Small Molecular Weight Soybean Protein-Derived Peptides Nutriment

Subscriber access provided by Kaohsiung Medical University

Article

Small molecular weight soybean protein-derived peptides nutriment attenuates rat burn injury-induced muscle atrophy by modulation of ubiquitin-proteasome system and autophagy signaling pathway Fen Zhao, Yonghui Yu, Wei Liu, Jian Zhang, Xinqi Liu, Huinan Yin, and Liuling Ying J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05387 • Publication Date (Web): 01 Mar 2018 Downloaded from http://pubs.acs.org on March 2, 2018

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

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 34

Journal of Agricultural and Food Chemistry

1

TITLE

2

Small molecular weight soybean protein-derived peptides nutriment attenuates rat burn injury-induced

3

muscle atrophy by modulation of ubiquitin-proteasome system and autophagy signaling pathway

4 5

AUTHORS

6

Fen Zhao†, Yonghui Yu*‡, Wei Liu‡, Jian Zhang†, Xinqi Liu*†, Lingying Liu‡, Huinan Yin‡

7

†

8

Technology Research Center of Food Additives, Beijing Technology and Business University (BTBU),

9

Beijing 100048, China

10

‡

11

*

Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Engineering and

Burn Institute, the First Affiliated Hospital of PLA General Hospital, Beijing 100048, China

These two authors contributed equally to this work and were both corresponding authors.

12 13

ABSTRACT

14

This article describes results of the effect of dietary supplementation with small molecular weight

15

soybean protein-derived peptides on major rat burn injury-induced muscle atrophy. As protein nutrients

16

have been previously implicated to play an important role in improving burn injury outcomes, optimized

17

more readily absorbed small molecular weight soybean protein-derived peptides were evaluated. Thus,

18

the quantity, SDS-PAGE patterns, molecular weight distribution, and composition of amino acids of the

19

prepared peptides were analyzed, and a major full-thickness 30% total body surface area (TBSA) burn-

20

injury rat model was utilized to assess the impact of supplementation with soybean protein-derived

21

peptides on initial systemic inflammatory responses as measured by interferon-gamma (IFN-γ),

22

chemokine (C-C motif) ligand 2 (CCL2, also known as MCP-1), chemokine (C-C motif) ligand 7 (CCL7,

23

also known as MCP-3) and generation of muscle atrophy as measured by tibialis anterior muscle (TAM)

24

weight relative to total body weight. Induction of burn injury-induced muscle atrophy ubiquitin-

25

proteasome system (UPS) signaling pathways in effected muscle tissues were determined by Western blot

26

protein expression measurements of E3 ubiquitin-protein ligase TRIM-63 (TRIM63, also known as

ACS Paragon Plus Environment


27

MuRF1) and F-box only protein 32 (FBXO32, also known as atrogin-1 or MAFbx). In addition, induction

28

of burn injury-induced autophagy signaling pathways associated with muscle atrophy in effected muscle

29

tissues were assessed by immunohistochemical analysis as measured by microtubule-associated proteins 1

30

light chain 3 (MAP1LC3, or commonly abbreviated as LC3) and beclin-1 (BECN1) expression, as well as

31

relative induction of cytoplasmic-liberated form of MAP1LC3 (LC3-I) and phagophore and

32

autophagosome membrane-bound form of MAP1LC3 (LC3-II), and BECN1 protein expression by

33

Western blot analysis. Nutrient supplementation with small molecular weight soybean protein-derived

34

peptides resulted a significant reduction in burn injury-induced inflammatory markers, muscle atrophy,

35

induction of TRIM63 and FBXO32 muscle atrophy signaling pathways and induction of autophagy

36

signaling pathways LC3 and BECN1 associated with muscle atrophy. These results implicated that small

37

molecular weight soybean-derived peptides dietary supplementation could be used as an adjunct therapy

38

in burn injury management to reduce the development or severity of muscle atrophy for improved burn

39

patient outcomes.

40 41

KEYWORDS

42

soybean protein-derived peptides, burn, muscle atrophy, ubiquitin-proteasome system, autophagy

43 44

ABBREVIATIONS

45

TBSA, total body surface area; IL-1β, interleukin-1 beta; IFN-γ, interferon-gamma; CCL2 (also known as

46

MCP-1), chemokine (C-C motif) ligand 2; CCL7 (also known as MCP-3), chemokine (C-C motif)

47

ligand 7; TAM, tibialis anterior muscle; UPS, ubiquitin-proteasome system; TRIM63, tripartite motif-

48

containing 63; FBXO32, F-box only protein 32; MAP1LC3 (or commonly abbreviated as LC3),

49

microtubule-associated proteins 1 light chain 3; BECN1, beclin-1; LC3-I, cytoplasmic-liberated form of

50

MAP1LC3; LC3-II, phagophore and autophagosome membrane-bound form of MAP1LC3; β-Actin,

51

beta-actin; TNF-α, tumor necrosis factor-alpha; IL-6, interleukin-6; BW, body weight; SPI, soy protein

52

isolate; SDS-PAGE, sodium dodecyl sulfate polyacrylamide-gel electrophoresis; GFC, gel filtration


Page 2 of 34

Page 3 of 34


53

chromatography; HAA, hydrophobic amino acids; BCAA, branched-chain amino acids; AAA, aromatic

54

amino acids.

55 56

INTRODUCTION

57

Severe non-fatal burn injuries are a prevalent and burdensome global health problem with a reported

58

frequency of annual thermal burn injuries alone of 67 million.1 Burn injuries are one of the most

59

expensive conditions to treat2 as they usually involve prolonged hospital stays, require multiple surgical

60

treatments and specialized care along with lengthy rehabilitation treatments.3 Furthermore, severe major

61

burn injuries often result in life long disabilities and loss of productivity, in 2013 estimated to represent

62

1.2 million years lived with disability and 12.3 million disability adjusted life years.4

63

Burn injury-induced muscle atrophy is a considerable problem impacting functional recovery, morbidity

64

and disability of severe burn-injured patients and is associated with poorer prognosis and quality of life.5

65

The development of muscle atrophy in burn patients has been associated with prolonged hospitalization

66

and delayed wound healing6 and can last for years after burn wounds have healed.7 In addition, the

67

development of muscle atrophy is a positive risk factor for death, increasing the chance that death will

68

result from an underlying condition.8 Thus, developing enhanced burn injury management interventions

69

that attenuate the development, progression or severity of muscle atrophy represents an opportunity to

70

improve functional recovery, reduce burden of care, morbidity and mortality of severe burn-injured

71

patients and improve global health care.

72

Muscle atrophy is wasting of normal mature muscle tissue to that of a reduced size. It can be considered

73

pathological when the normal homeostatic balance of tissue degradation and synthesis is altered to favor

74

protein degradation over protein synthesis pathways.9 Muscle atrophy in burn patients is complex and

75

cannot merely be attributed to disuse of muscles related to inactivity under conditions of bedrest

76

confinement. Rather, systemic induction of a hypermetabolic state,10 involving the activation of pro-

77

inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ),

78

interleukin-6 (IL-6) and interleukin-1 beta (IL-1β) contributes to the development of muscle atrophy in



79

burn patients.11 Activation of inflammatory cytokines mediate the induction of proteolysis and catabolism

80

of myofibrillar proteins through activation of the ubiquitin-proteasome system (UPS) protein degradation

81

pathways12-15 and autophagy16-19 producing skeletal muscle atrophy. Clinical manifestations include

82

weakness, fatigue, rapid loss of skeletal muscle mass and body weight and is associated with the

83

development of anemia, hyperglycemia, increased heart rate, heartbeat irregularities and poor immune

84

function.7

85

Even though muscle atrophy poses a significant clinical problem, due to the complexity of muscle atrophy,

86

currently there are limited rationally designed therapies in this area. Current therapeutics include anabolic

87

proteins such as growth hormones, insulin, insulin growth factor-1, insulin growth factor binding protein;

88

anabolic steroids such as oxandrolone or testosterone; and anti-catabolic agents such as adrenergic

89

antagonist propranolol or metoprolol, and these agents appear to be effective only for patients who are

90

catabolic.20 Furthermore, making new innovative therapeutic treatment options clinically available has

91

become largely impractical with the requirement for tremendous investment of financial resources to

92

comply with regulatory agency approval requirements, the additional demand of long time periods to

93

complete studies, prepare applications and processing yielding limited success rates making investments

94

in doing so profoundly unattractive.5 Thus, the development of new more practical and cost-effective

95

approaches to improve patient outcomes and quality of life is required.

96

As evidence continues to mount regarding various bioactivities and physiological effects of constituents

97

derived from food, the use of optimized functional nutrients represents an attractive intervention to aid in

98

the management of various disease states. Particularly, muscle atrophy is a condition resulting from a lack

99

of adequate protein nutrients to supply the body’s increased demand and thus optimized high-quality

100

protein nutriment may provide adequate treatment and reduce the need for additional pharmacologic

101

interventions associated with increased cost, risk of side effects and adverse interactions and reactions.

102

Soybean proteins and derivatives thereof, have been shown to have anti-hypertensive, immunomodulatory,

103

neuroactive, antimicrobial, mineral and hormonal regulating bioactivities.21-24 However, few studies have

104

been reported on the effects of soybean proteins in burn-induced muscle atrophy. Even more, soybean


Page 4 of 34

Page 5 of 34


105

protein-derived peptides, obtained by enzymatic hydrolysis soybean protein and further separated to yield

106

more easily absorbed small molecular weight peptides, have greatly enhanced the utilization value of

107

soybean protein25 and represent a novel interventional option in the management of burn injury-induced

108

muscle atrophy. Thus, we have investigated the hypothesis that small molecular weight soybean protein-

109

derived peptides can attenuate burn-induced muscle atrophy.

110

To test this hypothesis, a rat model of major full-thickness burn 30% TBSA injury11 with severity

111

classification in accordance with the American Burn Association, was employed. Four experimental

112

groups were examined including sham uninjured rats administered phosphate buffered saline (PBS) oral

113

supplementation, sham uninjured rats administered soybean protein-derived peptides supplementation,

114

burn injured rats administered PBS supplementation and burn injured rats administered soybean protein-

115

derived peptides supplementation.

116

Induction of an inflammatory response, a precursor of burn injury-induced muscle atrophy, was evaluated

117

days 3, 7 and 14 post-sham or burn injury, measured by assessing systemic IFN-γ, chemokine (C-C motif)

118

ligand 2 (CCL2, also known as MCP-1), chemokine (C-C motif) ligand 7 (CCL7, also known as MCP-3).

119

Degree of muscle atrophy was assessed by measuring tibialis anterior muscle (TAM) weight relative to

120

total body weight and induction of burn injury-induced muscle atrophy UPS signaling pathways were

121

measured by Western blot analysis of E3 ubiquitin-protein ligase TRIM-63 (TRIM63, also known as

122

MuRF-1) and F-box only protein 32 (FBXO32, also known as atrogin-1/MAFbx) protein expression.

123

Local muscle atrophy related autophagy was assessed in sham and burn-injured muscle tissue by

124

immunohistochemical analysis as measured by microtubule-associated proteins 1 light chain 3

125

(MAP1LC3, abbreviated as LC3) and beclin-1 (BECN1) expression. Relative induction of autophagy

126

signaling pathways in burn-injured muscle tissue were measured by Western blot protein expression

127

analysis and quantification of cytoplasmic-liberated form of MAP1LC3 (LC3-I) and phagophore and

128

autophagosome membrane-bound form of MAP1LC3 (LC3-II) relative to total LC3 and BECN1 relative

129

to beta-actin (β-Actin).

130



131

MATERIALS & METHODS

132

Materials. Soy protein isolate (SPI, 92% protein) preparing for peptides were purchased from Wilmar

133

International Ltd. (Qinhuangdao, China). 4 kinds of selective commercial proteases used for enzymolysis

134

of SPI were provided by Novozymes (Beijing, China). Prestained broad-range molecular weight standard

135

were purchased from Genview (Glenview, USA), Laemmli sample buffer was purchased from Bio-Rad

136

(Hercules, USA), Tris, glycine, SDS, 30% acrylamide/Bis solution, and urea were purchased from

137

Solarbio (Beijing, China), ammonium persulfate, β-mercaptoethanol, and tetramethylethylenediamine

138

(TEMED) were purchased from Amersco (Framingham, USA). Standards of insulin, bacitracin, Gly-Gly-

139

Tyr-Arg, and Gly-Gly-Gly, were supplied by Sigma-Aldrich Co., Ltd. (St. Louis, MO). HPLC grades of

140

acetonitrile, methanol and trifluoroacetic acid were purchased from Fisher Scientific Ltd. (Ottawa, ON,

141

Canada). Standards of all kinds of amino acid were from Biochrom Ltd. (Cambourne, England). Analysis,

142

deionized water purified with a Milli-Q system (Millipore, Bedford, USA) was used. Antibody specific to

143

atrogin-1/Fbx32 (ab74023) was purchased from Abcam (Shanghai, China). MuRF-1 (sc-27642) antibody

144

was from Santa Cruz Biotechnology (Shanghai) Co., Ltd. (Shanghai, China). Antibodies of LC3 (#4599S),

145

BECN1 (#3738S), and β-Actin (#12620) were obtained from Cell Signaling Technology, Inc. (Shanghai,

146

China). β-Actin was used as internal control.

147

Preparation of soybean peptides. The small molecular weight soybean protein-derived peptides were

148

kindly provided from Nutrily Biotechnology, Ltd. (Anyang, Henan, China) and prepared as previously

149

outlined26. As given in Figure 1, SPI (100 g) was dissolved in water and hydrolyzed by 4 kinds of

150

selective commercial proteases at 55℃ for 4 h. Combining with ultrafiltration membrane and metal film

151

separation, peptides were separated from enzyme hydrolysis solution of soybean proteins. After

152

sterilization and spray drying, peptides (60 g) were prepared as light yellow fine granules with faster

153

solvability and better solubility comparing with soybean proteins.

154

Sodium Dodecyl Sulfate Polyacrylamide-Gel Electrophoresis (SDS-PAGE). To monitor the pattern of

155

peptides, SDS-PAGE was performed on the basis of the method outlined by Laemmli.27,28 with slight


Page 6 of 34

Page 7 of 34


156

modifications. Each sample was dissolved in DDW (∼2.67 mg protein/mL) containing 480 mg/ml urea,

157

and an aliquot (300 µL) was mixed 3:1 (v/v) with Laemmli buffer under reducing conditions (0.71 M β-

158

mercaptoethanol) and boiled for 5 min. Prestained broad-range MW standard, SPI, enzymatic hydrolysate,

159

and peptides (10 µL, containing ∼20 µg protein) were loaded onto 18-well hand-cast 12% acrylamide and

160

5% stacking gels. The gels were electrophoresed at a constant voltage of 100 V for approximately 1.5 h.

161

The gels were stained using Coomassie Brilliant Blue for another 2 hours followed by destaining.

162

Molecular Imager Gel Dox XR system (Bio-Rad Laboratories) was used to scan the gels.

163

Molecular weight distribution. To determine the distribution of molecular weight of soybean peptide, a

164

Shimadzu LC20A instrument was performed on by gel filtration chromatography (GFC). The column

165

used was a TSK-GEL G2000SWXL column (5 µm, 7.8 × 300 mm). Peptides were prepared for 1 µg/mL

166

and standards for different molecular weight were all prepared for 1 mg/mL. The samples and standards

167

were all filtered with a 200-mesh screen through a 0.22-µm filter membrane before injection. Peptides

168

were separated on a 30 min (water/acetonitrile/trifluoroacetic acid 80:20:0.1) isocratic elution. The flow

169

rate was set at 0.5 mL/min, and the detection wavelength was 220 nm. The injection volume was 20 µL.

170

Amino acid composition. For comparing with SPI and the peptides, the amino acid composition of SPI

171

and the peptides used in the experiment were both assayed in triplicate by using an Amino Acid Analyzer

172

(Biochrom 30+ Amino Acid Analyzer, BioChrom Ltd., England) after hydrolysis by 6 N HCl (110 °C)

173

for 24 h with a Na cation-exchange column (8 µm, 4.6×200 mm). Amino acids were post column

174

derivatized with ninhydrin reagent and detected by absorbance at 440 (proline) or 570 (all the other amino

175

acids) nm and expressed as milligrams per 100 g of sample powders.

176

Animal and experimental design. All studies adhered to procedures consistent with the International

177

Guiding Principles for Biomedical Research Involving Animals issued by the Council for the

178

International Organizations of Medical Sciences (CIOMS) and were approved by the Institutional Animal

179

Care and Use Committee at the First Affiliated Hospital to PLA General Hospital. A total of 60 Six-

180

week-old Wistar rats (230-290 g) from Peking University Laboratory Animal Centre were housed at room

181

temperature (22-24˚C) in 12-hour light/dark cycles. Rats were anesthetized by intraperitoneal injection of



182

2 mL/kg body weight of ketamine/xylazine mixture (7.5 mL 100 mg/mL ketamine (Parnell Laboratories,

183

Auckland, New Zealand), 5.0 mL 20 mg/mL Ilium-Xylazine-20 (Troy Laboratories, Sydney, Australia)

184

and 7.5 mL MilliQ water (Millipore, Billerica, MA, USA). Dorsal rat hairs were shaved with an electric

185

razor and the rats were divided randomly and equally in four groups: (1) Sham Injury + PBS

186

Supplementation, (2) Sham Injury + Peptides Supplementation, (3) Burn Injury + PBS Supplementation

187

and (4) Burn Injury + Peptides Supplementation. The 30% TBSA thermal full-thickness third-degree burn

188

injury model has previously been established and described (11). In brief, the sham injury rat groups 1

189

and 2 back skins were placed in water at 37°C for 12 seconds. In burn injury groups 3 and 4, back skins

190

were placed in 94˚C water for 12 seconds. Immediately following injury, administration of balanced salt

191

solution injection (40 mL/kg body weight) to prevent shock and 1% tincture of iodine treatment to the

192

burn area to prevent infection, where burn injured area was left open. Rats in groups 1 and 3 were

193

intragastrically administered 2 mL 1x PBS once a day and groups 2 and 4 were intragastrically

194

administered low molecular weight soybean protein-derived peptides (0.33 g/kg body weight) constituted

195

2 mL 1x PBS.

196

Inflammatory cytokine measurements. Rats from each group were euthanized on day 3, 7, or 14 after

197

injury protocol and approximately 2 mL blood from each animal was collected in a separate heparin-

198

containing separating gel vacuum tube, thoroughly mixed to prevent clotting. Rat Chemokine/Cytokine

199

Panel 22 plex (eBioscience, Vienna, Austria) was used to measure plasma cytokine concentrations of

200

IFN-γ, CCL2 (MCP-1) and CCL7 (MCP-3) levels following the manufacturer’s instructions, measured

201

using a LuminexTM 200 (Luminex, Shanghai, China).

202

Immunohistochemical analysis. A total of 36 muscle tissue samples from sham or burn-injured areas

203

were collected from each group at day 3, 7, and 14 and fixed in 4% paraformaldehyde for 24 hours at

204

room temperature, embedded in paraffin and sliced in 5 µm thick perpendicular plane sections to the

205

incision, de-paraffinized in dimethylbenzene and rehydrated. Sections were incubated with specific

206

antibodies LC3 (#4599S; Cell Signaling Technology, Inc., Shanghai, China) and BECN1 (#3738S; Cell

207

Signaling Technology, Inc., Shanghai, China), followed by incubation with the corresponding secondary


Page 8 of 34

Page 9 of 34


208

FITC-labeled antibody. Protein expression levels of LC3 and BECN1 were evaluated in 5 randomly

209

selected fields of each slide using an inverted fluorescent microscope (Leica, Wetzlar, Germany) and

210

images were captured. Quantitative analysis of fluorescent density was measured using Image Pro Plus

211

software (Media Cybernetics, Rockville, MD, USA)

212

Western blot analysis. Total protein was extracted from sham or burn-injured tissue using

213

radioimmunoprecipitation assay (RIPA) lysis buffer (MACGENE, Beijing, China). Protein concentration

214

was measured using a BCA Protein Assay Kit (Thermo Fisher Scientific, Shanghai, China).

215

Approximately 30-60 µg of total protein was examined by SDS-PAGE Western blot followed by antibody

216

detection of TRIM63 (MuRF-1; sc-27642; Santa Cruz Biotechnology (Shanghai Co., Ltd., Shanghai,

217

China) FBXO32 (atrogin-1/MAFbx; ab74023; Abcam, Shanghai, China), BECN1 (Beclin-1; #3738S;

218

Cell Signaling Technology, Inc., Shanghai, China), LC3 (#4599S; Cell Signaling Technology, Inc.,

219

Shanghai, China), LC3-I (#4599S; Cell Signaling Technology, Inc., Shanghai, China), LC3-II (#4599S;

220

Cell Signaling Technology, Inc., Shanghai, China) and β-Actin (#12620; Cell Signaling Technology,

221

Danvers, MA, USA), followed by appropriate secondary antibodies and chemiluminescence detection, as

222

previously described.29

223

Statistical analysis. All data are expressed as the mean ± standard deviation (± SD) and were analyzed

224

using Wilcoxon signed-rank test for densitometric data, factorial design ANOVA was applied and Holm-

225

Bonferroni method post-hoc analysis (α = 0.05). The differences were considered to be statistically

226

significant at p ≤0.05.

227 228

RESULTS AND DISCUSSION

229

Electrophoresis Patterns. The electrophoresis patterns of prestained broad-range MW standard, peptides,

230

enzymatic hydrolysate, and SPI were shown in Figure 2. The pattern of SPI showed characteristic protein

231

bands of β-conglycinin and glycinin, which are corresponding to α′ subunit of β-conglycinin, α subunit of

232

β-conglycinin, β subunit of β-conglycinin, glycinin A3 chain, glycinin A1,2,4 chains, and glycinin basic

233

chains (Figure 2, lane 4). Following the hydrolysis of SPI by 4 kinds of enzymes, only very slight β



234

subunit of β-conglycinin, glycinin A1,2,4 chains, and glycinin basic chains remained, while α′ subunit of

235

β-conglycinin, α subunit of β-conglycinin, and glycinin A3 chain disappeared, resulting a subsequent

236

release of peptides with narrow, but much lower, molecular weights (Figure 2, lane 3). The state of

237

enzymatic hydrolysate was shown in Figure 3 (a). On the other hand, the pattern of peptides only

238

displayed narrow and low molecular weights, with all the protein bands of β-conglycinin and glycinin

239

disappeared (Figure 2, lane 2). The state of peptides were shown in Figure 3 (c) and (d). The results of the

240

protein bands showed significantly high hydrolysis rate of SPI with complex enzymes. In this condition,

241

the α′ subunit of β-conglycinin, α subunit of β-conglycinin, and glycinin A3 chain could be completely

242

hydrolyzed, with β subunit of β-conglycinin, glycinin A1,2,4 chains, and glycinin basic chains almost

243

completely. Even there is a little unhydrolyzed residual SPI, after enzyme deactivation, ultrafiltration

244

membrane and metal film separation, sterilization, and spray drying, the high molecular weight proteins

245

could be removed, and light yellow fine granules with faster solvability and better solubility were then

246

obtained (Figure 3 (c)). The protein content of peptides (92%) was approximately equal to SPI (90%) and

247

the yield is above 60%. The electrophoresis patterns of peptides also showed that the molecular weight

248

was much smaller than SPI and concentrated in a very small range, which should be further determined

249

by gel filtration chromatography (GFC).

250

Molecular weight distribution. Based on the results of SDS-PAGE, a Shimadzu LC20A instrument was

251

performed on by gel filtration chromatography (GFC) in order to further determine the molecular weight

252

distribution of peptides. As shown in Figure 4, the molecular weights distributed between 186-1000 Da

253

were about 85.59% with 1000-2000 Da 12.12%. The molecular weight distribution was relatively narrow

254

and the molecular weights were much smaller than SPI, which was in accordance with the results shown

255

in Figure 2. The average molecular weight was about 720 Da and constituted with about 3~5 amino acids.

256

Amino acid composition. Amino acids in SPI and peptides were determined in order to evaluate the

257

nutritional value of peptides and verify the differences between SPI and peptides. Analyzed were the

258

following amino acids: aspartic acid, serine, glutamic acid, histidine, glycine, arginine, threonine, alanine,

259

proline, cysteine, tyrosine, valine, methionine, lysine, isoleucine, leucine, and phenylalanine. The result


Page 10 of 34

Page 11 of 34


260

was shown in Table 1. Arginine, glycine, aspartic acid, glutamic acid, proline, and lysine in peptides

261

increased compared with SPI. Meanwhile, histidine, serine, threonine, alanine, cysteine, tyrosine,

262

methionine, valine, isoleucine, leucine, and phenylalanine in peptides decreased compared with SPI.

263

Burn injury model induced muscle atrophy and systemic inflammatory cytokine production. Major

264

burns rapidly induce a significant local inflammatory response and an acute systemic response,30 playing

265

a central role in the complex dynamics of burn injury-induced muscle atrophy. Excessive or prolonged

266

inflammation in burn-injured patients increase risk for the development of hypermetabolic states and

267

associated muscle loss, shock and multiple organ dysfunction syndrome.10 After burn injury, the

268

expressions of pro-inflammatory cytokines and chemokines are increased.31 Increased activation of the

269

IL-1ß cytokine is an important mediator of the inflammatory response that causes a number of different

270

inflammatory syndromes.32 IFN-γ is also an important auto-inflammatory activator that increases antigen

271

presentation on normal cells, lysosome activity of macrophages and is associated with a number of auto-

272

inflammatory and autoimmune diseases.33 The cytokines CCL2, also known as monocyte-chemotactic

273

protein-1 (MCP-1), and CCL7, also known as monocyte-chemotactic protein-3 (MCP-3), the most potent

274

known monocyte chemotactic factors leading to macrophage differentiation, are also upregulated in

275

several inflammatory syndromes.14 After burn injury and the induction of the inflammatory response,

276

more exogenous protein is needed to synthetize the additional proteins required to meet the increased

277

body demands. If exogenous protein is insufficient, myofibrillar degradation will accelerate and protein

278

synthesis will also decrease simultaneously resulting in rapid loss of muscle mass and body weight

279

observed in muscle atrophy. As such, the induction of muscle atrophy in the severe burn-injured rat model

280

was assessed by measuring TAM weight and total body weight, and the induction of inflammatory

281

responses after burn injury was assessed to indicate the establishment of the burn-induced muscle atrophy

282

model by measuring the expression levels of circulating inflammatory cytokines IFN-γ, CCL2 and CCL7

283

at 3, 7 and 14-days post sham or burn injury with and without administration of soybean protein-derived

284

peptides. Figure 5A shows the comparisons of TAM weights at days 3, 7 and 14 following sham or burn

285

injury in PBS and soybean protein-derived peptides administered rat groups, normalized to bodyweight in



286

Figure 5B. Burn injury significantly decreased TAM to body weight ratio in PBS administered rats at day

287

3, 7 and 14. However, at day 3, 7 and 14 soybean protein-derived peptides administered burn-injured rats

288

resulted in no significant difference from that of sham rats. Burn injury resulted in production of

289

significantly higher IFN-γ and CCL2 levels at days 3, 7 and 14 following burn injury as compared to

290

sham treatment groups administered PBS. Soybean protein-derived peptide supplementation in burn

291

injured rats reduced burn-injury induced increases in inflammatory cytokines IFN-γ and CCL2, resulting

292

in levels comparable to that of the sham, non-burn injured rats (Figure 6A and B). Burn injury resulted in

293

production of significantly higher and CCL7 levels at days 3, 7 and 14 following burn injury as compared

294

to sham treatment groups administered PBS. Soybean protein-derived peptide supplementation in burn

295

injured rats attenuated burn-injury induced increases in inflammatory cytokine CCL7 at days 7 and 14

296

(Figure 6C). Thus, taken together the burn-injury rat model established a significant systemic

297

inflammatory response and muscle atrophy, which was attenuated with the administration of soybean

298

protein-derived peptides.

299

Soybean protein-derived peptides nutriment attenuates induction of muscle-specific ubiquitin

300

ligases. Studies have shown that muscle atrophy was closely related to the UPS.12-15 In addition, our

301

previous studies have demonstrated an association between burn-induced muscle atrophy and UPS.29 In

302

this system, particular proteins are targeted for destruction by ligation of ubiquitin, targeting it for

303

destruction by the proteasome. Coupling to UPS ligases provides target protein specificity. Two key

304

muscle-specific UPS ligases, TRIM63 and FBXO32 are widely used as markers of accelerated proteolysis

305

and the atrophy process.5 Where FBXO32 protein has been shown to be highly expressed during muscle

306

atrophy and FBXO32 deficient mice are resistant to atrophy.34,35 Thus, increased TRIM63 and FBXO32

307

protein expressions are molecular indicators of muscle atrophy and have been assessed in this study.

308

Muscle tissue samples from sham experimental rat treatment groups and burn-injured rats with and

309

without soybean protein-derived peptides supplementation were evaluated at day 3 following burn injury

310

by Western blot analysis of TRIM63 and FBXO32 protein expression levels normalized to that of ß-actin

311

expression to determine the effect of peptides nutrient supplementation on UPS muscle atrophy markers


Page 12 of 34

Page 13 of 34


312

(Figure 7A). TRIM63 and FBXO32 protein expression in burn-injured muscle tissues of PBS

313

administered rats were dramatically higher than that of sham group rat muscle tissues (2-fold and 1.7-fold,

314

respectively) (Figure 7B), coinciding with TAM weight measurements (Figure 5A) indicating the

315

induction of muscle atrophy. In addition, consistent with the TAM weight measurements, administration

316

of peptides in burn-injured rats eliminated burn injury-induced increases in muscle atrophy indicators

317

TRIM63 and FBXO32 to levels that not statically different than that of the non-burn-injured sham groups.

318

Thus, burn injury induced UPS increases in TRIM63 and FBXO32 protein expression markers of muscle

319

atrophy which was attenuated by the administration of soybean protein-derived peptides nutrient

320

supplementation, correlating with TAM weights as a muscle atrophy metric.

321

Soybean protein-derived peptides nutriment decreases expression of autophagic proteins. Muscle

322

atrophy has been demonstrated to utilize autophagy16-17 as a mechanism for turnover of long-lived

323

proteins to support survival under conditions of amino acid deprivation.36,37 Autophagy is an

324

evolutionarily conserved catabolic mechanism that orderly disassembles unnecessary or dysfunctional

325

cellular components or unused proteins for lysosomal degradation or intracellular recycling of cellular

326

components in order to maintain energy homeostasis.38 In conditions of hypermetabolic induced

327

imbalances, where nutrient supply cannot meet demands of the body, breakdown of cellular components

328

promotes cellular survival by maintaining cellular energy levels. On a molecular level, following amino

329

acid sensing, growth factor signals or reactive oxygen species protein kinase activation results in

330

activation of BECN1 and the generation of phosphatidylinositol 3-phosphate docking sites for UPS

331

conjugates to bind on the forming double membrane known as an autophagosome around the cytoplasmic

332

target to mark it for destruction.39 Cleavage of diffuse cytosolic LC3 to expose the cytosolic C-terminus

333

glycine residue (LC3-I) occurs and is converted by UPS to the punctate LC3-II form in which the C-

334

terminus is covalently linked to phosphatidylethanolamine (PE) to the autophagosome membrane

335

surrounding the degradation target for capture. The autophagosome then fuses with the lysosome for

336

proteolytic degradation of the target by acidic lysosomal hydrolases or recycling of building blocks

337

through the action of permease vesicular release.39



338

Specifically, BECN1 expression correlates with autophagic responses to nutrient deprivation40 and

339

suppression of BECN1 expression has been shown to impair autophagy and sensitize cells to nutrient

340

depravity induced apoptosis41 and therefore is considered a marker of autophagy. Downstream of BECN1,

341

the generation and turnover of LC3 is used as an index of autophagosome formation and induction of

342

autophagy.39 Consequently, to investigate the effect of soybean protein-derived peptides on the induction

343

of autophagy associated with the development of muscle atrophy, sham and burn-injured muscle tissues

344

from rats treated with and without peptides were evaluated 3, 7 and 14 days after sham or burn-injury by

345

immunohistochemical analysis of autophagic BECN1 and LC3 protein expression. Figure 8A

346

demonstrates an increase in muscle cells expressing BECN1 in burn-injured rats administered PBS as

347

compared to sham rat treatment groups, most markedly at day 7, which was almost entirely mitigated by

348

supplementation with peptides. Similarly, muscle cell expression of LC3 was increased in burn-injured

349

rats from that of the sham experimental group and the administration of peptides moderated this response

350

(Figure 8B). The quantitative analysis of relative fluorescence density of BECN1 (Figure 8C) and LC3

351

(Figure 8D) were also measured. The relative fluorescence density of LC3 and BECN1 in burn-injured

352

rats administered PBS were significantly obvious than the sham groups. However, the groups with

353

peptides administration had significantly lower relative fluorescence density than the burn-injured group

354

administrated with PBS. The results indicated that peptides could decrease the fluorescence density of

355

LC3 and BECN1 obviously.

356

It is also well established that conversion of cytosolic LC3-I form to the autophagosome membrane bound

357

LC3-II form is closely correlated with the formation of autophagosomes42-44 where accumulation of LC3-

358

II is used as a marker for activation of the autophagic pathway.45,46 Therefore, to further investigate the

359

effect of soybean protein-derived peptides on the autophagic response dynamics in burn-injured rats and

360

quantify protein expression levels, total protein was extracted from the muscle tissues of sham and burn-

361

injured rats treated with and without peptides supplementation at day 3 post-sham or burn injury and were

362

evaluated by Western blot analysis for LC3-I form, LC3-II form, and total LC3 was calculated as the sum

363

of LC3-I form and LC3-II form, BECN1 and ß-actin protein expression levels (Figure 9A). Protein


Page 14 of 34

Page 15 of 34


364

expression levels were quantified by densitometric measurements and relative protein expression levels

365

were calculated based on the ratio of LC3-II to LC3-I and BECN1 normalized to ß-actin levels (Figure

366

9B). The ratio of LC3-II to LC3-I and the expression of BECN1 were increased by burn injury as

367

compared to sham experimental treatment groups (1.9-fold and 1.6-fold, respectively), which was

368

attenuated by the administration on peptides to that of sham group levels, thus confirming the results of

369

immunohistochemical staining and further demonstrating increased conversion of the LC3-II form.

370

This evidence indicates that the burn injury model induced systemic expression of inflammatory

371

cytokines, induced muscle atrophy measured as TAM, body weight and ratio of TAM to body weight

372

ratio reductions and the induction of UPS muscle atrophy proteins TRIM63 and FBXO32 and autophagy

373

as measured by increased BECN1, total LC3 expression and the conversion of LC3-I to LC3-II. Burn

374

injury-induced inflammatory cytokines, muscle atrophy and autophagy were all attenuated by the

375

administration of soybean protein-derived peptides nutrient supplementation, correlating with muscle

376

atrophy as measured by TAM to body weight ratios.

377

This data provided compelling evidence that soybean protein-derived small molecular weight peptides

378

nutrient supplementation under the experimental conditions reduced systemic inflammatory responses

379

induced by major 30% TBSA full-thickness burn-injury, induction muscle-specific UPS and autophagy of

380

burn-injured muscle tissue and the development of muscle atrophy in a rat model. As such, dietary

381

supplementation of humans with soybean protein-derived small molecular weight peptides represents a

382

novel and attractive potential adjunctive intervention for burn injury treatment to reduce inflammatory

383

response and hypermetabolic activation resulting in autophagy and related muscle atrophy for improve

384

burn injury outcomes.

385

More importantly, this study proves that burn injury-induced inflammation, UPS activation, autophagy

386

and muscle atrophy can be diminished by soybean protein-derived peptides nutrient supplementation

387

under the experimental conditions. Therefore, providing more usable circulating levels protein building

388

blocks through dietary supplementation with a more readily and easily absorbed low molecular weight

389

preparation form of protein nutrients may reduce the protein nutrient debt registered by the body, thus



390

reducing the induction of various complementary and coordinated local and systemic emergency

391

condition-like responses that become maladaptive when induced at high levels or for prolonged periods of

392

time. Then which components in the peptides had the predominant contributions? As we all know,

393

mixture obtained from enzymatic hydrolysis is composed of many components, sometimes having main

394

component, and sometimes not. However, the main component may not certainly be the active component,

395

which should be separated, purified, and further verified, and sometimes they will lose bioactivities after

396

being purified instead. Meantime, some minor components may have strong bioactivities even in a very

397

low concentration.47 In addition, from the nutritional point of view and macroscopic scale, good

398

equilibrium of the essential amino acids and fast absorption rate are key factors to rapidly provide

399

proteins for body’s needs, and certain single peptide may not reach the good equilibrium of amino acids.

400

Thus, for people under a negative nitrogen balance, the collective effect from various peptide chains

401

rather than from a single bioactive peptide should have the predominant contributions. Implications of

402

this assertion for applications in the treatment of other medical conditions are broad.

403

In addition to the encouraging results of this study for burn injury applications, this research supports the

404

considerable potential of enhancing patient outcomes through practical and cost-effective functional

405

nutriment optimization interventions. In fact, support for the important role of dietary nutriment has been

406

documented, where unhealthy dietary nutrition has been linked to either the development, aggravation or

407

promotion of disease progression of many medical conditions.48-50 However, well-designed scientific

408

investigations of functional nutrient efficacy as adjunctive interventions in the control, prevention and

409

development of diseases and complications as well as the ability to ameliorate of side effects, improve

410

efficacy or reduce dosing of current therapeutics are limited, preventing the realization of the immense

411

potential impact and tremendous therapeutic value that the development and implementation of such low-

412

cost and practical solutions can provide. In addition, optimization of functional nutrients to enhance

413

physiological benefits have been even more inadequate. In this experiment, we did not conduct the group

414

that amino acid mixture with same composition. One reason is that different amino acids have different

415

solubility, preparing aqueous solution that equated with the peptides in the composition of amino acids is


Page 16 of 34

Page 17 of 34


416

hard, especially for some essential amino acids such as branched-chain amino acids and aromatic amino

417

acids which have poor solubilities. In fact, we tried many times in our preliminary experiment but many

418

amino acids could not dissolve together in the water. Meanwhile, peptides used in our experiment were

419

with excellent characteristics of high solubility. Thus, even if we just mixed different amino acids

420

together as the amino acids composition of peptides in water and administrated, the actual dose taken was

421

not equal. Another reason is that small molecular weight peptides will be degraded in the body and

422

absorbed not only in the form of amino acids, but also shorter chain peptides. Thus, the group that amino

423

acid mixture with same composition probably will not have such obvious effects as the peptides used in

424

our experiment. Therefore, in a broader context, we hope this work not only leads to improved burn-

425

injured patient outcomes but inspires more significant dedicated resources and work in establishing

426

scientifically valid efficacy studies for functional nutriment interventions and optimization thereof to

427

contribute to practical, accessible and cost-effective solutions leading to improved health, wellness and

428

increase year of productive lives lived.

429

Our results showed that soybean protein-derived small molecular weight peptides mitigated major severe

430

30% TBSA full-thickness burn injury-induced muscle atrophy response by modulating systemic induction

431

of inflammatory response and local activation of UPS and autophagy signaling cascades at the site of burn

432

injury in a rat model. This study provided novel evidence suggesting nutritional supplementation with

433

soybean protein-derived small molecular weight peptides could reduce severe burn injury-induced muscle

434

atrophy occurrence in patients and improve burn injury outcomes. Thus, implicating soybean protein-

435

derived peptides nutriment as a practical and cost-effective intervention with a very minor safety and

436

adverse effect risk profile for use in the treatment of severe burn injured patients to prevent the

437

development of muscle atrophy.

438 439

FINANCIAL SUPPORT

440

This research was supported by the National Key R&D Program of China (2016YFD0400401), National

441

Natural Science Foundation of China (NSFC81471873, NSFC81571894), Beijing Natural Science



442

Foundation (7172210) and Capital Health Project (Z141100002114011), and Beijing Technology and

443

Business University Young Teacher Funding (QNJJ2015-25).

444 445

AUTHOR INFORMATION

446

Corresponding Author

447

Xinqi Liu, Ph.D., Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing

448

Engineering and Technology Research Center of Food Additives, Beijing Technology and Business

449

University (BTBU), 11 Fucheng Road, Beijing 100048, China. Tel: +86-10-68984481 E-mail:

450

[email protected];

451

Yonghui Yu, Ph.D., Burn Institute, the First Affiliated Hospital of PLA General Hospital, Beijing 100048,

452

China. E-mail: [email protected].

453 454

CONFLICTS OF INTEREST

455

The authors declare no competing financial interest.

456 457

ACKNOWLEDGEMENTS

458

We thank Dr. Rebecca Scotland for her assistance in writing of this manuscript.

459 460

AUTHORS’ CONTRIBUTIONS

461

Conception and Design: Fen Zhao, Yonghui Yu, Xinqi Liu

462

Acquisition of Data: Fen Zhao, Yonghui Yu

463

Analysis and interpretation of data: Fen Zhao, Yonghui Yu, Jian Zhang, Wei Liu, Xinqi Liu,

464

Huinan Yin, Lingying Liu

465

Writing, review, and/or revision of the manuscript: Fen Zhao, Yonghui Yu, Xinqi Liu


Page 18 of 34

Page 19 of 34


466 467

REFERENCES

468

1.

Vos, T.; Allen, C.; Arora, M.; Barber, R. M.; Bhutta, Z. A.; Brown, A.; et al. Global, regional, and

469

national incidence, prevalence, and years lived with disability for 310 diseases and injuries, 1990-

470

2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet 2016, 388, 1545-

471

1602.

472

2.

burn centre: The Gulhane experience. Ann. Burns Fire Disasters 2011, XXⅣ, 9-13.

473 474

Sahin, I.; Ozturk, S.; Alhan, D.; Açıkel, C.; Isik, S. Cost analysis of acute burn patients treated in a

3.

DeKoning, E. P. Thermal Burns. In Emergency Medicine: A Comprehensive Study Guide, 8th ed.;

475

Tintinalli, J. E., Stapczynski, J. S., Ma, O. J., Yealy, D. M., Meckler, G. D., Cline, D. M., Eds.;

476

McGraw-Hill Education: New York, NY, 2016, 1398-1404.

477

4.

Haagsma, J. A.; Graetz, N.; Bolliger, I. The global burden of injury: incidence, mortality, disability-

478

adjusted life years and time trends from the Global Burden of Disease study 2013. Inj. prev. 2016, 22,

479

3-18.

480

5.

481 482

Cohen, S.; Nathan, J. A.; Goldberg, A. L. Muscle wasting in disease: molecular mechanisms and promising therapies. Nat. Rev. Drug Discov. 2015, 14, 58-74.

6.

Hart, D. W.; Wolf, S. E.; Chinkes, D. L.; Gore, D. C.; Mlcak, R. P.; Beauford, R. B.; Obeng, M. K.;

483

Lal, S.; Gold, W. F.; Wolfe, R. R.; Herndon, D. N. Determinants of skeletal muscle catabolism after

484

severe burn. Ann. Surg. 2000, 232, 455-465.

485

7.

486 487

pharmacotherapy. Expert Opin. Pharmaco. 2012, 13, 2485-2494. 8.

488 489

Rojas, Y.; Finnerty, C. C.; Radhakrishnan, R. S.; Herndon, D. N. Burns: an update on current

Lainscak, M.; Podbregar, M.; Anker, S. D. How does cachexia influence survival in cancer, heart failure and other chronic diseases? Curr. Opin. Support. Pa. 2007, 1, 299-305.

9.

Sandri, M. Signaling in muscle atrophy and hypertrophy. Physiology 2008, 23, 160-170.



490 491

10. Hart, D. W.; Wolf, S. E.; Chinkes, D. L.; Gore, D. C.; Mlcak, R. P.; Beauford, R. B. Determinants of skeletal muscle catabolism after severe burn. Ann. surg. 2000, 232, 455-465.

492

11. Yu, Y. H.; Chai, J. K.; Zhang, H. J.; Chu, W. L.; Liu, L. Y.; Ma, L.; Duan, H. J.; Li, B. L.; Li, D. W.

493

miR-194 Promotes burn-induced hyperglycemia via attenuating IGF-IR expression. Shock 2014, 42,

494

578-84.

495 496

12. Jagoe, R. T.; Lecker, S. H.; Gomes, M.; Goldberg, A. L. Patterns of gene expression in atrophying skeletal muscles: response to food deprivation. FASEB J. 2002, 16, 1697-1712.

497

13. Lecker, S. H.; Solomon, V.; Mitch, W. E.; Goldberg, A. L. Muscle protein breakdown and the

498

critical role of the ubiquitin-proteasome pathway in normal and disease states. J. Nutr. 1999, 129,

499

227S-237S.

500

14. Gomes, M. D.; Lecker, S. H.; Jagoe, R. T.; Navon, A.; Goldberg, A. L. Atrogin-1, a muscle-specific

501

F-box protein highly expressed during muscle atrophy. P. Natl. Acad. Sci. USA. 2001, 98, 14440-

502

14445.

503

15. Sacheck, J. M.; Hyatt, J. K.; Raffaello, A.; Jagoe, R. T.; Roy, R. R.; Edgerton, V. R.; Lecker, S. H.;

504

Goldberg, A. L. Rapid disuse and denervation atrophy involve transcriptional changes similar to

505

those of muscle wasting during systemic diseases. FASEB J. 2007, 21, 140-155.

506

16. Zhao, J.; Brault, J. J; Schild, A.; Cao, P.; Sandri, M.; Schiaffino, S.; Lecher, S. H.; Goldberg, A. L.

507

FoxO3 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal

508

pathways in atrophying muscle cells. Cell Metab. 2007, 6, 472-483.

509

17. Mammucari, C.; Milan, G.; Romanello, V.; Masiero, E.; Rudolf, R.; Piccolo, P. D.; Bruden, S. J.;

510

Lisi, R. D.; Sandri, C.; Zhao, J.; Goldberg, A. L.; Schinaffino, S.; Sandri, M. FoxO3 controls

511

autophagy in skeletal muscle in vivo. Cell Metab. 2007, 6, 458-471.

512 513

18. Piccirillo, R.; Goldberg, A. L. The p97/VCP ATPase is critical in muscle atrophy and the accelerated degradation of muscle proteins. EMBO J. 2012, 31 (15), 3334-3350.

514

19. Chou, T. F.; Brown, S. J.; Minond, D.; Nordin, B. E.; Li, K.; Jones, A. C.; Chase, P.; Porubsky, P. R.;

515

Stoltz, B. M.; Schoenen, F. J.; Patricelli, M. P.; Hodder, P.; Rosen, H.; Deshaies, R. J. Reversible


Page 20 of 34

Page 21 of 34


516

inhibitor of p97, DBeQ, impairs both ubiquitin-dependent and autophagic protein clearance pathways.

517

P. Natl. Acad. Sci. USA. 2011, 108, 4834-4839.

518 519 520 521 522 523

20. Pereira, C.; Murphy, K.; Jeschke, M.; Herndon, D. N. Post burn muscle wasting and the effects of treatments. Int. J. Biochem. Cell Biol. 2005, 37, 1948-1961. 21. Mejia, E.; Lumen, B. O. Soybean bioactive peptides: A new horizon in preventing chronic diseases. Sex. Reprod. Menopause 2006, 4, 91-95. 22. Wang, W.; Mejia, E. G. A new frontier in soy bioactive peptides that may prevent age-related diseases. Compr. Rev. Food Sci. F. 2010, 4, 63-78.

524

23. Margatan, W.; Ruud, K.; Wang, Q.; Markowski, T.; Ismail, B. Angiotensin converting enzyme

525

inhibitory activity of soy protein subjected to selective hydrolysis and thermal processing. J. Agric.

526

Food Chem. 2013, 61, 3460-3467.

527

24. Rayaprolu, S. J.; Hettiarachchy, N. S.; Chen. P.; Kannan, A.; Mauromostakos, A. Peptides derived

528

from high oleic acid soybean meals inhibit colon, liver and lung cancer cell growth. Food Res. Int.

529

2013, 50, 282-288.

530 531

25. Gibbs, B. F.; Zougman, A.; Masse, R.; Mulligan, C. Production and characterization of bioactive peptides from soy hydrolysate and soy-fermented food. Food Res. Int. 2004, 37, 123-131.

532

26. Liu, X. Q.; Scotland, R. L. Isolation of plant oligopeptides and uses thereof. WO 2016009364-A1 [P].

533

27. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.

534

Nature 1970, 227, 680-685.

535

28. Margatan, W.; Ruud, K.; Wang, Q.; Markowski, T.; Ismail, B. Angiotensin Converting Enzyme

536

Inhibitory Activity of Soy Protein Subjected to Selective Hydrolysis and Thermal Processing. J.

537

Agric. Food Chem. 2013, 61, 3460−3467.

538

29. Yu, Y. H.; Li, X.; Liu, L. Y.; Chai, J. K.; Zhang, H. J.; Chu, W. L.; Yin, H. N.; Ma, L.; Duan, H. J.;

539

Xiao, M. J. MiR-628 promotes burn-induced skeletal muscle atrophy via targeting IRS1. Int. J. Biol.

540

Sci. 2016, 12, 1213-1224.



541

30. Pruitt, Jr. B. A.; Wolf, S. E.; Mason, Jr. A. D. Epidemiological, Demographic, and Outcome

542

Characteristics of Burn Injury. In Total Burn Care, 4th. ed.; Herndon, D. N., Eds.; Saunders: St.

543

Louis, MO, 2012; 23.

544

31. Duan, H. J.; Chai, J. K.; Sheng, Z. Y.; Yao, Y. M.; Yin, H. N.; Liang, L. M.; Shen, C. N.; Lin, J.

545

Effect of burn injury on apoptosis and expression of apoptosis-related genes/proteins in skeletal

546

muscles of rats. Apoptosis 2009, 14, 52-65.

547

32. Zhang, H. Anti-IL-1β therapies. Recent Pat. DNA Gene Seq. 2011, 5, 126-135.

548

33. Schoenborn, J. R.; Wilson, C. B. Regulation of interferon-gamma during innate and adaptive

549 550 551

immune responses. Adv. Immunol. 2007, 96, 41–101. 34. Wada, T.; Yokoyama, H.; Matsushima, K.; Kobayashi, K. Monocyte chemoattractant protein-1: does it play a role in diabetic nephropathy. Nephrol. Dial. Transpl. 2003, 18, 457-459.

552

35. Bodine, S. C.; Latres, E.; Baumhueter, S.; Lai, V. K.; Nunez, L.; Clarke, B. A.; Poueymirou, W. T.;

553

Panaro, F. J.; Na, E.; Dharmarajan, K.; Pan, Z. Q.; Valenzuela, D. M.; DeChiara, T. M.; Stitt, T. N.;

554

Yancopoulos, G. D.; Glass, D. J. Identification of ubiquitin ligases required for skeletal muscle

555

atrophy. Science 2001, 294, 1704–1708.

556 557 558 559 560 561 562 563

36. Dunn, W. A. Autophagy and related mechanisms of lysosome-mediated protein degradation. Trends Cell Biol. 1994, 4, 139-143. 37. Klionsky, D. J.; Emr, S. D. Autophagy as a regulated pathway of cellular degradation. Science 2000, 290, 1717-1721. 38. Abeliovich, H.; Klionsky, D. J. Autophagy in yeast: mechanistic insights andphysiological function. Microbiol. Mol. Biol. R. 2001, 65, 463-479. 39. Nedelsky, N. B.; Todd, P. K.; Taylor, J. P. Autophagy and the ubiquitin-proteasome system: Collaborators in neuroprotection. BBA-Mol. Basis Dis. 2008, 1782, 691-699.

564

40. Liang, X. H.; Jackson, S.; Seaman, M.; Brown, K.; Kempkes, B.; Hibshoosh, H.; Levine, B.

565

Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature 1999, 402, 672-676.


Page 22 of 34

Page 23 of 34


566

41. Boya, P.; Gonzalez-Polo, R. A.; Casares, N.; Perfettini, J. L.; Dessen, P.; Larochette, N.; Metivier, D.;

567

Meley, D.; Souquere, S.; Yoshimori, T. Inhibition of macroautophagy triggers apoptosis. Mol. Cell.

568

Biol. 2005, 25, 1025-1040.

569 570

42. Mizushima, N.; Yoshimori, T.; Levine, B. Methods in mammalian autophagy research. Cell 2010, 140, 313-326.

571

43. Kabeya, Y.; Mizushima, N.; Ueno, T.; Yamamoto, A.; Kirisako, T.; Noda, T.; Kominami, E.;

572

Ohsumi, Y.; Yoshimori, T. LC3, a mammalian homologue of yeast apg8p, is localized in

573

autophagosome membranes after processing. EMBO J. 2000, 19, 5720-5728.

574 575

44. Tanida, I.; Ueno, T.; Kominami, E. LC3 conjugation system in mammalian autophagy. Int. J. Biochem. Cell B. 2004, 36, 2503-2518.

576

45. Mizushima, N. Methods for monitoring autophagy. Int. J. Biochem. Cell B. 2004, 36, 2491-2502.

577

46. Kirkegaard, K.; Taylor, M. P.; Jackson, W. T. Cellular autophagy: surrender, avoidance and

578

subversion by microorganisms. Nat. Rev. Microbiol. 2004, 2, 301-314.

579

47. Wu, J. H.; Huo, J. Y.; Huang, M. Q.; Zhao, M. M.; Luo, X. L.; Sun, B. G. Structural characterization

580

of a tetrapeptide from sesame flavor-type Baijiu and its preventive effects against AAPH-induced

581

oxidative stress in HepG2 cells. J. Agric. Food Chem. 2017, 65, 10495-10504.

582 583 584 585 586 587

48. Sikand, G.; Kashyap, M. L.; Yang, I. Medical nutrition therapy lowers serum cholesterol and saves medication costs in men with hypercholesterolemia. J. Am. Diet. Assoc. 1998, 98, 889-894. 49. Copperman, N.; Jacobson, M. S. Medical nutrition therapy of overweight adolescents. Arch. Pediatr. Adolesc. Med. 2003, 14, 11-21. 50. Novaković, B.; Grujicić, M.; Trajković-Pavlović, L. Medical nutrition prevention and medical nutrition therapy of lipid metabolism disorder. Med. Pregl. 2009, 62, Suppl 3, 95-100.

588 589 590



591

FIGURES

592 593

Figure 1. Preparation of peptides from SPI.

594

595 596

Figure 2. SDS-PAGE visualization of SPI hydrolysis pattern. The amount of protein loaded in each lane

597

is ∼20 µg. Lanes: 1, molecular weight marker; 2, peptides; 3, enzymatic hydrolysate; 4, SPI.

598


Page 24 of 34

Page 25 of 34


599 600

Figure 3. Molecular weight distribution of peptides. Standards were insulin, bacitracin, Gly-Gly-Tyr-Arg,

601

and Gly-Gly-Gly, respectively.

602

603 604

Figure 4. Product states of different preparation process: (a) enzymatic hydrolysate, (b) centrifugation of

605

enzymatic hydrolysate, (c) peptides, (d) peptides dissolved in water.

606



Page 26 of 34

607 608

(A)

(B)

609

Figure 5. Soybean protein-derived peptides supplementation attenuates burn injury-induced muscle

610

atrophy. (A) Tibialis anterior muscle (TAM) weight of sham and 30% TBSA burn injury rats

611

administered either PBS or soybean protein-derived peptides (Sham + PBS, Sham + Peptides, Burn +

612

PBS and Burn + Peptides, respectively) were measured 3, 7 and 14-days post-sham or burn injury (n=5).

613

(B) Relative TAM to body weight ratios were calculated (% TAM weight of total body weight) for each

614

of the specified time-points post-sham or burn injury (n=5) and compared. The star symbol (*) indicates a

615

significant difference between sham experimental group administered PBS, the ‡ symbol represents a

616

significant difference between burn-injured experimental group administered PBS, and the symbol (#)

617

indicates no significant difference between sham and burn-injured groups administered peptides

618

(ANOVA, p

Small Molecular Weight Soybean Protein-Derived Peptides Nutriment

Recommend Documents