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Bioactive Constituents, Metabolites, and Functions

Nutshell extracts of Xanthoceras sorbifolia: a new potential source of bioactive phenolic compounds as natural antioxidant and immunomodulator Li Zhao, Xing Li, Fei Zhang, Juan-Juan Han, Ting Yang, Ze-Qing Ye, Zhe-Zhi Wang, and Yuan Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05590 • Publication Date (Web): 03 Apr 2018 Downloaded from http://pubs.acs.org on April 3, 2018

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Journal of Agricultural and Food Chemistry

Nutshell extracts of Xanthoceras sorbifolia: a new potential source of bioactive phenolic compounds as natural antioxidant and immunomodulator

Li Zhao†, Xing Li†, Ze-Qing Ye, Fei Zhang, Juan-Juan Han, Ting Yang, Zhe-Zhi Wang*, and Yuan Zhang*

1

National Engineering Laboratory for Resource Development of Endangered Crude Drugs in Northwest China, Key Laboratory of the Ministry of Education for

Medicinal Resources and Natural Pharmaceutical Chemistry, College of Life Sciences, Shaanxi Normal University, Xi’an 710119, P. R. China

1

ACS Paragon Plus Environment


1

ABSTRACT

2

Nutshell of Xanthoceras sorbifolia, a waste product in the production of edible oil, is

3

rich in health-promoting phenolic acids. However, the individual constituents,

4

bioactivities, and mechanism of action are largely unknown. In this study, 20 phenolic

5

compounds were characterized in nutshell extracts (NE) of X. sorbifolia by GC-MS.

6

Four established in vitro studies showed that NE has significant antioxidant potential.

7

Results in vivo indicated that oral administration of NE eﬀectively ameliorated

8

clinical disease severity of experimental autoimmune encephalomyelitis (EAE) and

9

reduced the neuroinflammation and the central nervous system (CNS) demyelination.

10

The underlying mechanism of NE-induced effects involved decreased penetration of

11

pathogenic immunocyte into the CNS, a reduced production of proinflammatory

12

cytokines and factors, and suppressed differentiation of Type 1 T helper (Th1) and

13

Th17 cells through the JAK/STAT pathway. Taken together, our studies showed that

14

X. sorbifolia nutshell, considered a waste material in the food industries, is a novel

15

source of natural antioxidants and immunomodulator.

16 17

KEYWORDS:

18

Xanthoceras sorbifolia, nutshell extracts, phenolic constituents, antioxidant activities,

19

experimental autoimmune encephalomyelitis, JAK/STAT pathway

2


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INTRODUCTION

21

Multiple sclerosis (MS) is known to be a neuroinflammatory demyelinating disease

22

(1). Experimental autoimmune encephalomyelitis (EAE), also called Experimental

23

allergic encephalomyelitis, is a well-accepted animal model to study MS (1, 2).

24

Demyelination and inflammation of the CNS in MS/EAE impair physical and

25

cognitive abilities, eventually resulting in death (3). Till now the pathogenesis of MS

26

has not been fully elucidated, however, it is generally accepted that, in MS and EAE,

27

auto-reactive CD4+ T cells crossed the broken blood-brain-barriers (BBB), induced

28

abnormal inflammatory reactions, and resulted in the tissue damage of the CNS (4).

29

Among these CD4 positive T cells, Th17 cells that secreting IL-17 and GM-CSF are

30

considered to be one of the most pro-inflammatory and encephalitogenic subsets

31

resulting in autoimmune and inflammatory diseases (4, 5). In addition, in both MS and

32

EAE models, there is a close correlation between oxidative stress and progression of

33

neuroinflammation (6-11). Accumulating evidence strongly supports the idea that the

34

CNS damage was induced by the over-reactive oxygen species (ROS) in MS (6, 12).

35

Therefore, therapeutic strategy that focuses on improving antioxidant potential and

36

modulation of the immune response would provide a new option for the prevention

37

and treatment of this chronic disease.

38

Diets accumulated plentiful phenolic compounds have shown a variety of

39

bioactive properties including antioxidant, neuroprotective, and anti-inflammatory

40

effects (13). Accordingly, dietary intake of naturally occurring phenolic compounds

41

with specific antioxidant and immunomodulation activity could not only enhance the 3



42

nutraceutical value and health benefits of the food, but also protect against chronic

43

diseases like MS. It would therefore be of great interest to the modern-day food

44

industry in its search for novel and economical sources rich in such bioactive

45

compounds, which can be used either as nutraceuticals or in functional foods to fight

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against and prevent these diseases.

47

Xanthoceras sorbifolia, an unusual tree species belonging to the family of

48

Sapindaceae, is distributed widely throughout the north of China and has a lifespan of

49

more than 200 years (14). This species is an economically and pharmaceutically

50

important energy crop with more than 50% oil in its seeds, which not only serve as

51

nutritious nuts but are also used to produce edible oil and biofuels (14). Currently,

52

interest is mainly focused on producing oil from the seeds, a process that generates a

53

large amount of residue including nutshells. Chemical studies of X. sorbifolia fruits

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and seeds have shown that they contain a variety of compounds, including flavonoids,

55

triterpenoids, and sterols (15, 16). Pharmacological research has shown that these

56

components have neuroprotective, anti-HIV, anti-oxidant, anti-inflammatory,

57

anti-tumour, and anti-Alzheimer effects (17-19), indicating the potential of X.

58

sorbifolia as a treatment for autoimmune diseases. Given that by-products represent

59

approximately 50% of total biomass of the seeds, and that they also contain bioactive

60

constituents with diverse pharmacologic properties, these wastes are a valuable source

61

of natural compounds that have potential as candidate drugs with novel mechanisms

62

of action in the treatment of autoimmune diseases.

63

The main goal of our work was to provide complete scientific information on X. 4


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Sorbifolia seeds and to prevent wasting this resource after the extraction process in

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the oil industry. As part of our ongoing search for antioxidant and immunomodulation

66

agents derived from medicinal and food plants(3, 5), our study provides for the first

67

time information on the phenolic profile, in vitro antioxidant activities, and in vivo

68

anti-inflammatory activities of the residue of X. Sorbifolia seeds, which, after

69

extraction of the oil, could serve as an alternative new source of bioactive components

70

in the food and pharmaceutical industries.

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MATERIALS AND METHODS

72

Reagents. Chemicals are supplied from MilliporeSigma (St. Saint Louis, MO,

73

USA), unless stated otherwise. Reagents of Silylation [TMCS (trimethylchlorosilane)

74

and BSTFA (Ν,Ο-bis(trimethylsilyl)trifluoroacetamide)] were manufactured by

75

Merck KGaA (Darmstadt, Germany). Standards [Benzoic acid, Isoeugenol,

76

tran-Cinnamic acid, p-Hydroxybenzoic acid, Gentisic acid, Vanillic acid, Gallic acid,

77

p-Coumaric acid, o-Hydroxycinnamic acid, o-Phthalic acid, Ferulic acid, Cinnamic

78

acid, Caffeic acid, Sinapic acid, Quercetin, (+)-Catechin, Hydroxytyrosol,

79

p-Hydroxyphenylacetic acid, and (-)-Epicatechin] were ordered from MilliporeSigma.

80

The standard stock solution were prepared in methanol and kept at -18 °C in dark.

81

Test extracts were dissolved in 0.9% saline and prepared immediately before

82

administration to the animals.

83

Plant material. Mature seeds of Xanthoceras sorbifolia Bunge (Figure 1A) were

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collected from Xi’an Botanical Garden of Shannxi Province at the harvest stage (Sep.,

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2007). The plant material was authenticated by Dr. Yi Ren at the National 5



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Engineering Laboratory for Resource Development of Endangered Crude Drugs in

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Northwest China, Xi’an, China. The voucher specimen No. XS070918 was deposited

88

thereby.

89

Nutshell extract (NE) preparation and derivatization. Air-dried nutshells of X.

90

sorbifolia were ground into a fine powder in a mechanical grinder and a mesh of

91

2mm-diameter were used. One gram powder was defatted with petroleum ether, and

92

extracted with 70% aqueous methanol with BHT (40 ml, 1.0 g/l). Then, 6 M HCL (10

93

ml) was added carefully, kept at 35 °C for 16 h, and stirring frequently. The cooling

94

supernatant was collected, filtered, and then extracted with 10 ml ethyl acetate for 3

95

times. This extract was evaporated to dryness on a rotovap. The resulted dried

96

nutshell extracts (NE) were kept at 4 °C in dark for future investigation.

97

For component analysis, 100 µg of the dried NE was dissolved in pyridine (1ml).

98

Then, a mixture of 100 µl of TMCS and 200 µl of BSTFA were added to the screw

99

cap glass tubes containg10 µl of the pyridine solution. The mixture solution were

100

incubated at 80 °C for 45 min for silylation. Because the hypersensitivity of

101

trimethylsilyl (TMS) derivatives to moisture, all the procedure mentioned above

102

should be kept under the anhydrous conditions. The silylated mixture was directly

103

analyzed

104

chromatography-flame ionization detection (GC-FID).

by

gas

chromatography-mass

spectrometry

(GC-MS)

and

gas

105

Determination of total phenolics and phenolic compounds of NE. Colorimetric

106

Folin-Ciocalteu method was used to determinate the total phenolic contents (20).

107

Qualitative and semi-quantification analysis of silylated phenolic compounds in NE 6


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were tested by using the GC-FID (Agilent 6890N) and GC-MS system (SHIMADZU

109

QP2010) according to our previous study (21). In the splitless mode, 2 µl of silylated

110

mixture was injected into the column. The oven temperature was programmed as

111

follows: (1) 80 °C for 1 minute; (2) 80 °C to 120 °C, at a rate of 5 °C per minute; (3)

112

120 °C to 240 °C, at a rate of 10 °C per minute; (4) 240 °C to 280 °C, at a rate of

113

20 °C/minute; (5) 280 °C final hold for 5 minute.

114

The percentage of the silylated phenolic compounds was analyzed using the

115

normalization method from the areas of FID (semi-quantification). GC-MS was

116

performed with the same operating conditions as GC-FID analysis. Components were

117

identified by comparing the retention times (RT) with those of standards, as well as

118

matching their recorded mass spectra with the libraries of

119

Standards and Technology (NIST05.LIB and NIST05s.LIB) .

National Institute of

120

In vitro determination of antioxidant activity. Antioxidant activity of NE of X.

121

sorbifolia was evaluated using four antioxidant test systems (DPPH radical

122

scavenging activity, •O2- scavenging activities, β-carotene bleaching assay, and ferric

123

ion reducing power) as has been described in our previous study (21).

124

In vivo assessment of the effects of NE on EAE mice. EAE was induced as

125

described in our previous studies (3, 4). EAE mice were randomly divided into two

126

treatment groups: PBS-treated control group and NE-treated group. Different dose of

127

NE (50, 100, and 150 mg/kg/d) was oral gavaged daily starting from the first day of

128

post-immunization (disease prevention), 10 days post-immunization (onset), as well

129

as 15 days post-immunization (peak). Dose optimization study was performed and the 7



130

suitable dosage was used for the further experiment (Figure 3A). Histopathological

131

analysis, mononuclear cell (MNC) preparation, ELISA, flow cytometry, western blot

132

and quantitative real-time PCR analysis were preformed according to our previous

133

studies (3, 4).

134

Statistical analysis. All data are presented as mean ± standard deviation (SD). For

135

the in vitro data, EC50 values were calculated by regression analysis. Data analyses

136

were performed with GraphPad Prism 6 (GraphPad, La Jolla, CA). , data were

137

analyzed by Analysis of variance (ANOVA) with Tukey’s multiple comparisons test

138

were used when comparing multiple groups. The value of p 0.05 was considered

139

statistically significant.

140 141

RESULTS AND DISCUSSION

142

Total phenolic and individual phenolic compound analysis of NE. According to

143

our extraction procedure, NE of X. sorbifolia gave brick-red powder in yields of 9.16

144

± 0.31 % (w/w) on the basis of the dry weight of crush nutshell (Figure 1B). The odor

145

was very mild. The amount of total phenolic of NE was 69.52 ± 2.66 mg GAE/g

146

extract. We next investigated the compound composition and quantitation of NE using

147

GC-MS after silylation. The total ion chromatogram (TIC) for this analysis is shown

148

in Figure 1C. Retention time (RT) and mass spectra of the major phenolic compounds

149

are listed in Table 1. A total of 20 constituents were determined by GC-FID and

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GC-MS, separately, representing 84.62 % of NE (Table 1). Major phenolic

151

compounds in NE are caffeic acid (20.05%), p-hydroxyphenylacetic acid (9.32%), 8


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p-coumaric acid (8.84%), and o-hydroxycinnamic acid (8.29%). So far as could be

153

ascertained from our literature survey, data on the phenolic composition of X.

154

sorbifolia are lacking. Only one paper (16) characterized 23 bioactive phenols from

155

the leaves of X. sorbifolia as the potential neuroinflammation inhibitors. Our data fill

156

the gap in the literature deficiency and describe for the first time the phenolic profile

157

of X. sorbifolia seed extracts. Prior reports indicated that phenolic content and

158

antioxidant potential have a positive correlation in the plant crude extracts (21).

159

Therefore, the high contents of total phenolics in NE indicated its strong antioxidant

160

properties, which could be exploited as phenolic products in the food and

161

pharmaceutical field in the future.

162 163

In vitro antioxidant activity of NE. Given that ROS play a critical role in the MS

164

pathology, several studies have focused on antioxidant therapies that are beneficial in

165

animal models for MS (6, 12). Of the phenolic compounds identified in NE of X.

166

sorbifolia, caffeic acid, coumaric acid, and their derivatives have been previously

167

shown to exhibit obviously antioxidant activities (22). Because of the synergistic

168

effect of various compounds, crude extracts usually have a more powerful antioxidant

169

potential and a greater number of biological activities than the individual

170

substances(23, 24),. Therefore, crude extracts are currently used for protection against

171

spoilage and oxidation in both the pharmacological and food industry (25). In this

172

study, both the lipophilic and hydrophilic antioxidant experiments were applied to

173

fully display the antioxidant effects of NE (Figure 2, Table 2), and NE showed 9



174

significant antioxidant potential in the four assays.

175

As shown in Figure 2A, NE and the positive standards (VC, VE and BHT)

176

demonstrated a strong ability to scavenge DPPH radicals in a dose-dependent manner.

177

With regard to scavenging capacity, IC50 of NE (6.22 ± 0.16 µg/mL) was similar to

178

VC (6.12 ± 0.21 µg/mL) and BHT (6.82 ± 0.46 µg/mL) (p = 0.066), which was

179

2.1-times greater than VE (12.51 ± 0.65 µg/mL) (Table 2).

180

The scavenging effect of NE toward •O2- was dose related (Figure 2B). At low

181

concentrations (0.02-0.05 mg/mL), VC exhibited a better scavenging rate than NE.

182

However, the maximum •O2- scavenging rate of VC and NE reached nearly 100% and

183

was comparable (p > 0.05), suggesting a similar scavenging capacity. The IC50 of

184

•O2- s scavenging activity of NE was found to be 46.33 ± 3.50 µg/mL, whereas that of

185

VC was 38.69 ± 4.22 µg/mL (Table 2).

186

As shown in Figure 2C, NE demonstrated significantly linoleate-derived free

187

radicals scavenging effect and decreased the β-carotene bleaching. In the control

188

group, the absorption value at 470 nm was reduced to a minimal value of 0.153±

189

0.011 after 120 min, while NE was still stayed at 0.401± 0.029. These results indicate

190

that NE significantly inhibited oxidation of linoleic acid. The antioxidant activities of

191

the test samples in this system were decreased as follows: BHT > VE > NE (Table 2).

192

In a reducing power assay, all samples shown their antioxidant activates in a

193

dose-dependent manner (Figure 2D). According to the results shown in Table 2, the

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reducing power of NE was as strong as that of BHT (p > 0.05), a widely used

195

commercial antioxidant, indicated that NE have good electron donating capacities, 10


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although it was still slightly less effective than VC.

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Our study demonstrated that, for the first time, the phenolic composition as well

198

as the antioxidant capacities of NE, the crude extracts of the defatted residue of X.

199

Sorbifolia seeds. And the results indicated that the investigated extracts could be

200

utilizing as the potential phenolic antioxidants that should be specifically studied for

201

their effects on human health.

202 203

Oral NE effectively enhanced clinical recovery from MOG-induced EAE.

204

Given that the important role of oxidative damage mediated CNS lesion, antioxidant

205

therapy represents an attractive treatment for neuroinflammation and/or inflammatory

206

autoimmune diseases (6, 12). To test the immunoregulation activity of NE for

207

treatment of neuroinflammatory disorders, we used EAE mouse model that

208

recapitulates human MS.

209

To determine the effects of NE administration on disease prevention or relapse,

210

oral treatment was started from day -5 post immunization (p.i.). Compared to

211

PBS-treated control mice, the clinical score records reflected a significantly delay of

212

disease onset and obviously inhibition of disease incidence (P < 0.01; Figure 3A). In

213

Figure 3A, 100% of mice in the PBS-treated group developed EAE around day 11 p.i.,

214

while only 39.6% of mice treated with NE (50 mg/kg/d) showed delayed disease onset

215

(~day 14 p.i.) and mild clinical symptoms. Furthermore, the loss of body weight of

216

EAE mice were also prevented by oral treatment of NE, in consistent with the

217

decreased clinical signs (Figure 3B). For dose optimization, 100 mg/kg/d were chosen 11



218

and used for the following experiments.

219

To further test whether oral administration of NE has beneficial effects on

220

ongoing EAE, in the therapeutic regimen, mice were fed with NE daily at the day 10

221

p.i. （onset）or at the day 15 p.i. （peak） of clinical EAE. Generally, severe clinical

222

symptoms were observed in the PBS-treated group, such as limp tail, wadding gait,

223

and/or paralysis of limbs. From Figure 3C we can see that the maximum clinical score

224

at ~ 18 days after EAE induction was 3.1 ± 0.56, while NE-treated mice showed 2.1 ±

225

0.52 of maximum EAE score accompanied by a reduced accumulative score.

226

Furthermore, NE treatment at the peak (day 16 p.i.) of clinical EAE also effectively

227

reduced disease severity and suppressed EAE progression (Figure 3D). Taken

228

together, these data indicated that NE has a significant therapeutic effect in EAE.

229

As we known, the safety, effective, and ideal selection in clinical treatment of

230

MS should prevent disease aggravation induced by neuroinflammation at the

231

induction and effector stages, halt the neurodegeneration and the related disability

232

mediated by axonal injury, as well as prevent disease relapse already underway. In

233

this regard, oral treatment of NE presents an important therapeutic agent, as it

234

effectively inhibited disease progression at the onset (induction phase), peak (effector

235

phase), and stable (chronic phase) of clinical EAE, and represents a promising

236

alternative to current therapies for MS treatment.

237 238

Oral NE reduced neuroinflammation and demyelination in EAE. As is well

239

known, EAE is an ideal experimental model that recapitulates several features of 12


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human MS (26). MS/EAE is induced by peripherally over-activated myelin-reactive

241

CD4+ T cells, as well as other immune cells, migrating into the CNS, and then

242

activating resident CNS immune cells (astrocytes and microglia cells) to produce

243

plentiful pro-inflammatory cytokines and chemokines. The immunological attacks in

244

the CNS of MS/EAE focus on the myelin sheaths, leading to the death of

245

oligodendrocytes, myelin loss, axons functionally compromised, and/or CNS lesion

246

(27). At the day of 30 post-immunization, to evaluate the therapeutic ability of NE on

247

the pathology of CNS, NE-treated and control mice were sacrificed. Lumbar spinal

248

cords were isolated for histological analysis. As shown in Figure 4A&B, NE-treated

249

mice showed significantly decreased inflammation (P= 0.006) and reduced

250

demyelination (P= 0.035) compared with the control group,. The total number of

251

MNCs infiltrating in the CNS also had a significantly reduced in the NE-treated group

252

(P = 0.0069; Figure 4C). To clarifying the underlying mechanism of NE function, the

253

expression level of cytokine and chemokine from the spinal cords of NE-treated and

254

control mice were assayed using Cytokines & Chemokines PCR Array (QIAGEN Inc).

255

As shown in Figure 4D, NE treatment substantially reduced expression of several

256

pro-inflammatory factors, including Il17a, Il17f, Il1a, Il1b, Il12b, Il21, Il23a, Il25

257

(Il17e), Il27, Il6, Ifng, Csf2 (GM-CSF), Csf3 (G-CSF), Tgfb1, and Tnf, while it

258

induced expression of some anti-inflammatory cytokines/neurotrophins such as Il10

259

and Lif. Consistent with these findings, expression of most chemokines we tested,

260

including Ccl1, Ccl2, Ccl20, Ccl7, Cx3cl1, Cxcl1, Cxcl10, Cxcl11, Cxcl12, and Cxcl5,

261

was significantly decreased (Figure 4D). Among them, the most robustly inhibited 13



262

was the IL-17 family (Figure 4D), the essential cytokine produced by T helper cells

263

(Th17 cells), which have strong pro-inflammation effects in the pathogenesis of EAE

264

and MS (28).

265

266

NE inhibited Th1 and Th17 cell subsets though the Janus kinase/STAT (Signal

267

transducer and activator of transcription) signaling pathway. To study the effect

268

of orally administered NE on the immune response, expression of surface markers

269

and/or cytokines in mononuclear cells from the peripheral and CNS of different EAE

270

groups were investigated by flow cytometry. The percentages and absolute numbers

271

of CD4 and CD8 positive T cells, both in the spleen and CNS of NE treatment group,

272

were decreased significantly (P < 0.05). Compared with PBS-treated control,

273

percentages of MOG-reactive Th1 (CD4+IFN-γ+) and Th17 (CD4+IL-17+) cells were

274

remarkably reduced by NE treatment in both peripheral and CNS (Figure 5A&B).

275

Nevertheless, the portions of Th2 (CD4+IL4+) and Treg (CD4+Foxp3+) cells were not

276

significantly changed (data not shown). When stimulated ex vivo, splenocytes of

277

NE-treated EAE mice produced less MOG-induced IFN-γ, IL-17 and GM-CSF in the

278

supernatant of splenic culture, while there was no effect on Th2/Treg cytokines IL-5

279

and IL-10 (Figure 5C). These results indicated that NE ameliorates clinical symptom

280

by suppressing Th1 and Th17 cells development, and perhaps by inducing

281

immunoregulatory cytokines production.

282

Typically, an important limitation in the use of small molecule compounds for

283

therapy is that their mechanism of action is not fully understood, a factor that adds to 14


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our misgivings about their use in clinical treatment. While our data suggest that NE

285

suppresses development of EAE by inhibiting pro-inflammatory factor secretion by

286

Th1 and Th17 cells (Figure 4D), how NE action in the differentiation of T cell subsets

287

has not yet been clarified. As the JAK/STAT signaling plays an important role in this

288

process (29), here we hypothesized that NE targeting JAK/STAT pathway to exert its

289

regulatory effects on Th1/Th17 development. Therefore, protein samples were

290

obtained from splenic CD4+ T cells purified from NE-treated or control EAE mice,

291

and key signaling molecules of the Janus Kinase/STAT pathway were evaluated by

292

immunoblot. STAT1

293

related to the Th1/Th17 cells polarization (28, 30). As shown in Figure 5D, p-STAT1

294

and p-STAT3 were significantly decreased in NE-treated group. Conversely, the level

295

of STAT5 and STAT6, which are associated to Treg and Th2 cell differentiation (31,

296

32), were not changed (data not shown). To further access whether the NE were

297

functional directed toward Th1 or Th17 cell differentiation, CD4+ T cells were

298

purified from NE- or PBS-treated EAE mice, then assayed by JAK/STAT signaling

299

pathway array (QIAGEN Inc). Among all the genes significantly influenced by NE

300

treatment, 5 were upregulated and 25 down-regulated significantly (p

Nutshell Extracts of

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