q.; Huang, D.-F.; Li, W.-J.; Xie, M.-Y., Signaling

507

pathway involved in the immunomodulatory effect of Ganoderma atrum

508

polysaccharide in spleen lymphocytes. J. Agric. Food. Chem. 2015, 63, 2734-2740.

509

(38) Liu, Q.; Zhang, Y.; Laskowski, J. S., The adsorption of polysaccharides onto

510

mineral surfaces: an acid/base interaction. Int. J. Miner. Process. 2000, 60, 229-245.

511

(39) Caffall, K. H.; Mohnen, D., The structure, function, and biosynthesis of plant cell

512

wall pectic polysaccharides. Carbohydr. Res. 2009, 344, 1879-1900.

513

(40) Wasser,

514

immunomodulating polysaccharides. Appl. Microbiol. Biotechnol. 2002, 60, 258-274.

515

(41) Dailey, M. O. The immune functions of the spleen. In The complete spleen,

516

Bowdler, A. J., Ed.; 2002, pp pp 51-69.

517

(42) Abbas, A. K., Basic immunology: Functions and disorders of the immune system

518

(2nd ed.). 2004; Vol. 32, p 1030-1045.

519

(43) Gutcher, I.; Becher, B., APC-derived cytokines and T cell polarization in

520

autoimmune inflammation. J Clin Invest. 2007, 117, 1119-1127.

521

(44) Leung, S.; Liu, X.; Fang, L.; Chen, X.; Guo, T.; Zhang, J., The cytokine milieu in

S.,

Medicinal

mushrooms

as

a

source

22


of

antitumor

and

Page 23 of 39


522

the interplay of pathogenic Th1/Th17 cells and regulatory T cells in autoimmune

523

disease. Cell. Mol. immunol. 2010, 7, 182-189.

524

(45) Liu, Z.; Xing, J.; Zheng, S.; Bo, R.; Luo, L.; Huang, Y.; Niu, Y.; Li, Z.; Wang, D.;

525

Hu, Y.; Liu, J.; Wu, Y., Ganoderma lucidum polysaccharides encapsulated in liposome

526

as an adjuvant to promote Th1-bias immune response. Carbohydr. Polym. 2016, 142,

527

141-148.

528

(46) Zhou, D.; Gao, S.; Wang, H.; Lei, T.; Shen, J.; Gao, J.; Chen, S.; Yin, J.; Liu, J.,

529

De novo sequencing transcriptome of endemic Gentiana straminea (Gentianaceae) to

530

identify genes involved in the biosynthesis of active ingredients. Gene 2016, 575,

531

160-170.

532

(47) Winslow, M. M.; Neilson, J. R.; Crabtree, G. R., Calcium signalling in

533

lymphocytes. Curr. Opin. Immunol. 2003, 15, 299-307.

534

(48) Rusnak, F.; Mertz, P., Calcineurin: form and function. Physiol. Rev. 2000, 80,

535

1483-1521.

536

(49) Hogan, P. G.; Chen, L.; Nardone, J.; Rao, A., Transcriptional regulation by

537

calcium, calcineurin, and NFAT. Genes & Dev. 2003, 17, 2205-2232.

538

(50) Huang, J.; Nie, Q.; Liu, X.; Zhang, S.; Nie, S.; Huang, D.; Wang, S.; Zhu, F.; Xie,

539

M., Ganoderma atrum polysaccharide modulates TNF-α secretion and mRNA

540

expression in macrophages of S-180 tumor-bearing mice. Food Hydrocoll. 2016, 53,

541

24-30.

542 543

23



544

FIGURE CAPTIONS

545

Figure 1. Effect of PSG-1 on the proliferation and cytokines production of T

546

lymphocytes. (A) Effect of PSG-1 on the proliferation of T lymphocytes. T

547

lymphocytes were stimulated with PSG-1 (160 µg/mL) and/or ConA (5 µg /mL) for

548

48 h, and the cells were measured by CCK-8 kit. Data were expressed as means ± SD

549

(n = 12), *P < 0.05, **P < 0.01 versus control group. (B) The level of IL-2. (C) The

550

production of IFN- γ. (D) The level of IL-12.Similar results were obtained in three

551

separate experiments, no statistical significance (ns), *P < 0.05 versus control group;

552

##

553

Figure 2. RNA-seq analysis and pathway analysis in PSG-1 stimulated T

554

lymphocytes. RNA-seq was performed on RNA isolated from T lymphocytes. C

555

represented control group and P represented the group with PSG-1 treatment. (A)

556

Principal component analysis (PCA) performed on whole transcriptome of T

557

lymphocytes. The different shapes in the figure represented different samples, and

558

color represented different groups (n = 3). (B) The top 20 canonical pathways

559

enriched by differentially expressed genes from the RNA-seq analysis comparing with

560

C group and P group highlighted by KEGG pathway analysis. The size of the dot in

561

the figure indicated the number of differentially expressed gene was annotated to the

562

pathway and the color represented P value of the pathway. (C) The top 7 significantly

563

enriched biological functions associated with differentially expressed genes in T

564

lymphocytes. (D) The fold change of relative mRNA expression level of Lcn2, H2-Aa,

565

Arrdc4, Adamtsl4, H2-Eb1, Smyd1, Tmem140, Gpr83 and Ecm1 measured by

566

RNA-seq and qPCR (n = 3).

567

Figure 3. PSG-1 stimulated Ca2+/CaN pathway in T lymphocytes. (A) Effect of

568

PSG-1 on the intracellular Ca2+ concentration in T lymphocytes. The cells were

P < 0.01 versus ConA alone group.

24


Page 24 of 39

Page 25 of 39


569

stimulated with PSG-1 (160 µg/mL) and/or ConA (5 µg/mL). After 48 h the cells

570

were collected and washed twice with PBS, and cultured with the fluorescent probe

571

Fluo-3/AM at 37 °C for 60 min. The Ca2+ level was analyzed by flow cytometry (n =

572

3). (B) Effect of PSG-1 on CaN activity in T lymphocytes. The CaN activity was

573

determined by the CaN activity assay kit (n = 3), *P < 0.05, **P < 0.01 versus control

574

group. (C) Heatmap representation of differentially expressed genes associated with

575

calcium pathway.

576

Figure 4. Effect of calcium channel inhibitor on proliferation and cytokines

577

production induced by PSG-1 in T lymphocytes. (A) The inhibitor of Ca2+ prevented

578

T lymphocytes proliferation. The cells were pretreated with verapamil (40 µM/mL)

579

for 30 min, then cells proliferation were detected by CCK-8 kit (n = 12), *P < 0.05,

580

**P < 0.01 versus control group,

581

C and D) The levels of IL-2, IFN-γ and IL-12 produced by T lymphocytes. The cells

582

from Balb/c mice were pretreated with verapamil (40 µM/mL) for 30 min, and then

583

cells were treated with PSG-1 (160 µg/mL) and/or ConA (5 µg/ml) for 48 h. The data

584

were expressed as mean ± SEM for three separate experiments. ##P < 0.01 vs PSG-1

585

in combination with ConA.

586

Figure 5. PSG-1 activated MAPKs in T lymphocytes. (A, C and E) The

587

immunoblotting bands of p-ERK, ERK, p-JNK, JNK, p-p38 and p38. (B, D and F)

588

Mean ± SED of immunoblotting bands of p-ERK/ERK, p-JNK/JNK, p-p38/p38 (n=3).

589

*P < 0.05 versus control group,

590

heatmap representation of differentially expressed genes associated with MAPK

591

pathway.

592

Figure 6. Effect of MAPKs inhibitors on proliferation and cytokines production

593

induced by PSG-1 in T lymphocytes. (A) The inhibitors of ERK, JNK and p38

##

P < 0.01 versus the group PSG-1 plus ConA. (B,

##

P < 0.01 versus PSG-1 alone group. (G) The

25



Page 26 of 39

594

restrained PSG-1 induced T-cell proliferation. T lymphocytes were pretreated with

595

PD98059, SP600125, or SB 203580 (40 µM/mL) for 30 min, then cells proliferation

596

was detected by CCK-8 kit (n = 12). **P < 0.01 versus control group;

597

versus group that PSG-1 combinated with ConA. (B, C and D) The production of IL-2,

598

IFN-γ and IL-12 in T lymphocytes. The cells (1×106 cells/well) were pretreated with

599

inhibitors for 30 min, and stimulated with PSG-1 (160 µg/mL) in presence ConA (5

600

µg/mL) for 48 h. Similar results were obtained in three separate experiments,

601

0.01 versus the group of PSG-1 plus ConA.

26


##

P < 0.01

##

P

Immunomodulatory Activity of Ganoderma atrum ... - ACS Publications

Recommend Documents