Characterization and Immunomodulatory Activity of a Novel Peptide

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Article

Characterization and Immunomodulatory Activity of a Novel Peptide, ECFSTA, from Wheat Germ Globulin Wenjia Wu, Mengmeng Zhang, Yao Ren, Xinze Cai, Zhina Yin, Xiaoai Zhang, Tian Min, and Hui Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01360 • Publication Date (Web): 21 Jun 2017 Downloaded from http://pubs.acs.org on June 24, 2017

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Journal of Agricultural and Food Chemistry

1

Characterization and Immunomodulatory Activity of a Novel

2

Peptide, ECFSTA, from Wheat Germ Globulin

3

Wenjia Wu†, Mengmeg Zhang†, Yao Ren§, Xinze Cai†, Zhina Yin†, Xiaoai

4

Zhang‡, Tian Min†, Hui Wu*,†

5

†

6

Guangzhou 510640, China

7

§

8

Chengdu 610065, China

9

‡

10

College of Food Sciences and Engineering, South China University of Technology,

College of Light Industry, Textile and Food Engineering, Sichuan University,

Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China

ABSTRACT

11

A novel peptide was extracted from wheat germ globulin and purified using

12

ion-exchange chromatography, gel filtration chromatography and semi-preparative

13

reverse phase-high performance liquid chromatography (RP-HPLC). The sequence of

14

the

15

immunomodulatory effects were evaluated, and the results showed that ECFSTA

16

could enhance phagocytosis of RAW 264.7 cells and significantly increase their

17

secretion of nitric oxide (NO), interleukins-6 (IL-6), tumor necrosis factors-α (TNF-α),

18

and reactive oxygen species (ROS). ECFSTA activated macrophages mainly through

19

the pattern-recognition receptors (PRRs) of toll-like receptor 2 (TLR2) and TLR4, and

20

the production of ROS simultaneously stimulating macrophages to produce TNF-α.

21

Thus, ECFSTA could be used as an immunomodulator and might be a promising

22

component of functional foods.

peptide

was

found

to

be

Glu-Cys-Phe-Ser-Thr-Ala

ACS Paragon Plus Environment

(ECFSTA).

Its


23

KEYWORDS: wheat germ globulin; peptide sequence; immunomodulatory activity;

24

signal transduction pathways

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44


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INTRODUCTION

46

Wheat germ is a by-product of the flour milling industry. Although it comprises

47

about 2.5 %-3.8 % of the total seed weight, it contains significant quantities of fat,

48

proteins, vitamins, and other bioactive compounds.1 Defatted wheat germ, the

49

by-product of wheat germ oil extraction, has been reported to be rich in proteins,2 and

50

several studies have indicated that wheat germ proteins have pharmacological

51

properties of antioxidant, antihypertension, calcium binding and immunomodulation.2,

52

3

53

have a well-balanced amino acid composition.4 Recently, many studies have focused

54

on albumin; however few studies have been performed on globulin, especially its

55

immunomodulatory activity.

Albumin and globulin are composed more than half of the wheat germ protein, and

56

The immune system comprises innate and adaptive immunity. The former is

57

considered the first line of defence against invasion. Macrophages, one of the most

58

important immunocytes, performs immune functions in both innate and adaptive

59

immunity.5 Macrophages are involved in many immune functions, including

60

phagocytosis

61

oxygen-dependent and oxygen-independent pathways, production of cytokines for

62

inflammation and presentation of antigens.6 The activation of macrophages is

63

mediated by pattern-recognition receptors (PRRs), and PRRs related to peptides

64

include scavenger receptors (SR), toll-like receptors (TLRs) and complement receptor

65

(CR). Through the stimulation of PRRs, nitric oxide (NO), reactive oxygen species

66

(ROS), and pro-inflammatory cytokines (such as interleukins [IL] and tumor necrosis

of

invaders,

elimination

and

killing


of

pathogens

through


67

factors[TNF]) are produced. 7

68

In our previous study, we have shown that alcalase hydrolysate of wheat germ

69

globulin had the strongest immunomodulatory activity;8 however the sequence of the

70

immunomodulatory peptide and the mechanism of its immunomodulatory effect are

71

still unknown. In the present study, alcalase hydrolysate was purified and

72

characterized, its immunomodulatory activity on RAW 264.7 cells was evaluated by

73

determining its capacity for phagocytosis; its production of NO, IL-6, TNF-α, and

74

ROS was analysed; and its PRRs on RAW 264.7 cells were investigated. The results

75

of this study are expected to provide beneficial information for further studies on

76

immunomodulatory peptides.

77

MATERIALS AND METHODS

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Materials and Chemicals. Defatted wheat germ flour was provided by

79

Mantianxue Co., Ltd. (Anyang, China). Alcalase (200 u/mg) was purchased from

80

Novozymes (Bagsvaerd, Denmark). Diethylaminoethyl (DEAE)-Sephrose Fast Flow

81

(FF) and Sephadex G-15 were purchased from GE Healthcare Bio-Sciences (Uppsala,

82

Sweden). Acetonitrile and trifluoroacetic acid (HPLC grade) were purchased from

83

Tedia Company (Fairfield OH, USA). RAW 264.7 murine macrophage cells were

84

gifted to us by the Traditional Chinese Medicine Hospital of Guangdong (Guangzhou,

85

China). Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum (FCS), and

86

penicillin-streptomycin were purchased from Gibco Life Technologies (Grand Island,

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NY, USA). Lipopolysaccharide (LPS), neutral red and N-acetyl-L-cysteine (NAC)

88

were purchased from Sigma Company (St. Louis, MO, USA). Vybrant Phagocytosis


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Assay Kit was purchased from Molecular Probes (Carlsbad, USA). The Griess

90

reagent

91

Enzyme-linked immunosorbent assay (ELISA) kits to detect mouse IL-6 and TNF-α

92

were purchased from Neobioscience Technology (Shenzhen, China). The ROS Assay

93

Kit was purchased from Solarbio Life Sciences (Beijing, China). Trizol was

94

purchased from Invitrogen Life Technologies (Carlsbad, USA). The First strand

95

cDNA Synthesis Kit and the Fast Start Universal SYBR Green Master (ROX) were

96

purchased from Roche Life Science (Basel, Switzerland). Primers for the inductive

97

nitric oxide synthase (iNOS), IL-6, TNF-α, Fa nicotinamide adenine dinucleotide

98

phosphate (FaNADPH), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

99

genes were synthesized at Sangon Biotech (Shanghai, China). Anti-TLR2 and

100

anti-TLR4, anti-SR and anti-CR3 were purchased from Abcam (Cambridge, UK). All

101

other reagents and chemicals used were of analytical grade.

was

purchased

from

Beyotime

Biotechnology

(Shanghai,

China).

102

Extraction of Globulin from Defatted Wheat Germ and Preparation of

103

Globulin Hydrolysate. Wheat germ globulin and its alcalase hydrolysate (AH) were

104

prepared based on the protocol described by Wu, et al,8 the protein content, as

105

determined by the micro-Kjeldahl method was 85.26 ± 2.73 % and the degree of

106

hydrolysate, as measured by the pH-stat method was 19.09 ± 0.09 %.

107

Ion-exchange Chromatography. A total of 50 mg AH was dissolved in 10 mL of

108

Milli Q water, and loaded onto an ion-exchange column (2.6 cm × 15 cm) packed

109

with pre-equilibrated DEAE Sepharose FF. Milli Q water, NaCl with differing

110

concentrations (0.05 M, 0.1 M, 0.3 M, and 0.5 M) was successively passed through



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the column at a flow rate of 1 mL/min. The effluent was monitored using an

112

ultraviolet-visible spectrophotometer (UV 752s, Lingguang, Shanghai, China) at 220

113

nm. The fractions with peak UV absorption were collected and dialysis was

114

performed against Milli Q water for 72 h. The samples were then freeze-dried

115

(Alpha-4, Martin Christ, Osterode, Germany) for the immunomodulating assay. The

116

fraction with the highest immunomodulatory activity was further purified.

117

Gel Filtration Chromatography. The pretreated Sephadex G-15 was packed into

118

a chromatography column (1.6 cm × 60 cm) and equilibrated using Milli Q water at a

119

flow rate of 0.4 mL/min overnight. Ten milligrams of the fraction obtained from

120

ion-exchange chromatography was dissolved in 1 mL of Milli Q water, and loaded

121

onto the gel filtration chromatography column. The eluent was obtained at a flow rate

122

of 0.4 mL/min with Milli Q water, and its absorbance was measured at 220 nm. The

123

fractions

124

immunomodulatory evaluation. The fraction with the highest immunomodulatory

125

activity was purified further.

with

peak

absorbance

were

collected

and

freeze-dried

for

126

Semi-preparative Reverse Phase-high Performance Liquid Chromatography

127

(RP-HPLC). The fraction described above was adjusted to 20 mg/mL with Milli Q

128

water and applied to a semi-preparative HPLC system (GX-281, Gilson,

129

Villiers-le-Bel, France) on a C18 column (300 SB, Agilent, Palo Alto, USA). The

130

mobile phase was 15 % acetonitrile solution containing 0.1 % trifluoroacetic acid, the

131

injection volume was 200 µL, and flow rate was 0.5 mL/min.

132

Identification of Immunomodulating Peptides by Matrix-assisted Laser


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Desorption/ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS).

134

The fractions obtained from the semi-preparative RP-HPLC were identified by

135

MALDI-TOF-MS. One microliter of the prepared fraction (2 mg/ml) was mixed with

136

excess matrix solution and dried on a MALDI plate. The plate was loaded onto the

137

sample chamber of the MALDI-TOF-MS spectrometer (Autoflex III Smabean, Bruker,

138

Karlsruhe, Germany) and the spectra were obtained in the positive reflector mode.

139

After the primary MS spectra were obtained, the fragment with the highest response

140

signal was lifted to obtain the second MS spectra. The sequence of the peptide was

141

analyzed using Bio Tools (version 3.0, Bruker, Karlsruhe, Germany) and manual

142

interpretation.

143

Culture of RAW 264.7 Cells. The cryopreserved cells were rapidly thawed in a 37

144

°C water bath and transferred into a 15-mL centrifuge tube with a large volume of the

145

medium (DMEM with 10 % FCS and 1 % penicillin-streptomycin). The cells were

146

centrifuged at 1200 g for 10 min. The supernatant was removed, and after the new

147

medium was added, the cells were fully blended, transferred into a petri dish, and

148

cultured in a 37 °C incubator with 5 % CO2.

149

Viability of RAW 264.7 Cells. The cells in logarithmic growth phase were

150

adjusted to a concentration of 5 × 105 cells/mL, seeded into a 96-well plate (100 µL in

151

each well), and cultured for 24 h. The medium was then removed, and the cells were

152

treated with 100 µL of each fraction from DEAE Sepharose FF and Sephadex G-15

153

(10, 20, 40, 80 and 160 µg/mL, dissolved in DMEM). DMEM was used as the

154

negative control. After a 24-h incubation, 10 µL of MTS was added to each well and



155

incubated for 1 h. The absorbance of the wells was measured at 490 nm using a

156

microplate absorbance reader (Infinite M1000 Pro, Tecan, Männedorf, Switzerland).

157

The viability rate was calculated as follows: OD value of sample treated cells - OD value of blank wells ×100 OD value of negative control cells - OD value of blank wells

158

Cell viability rate (%) =

159

Phagocytosis of Neutral Red Assay, Measurement of NO and Cytokines. The

160

cells in the logarithmic growth phase were seeded into a 96-well plates at a

161

concentration of 105 cells/well (100 µL per well), and continuously cultured for 24 h.

162

The medium was discarded and the cells were treated with fractions from DEAE

163

Sepharose FF and Sephadex G-15 at different concentrations (10, 20, 40, 80 and 160

164

µg/mL) for another 24 h. LPS (25 µg/mL, dissolved in DMEM) and DMEM were

165

used as positive and negative controls, respectively. The supernatant was collected,

166

and the level of NO was detected using the Griess reagent and measured at 540 nm.

167

The levels of IL-6 and TNF-α were detected by ELISA kits, a certain amount of

168

supernatant was added to the wells which coated with mouse IL-6 or TNF-α, after

169

incubated at 36 °C for 90 min, the wells were washed with wash buffer. Then 100 µL

170

of biotinylated anti-IL-6 or anti-TNF-a solution, streptavidin–horseradish peroxidase

171

(HRP), stabilised chromogen were successively added to the wells, incubated at 36 °C

172

for 60, 30 and 15 min, respectively, however, the washing operation was also needed

173

except stabilised chromogen procedure. Finally, 100 µL of stop solution was added

174

and measured at 450 nm immediately.

175

The cells on the bottom of the wells were treated with 0.075 % neutral red (100 µL

176

for each well), and incubated for 1 h. They were then washed thrice with phosphate


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buffer saline (PBS) and lysed with a mixture of ethanol and 1 M acetic acid (v/v= 1:1)

178

for 3 h at 25 °C. The absorbance of the wells was measured at 540 nm. The

179

phagocytosis rate was calculated as follows: OD value of sample treated cells - OD value of blank wells × 100 OD value of negative control cells - OD value of blank wells

180

Phagocytosis rate (%) =

181

Determination of ROS Formation. As described in a previous section, after the

182

cells were treated with purified fractions at 80 µg/mL, LPS (25 µg/mL) and DMEM

183

for 24 h, the supernatant was discarded. The cells were then treated with 100 µL of

184

2,7-dichlorodi-hydrofluorescein diacetate (DCFH-DA, 10µM) per well, and incubated

185

at 37 °C for 30 min in the dark. The supernatant was removed and the cells were

186

washed twice and then suspended with PBS. The fluorescence intensity was then

187

assessed using a microplate absorbance reader, with excitation and emission

188

wavelengths set at 485 nm and 530 nm, respectively.

189

Phagocytosis of fluorescein isothiocyanate (FITC)-labeled Escherichia. coli

190

(E.coli) Assay. The cells at the logarithmic growth phase were adjusted to 106

191

cells/mL, and seeded into 6-well plates (1.5 mL per well). After 24 h of culture, the

192

cells were stimulated with fraction obtained from RP-HPLC at 20, 40, and 80 µg/mL;

193

LPS (25 µg/mL) and DMEM were also added as positive and negative controls,

194

respectively. After 6 h of stimulation in a 37 °C incubator with 5 % CO2, the

195

supernatant was removed. FITC-labeled E. coli solution (1 mL) was added to each

196

well and continuously incubated for 30 min in the dark. After removing the

197

supernatant, the cells in each well were treated with 2 mL of pre-cooled PBS (4 °C)

198

for 10 min to stop phagocytosis and then washed thrice with PBS. The cells were



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suspended in PBS before the fluorescence intensity was measured using a flow

200

cytometer (FACSAria II, BD company, New York, USA). A fluorescence microscope

201

(Axiovert 200, Carl Zeiss, Oberkochen, Germany) was used to observe the phagocytic

202

effect after the washed cells were immobilized with 4 % paraformaldehyde.

203

Quantitative Polymerase Chain Reaction (QPCR) Analysis. As described

204

previously, after the cells were treated by fraction obtained from RP-HPLC (20, 40

205

and 80 µg/mL), LPS (25 µg/mL) and DMEM for 12 h, they were lysed with Trizol.

206

RNA was then extracted with the sequence of chloroform, isopropanol and 75 %

207

ethanol. After drying, the cells were dissolved in diethyl pyrocarbonate (DEPC) water.

208

RNA was transcribed to DNA using the First strand cDNA Synthesis Kit. The cDNA

209

encoding the genes for iNOs, IL-6, TNF-α, and FaNADPH were quantified by QPCR.

210

GAPDH was used as an internal reference. The primers for the different genes were as

211

follows:

212

5′-TGGTCAAACTCTTGGGGTTC-3′

213

5′-AGTTGCCTTCTTGGGACTGA-3′

214

5′-CAGAATTGCCATTGCACAAC-3′

215

5′-AGCCCCCAGTCTGTATCCTT-3′

216

5′-CATTCGAGGCTCCAGTGAAT-3′

217

5′-AATGAGGTGGCTGAGATGGA-3′

218

5′-TGGCATGGTGGAGATTCTGA-3′

219

5′-GAGTCAACGGATTTGGTCGT-3′

220

5′-GACAAGCTTCCCGTTCTCAG-3′ for

forward

5′-TTCCAGAATCCCTGGACAAG-3′ for

iNOS; and

for and

TNF-a;

FaNADPH; and

GAPDH.


forward

forward reverse

and for

reverse

reverse IL-6;

for

and

forward reverse forward reverse

Page 11 of 29


221

Investigation of Proposed Signal Transduction Pathways in RAW 264.7 Cells

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Activated by P3-1-1. After the cells were seeded into 96-well plates and incubated

223

for 24 h, the medium was discarded and 100 µL of antibodies (5 µg/mL) against

224

membrane receptors (including anti-TLR2, anti-TLR4, anti-SR and anti-CR3) and

225

NAC were added for 2 h before stimulation with fraction obtained from RP-HPLC

226

(80 µg/mL). The cells treated with only fraction obtained from RP-HPLC (80 µg/mL),

227

DMEM and LPS were used as control groups. After 24 h of incubation, the

228

supernatant was collected and the production of NO, IL-6, and TNF-α was assessed.

229

The formation of ROS was also measured.

230

Statistical Analysis. All data were expressed as mean ± standard deviation from at

231

least three independent experiments. Statistical analyses were performed using SPSS

232

19.0 software, and statistical significance was determined using one-way analysis of

233

variance. Differences were considered significant when p < 0.05.

234

RESULTS AND DISCUSSION

235

Purification and Identification of the Immunomodulatory Peptide from Wheat

236

Germ Globulin. Ion-exchange Chromatography and Immunomodulatory Evaluation.

237

Ion-exchange chromatography could separate samples according to their charges,

238

DEAE-sepharose FF is a weak anion-exchanger, when the hydrolysate was bonded to

239

the exchanger, fractions with positive charges could be eluted first. From this

240

procedure, many impurities and non-target products could be removed. The

241

ion-exchange chromatogram is shown in Figure 1A; four fractions (P1, P2, P3 and P4)

242

were obtained. The toxic effects of P1, P2, P3 and P4 on RAW 264.7 cells were



243

investigated. The survival rate of cells treated with the four fractions at concentrations

244

of 10, 20, 40, 80 and 160 µg/mL were all above 95 %, which indicated that the four

245

fractions were non-toxic under 160 µg/mL. However, P2 and P4 were toxic to the

246

cells at 320 µg/mL (Figure 1B); therefore the cells treated with 320 µg/mL of fractions

247

were abandoned in the following immunomodulatory evaluation. The effects of P1,

248

P2, P3, and P4 on phagocytosis of neutral red and their production of NO, IL-6,

249

TNF-α, and ROS are shown in Figure 1C-G. The results showed that P3 had the

250

strongest ability for phagocytosis of neutral red and for production of NO, IL-6, and

251

TNF-α at various concentrations; P3 also produced the highest amount of ROS at all

252

the time point studied at 80 µg/mL. Overall, P3 displayed the strongest

253

immunomodulatory effects on the phagocytosis rate (143.53 ± 4.10 %), NO

254

production (38.52 ± 1.09 µM) at 80 µg/mL, on IL-6 production (1483.04 ± 72.15

255

pg/mL) and TNF-α production (38.95 ± 1.11 ng/mL) at 160 µg/mL, on ROS

256

production (4.64 ± 0.06) at 48 min. Thus, P3 was selected for further purification

257

through sephadex G-15 filtration.

258

Sephadex G-15 Filtration Chromatography and Immunomodulatory Evaluation.

259

Gel filtration chromatography separates samples due to the molecular weight, and the

260

fractions with high molecular weight could be eluted first. As shown in Figure 2A,

261

two fractions, P3-1 and P3-2 were obtained. As shown in Figure 2B, P3-1 was

262

non-toxic to RAW 264.7 cells at concentrations from 10 to 320 µg/mL, while P3-2

263

was toxic to the cells at 320 µg/mL. The results of the immunomodulatory analysis

264

(Figure 2C-G) indicated that the two fractions at 20, 40, 80 and 160 µg/mL showed


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significantly higher (p < 0.05) immunomodulation than the blank control for

266

phagocytosis rate and for production of NO, IL-6 and TNF-α. P3-1 showed

267

significantly higher (p < 0.05) ability in most cases. The highest immunomodulatory

268

effects were observed for phagocytosis rate (141.95 ± 4.39 %), NO production (24.72

269

± 1.04 µM) of P3-1, for IL-6 production (833.47 ± 2.12 pg/mL) and TNF-α

270

production (20.42 ± 0.85 ng/mL) at 160 µg/mL of P3-1, and for ROS production (5.63

271

± 0.02) at 48 min. Thus, P3-1 was selected for RP-HPLC purification.

272

Semi-preparative RP-HPLC Purification and MALDI-TOF-MS Identification. P3-1

273

was further purified on a 300 SB C18 column in a semi-preparative RP-HPLC system.

274

Only one peak, P3-1-1, was obtained (Figure 3A). It was isolated and identified by

275

MALDI-TOF-MS. The primary and secondary MS spectra are shown in Figure 3B

276

and 3C, respectively. The molecular weight of the peptide was found to be 656Da and

277

the amino acid sequence was as follows: Glu-Cys-Phe-Ser-Thr-Ala (ECFSTA). This

278

novel peptide has never been reported before and was used for further

279

immunomodulatory evaluation.

280

When a body is infected by pathogens, macrophages first perform phagocytosis,

281

followed by production of NO and ROS (oxygen-dependent pathways) to kill

282

pathogens; cytokines (such as IL-6, TNF-α) are simultaneously secreted to participate

283

in the inflammation reaction. NO, ROS and cytokines are associated with PRRs. In

284

addition, ROS, apart from being cytotoxic to kill pathogens, are also involved in cell

285

signaling and regulation.9, 10 Many immunomodulating peptides have been reported in

286

recent years. A peptide isolated from the Alaska Pollock frame was purified by SP



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287

-Sephadex C-25 ion exchange column, Sephadex G-25, and semi-preparative

288

RP-HPLC; two peptides were obtained, their molecular weights were 584 Da and 470

289

Da,

290

Asn-Gly-Leu-Ala-Pro, respectively. They were both reported to possess high

291

lymphocyte proliferation activity (35.92 % and 32.96 %).11 A casein-derived peptide

292

Gln-Glu-Pro-Val-Leu and its digested product, Gln-Glu-Pro-Val, were reported to

293

significantly improve lymphocyte proliferation (by 1.47 and 1.41, respectively) and

294

cAMP production (by 2.04 and 4.26, respectively).12 Oral administration of

295

shark-derived protein hydrolysate (SPH) was shown to enhance the production of IgA

296

producing cells, IL-6, and TNF-α in the mice gut barrier.13 Another peptide

297

(Thr-Gly-Ala-Asp-Tyr) from Alaska Pollock hydrolysate was shown to enhance

298

humoral, cellular, and non-specific immunity in immunosuppressed mice; the

299

production of IL-2, IL-4, and IL-6 were found to be significantly increased.14 The

300

hydrophobic value was reported to correlation with immunomodulatory activity,8, 15

301

which may increase the interaction of peptide and cytomembrane in order to improve

302

the immunomodulation.16 In addition, rich in basic amino acids or hydrophobic amino

303

acid in terminals were also related to immunomodulatory activity.16, 17 In this study,

304

ECFSTA purified from alcalase hydrolysate had three hydrophobic amino acids (Phe,

305

Thr and Ala), and Ala was in carboxyl terminal, these features may contribute to the

306

immunomodulatory activity.

and

their

amino

acid

sequences

were

Asn-Gly-Met-Thr-Thr

and

307

Immunomodulatory Mechanism of P3-1-1. Phagocytosis of FITC-labeled E. coli.

308

To study the effect of P3-1-1 on phagocytosis of pathogens similar to those seen in


Page 15 of 29


309

infections, FITC-labeled E.coli were introduced and the capacity of RAW 264.7 cells

310

treated with P3-1-1 for phagocytosis was analyzed using a fluorescence microscope

311

and a flow cytometer. As seen in Figure 4A, the fluorescence intensity of cells treated

312

with P3-1-1 was dose-dependent and obviously higher than that of the blank controls.

313

The results of the flow cytometry (Figure 4B) revealed that the fluorescence intensity

314

of the treated cells was higher than that of the blank control; furthermore, as the

315

concentration of P3-1-1 increased, the intensity of fluorescence became stronger.

316

However, the flow cytometry result about LPS was not quite satisfied, since the

317

fluorescence intensity only slightly higher than the blank control; however, both the

318

neutral red and fluorescence microscope results indicated that the effect of LPS on

319

phagocytosis was not quite strong when compared to our peptide fractions. These

320

results showed that P3-1-1 could distinctly enhance the capacity of macrophages for

321

phagocytosis.

322

Effects on mRNA Expression of iNOS, IL-6, TNF-α, and FaNADPH. Figure 5A-D

323

show the mRNA expressions levels of iNOS, IL-6, TNF-α, and FaNADPH measured

324

using QPCR and illustrate the production of NO, IL-6, TNF-α, and ROS at the gene

325

level. These results showed that the mRNA expression levels of iNOS, IL-6, TNF-α,

326

and FaNADPH in the treated cells were significantly higher (p < 0.05) than those in

327

the blank controls at 20, 40 and 80 µg/mL. All mRNA levels showed dose-dependent

328

variations.

329

Proposed Signal Transduction Pathways in RAW 264.7 Cells Activated by P3-1-1.

330

In order to investigate the immunomodulatory effect of P3-1-1 on PRRs and ROS



331

production pathways of macrophages, the cells were pre-treated with the antibodies

332

against receptors (anti-TLR-4, anti-TLR2, anti-SR, and anti-CR3) and ROS inhibitor

333

NAC. The results are shown in Figure 6A-D. Cells that were pre-treated with

334

anti-TLR4 and anti-TLR2 showed significantly lower (p < 0.05) production of NO

335

than the cells that were only treated with P3-1-1. The highest decrease in NO was

336

observed after anti-TLR2 pre-treatment, although the decrease was insignificant after

337

pre-treatment with anti-TLR4 (Figure 6A). Unlike NO, the decrease in IL-6 and

338

TNF-α after pre-treatment with antibodies and NAC were significantly lower (p

Characterization and Immunomodulatory Activity of a Novel Peptide

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