TITLE Small molecular weight soybean protein-derived peptides

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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

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Journal of Agricultural and Food Chemistry

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TITLE

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

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

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AUTHORS

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

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†

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

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Beijing 100048, China

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‡

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*

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.

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ABSTRACT

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

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

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

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

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

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

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

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

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

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

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

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

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

ACS Paragon Plus Environment


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

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

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

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

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

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

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

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

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

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

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

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

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patient outcomes.

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KEYWORDS

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

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ABBREVIATIONS

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

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

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

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

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

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

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

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


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

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amino acids.

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INTRODUCTION

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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patients and improve global health care.

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

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

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

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

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

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

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



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

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

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

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

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

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function.7

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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derived peptides can attenuate burn-induced muscle atrophy.

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

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

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

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

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

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derived peptides supplementation.

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

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

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

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

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

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

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

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

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

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

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

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

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

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to beta-actin (β-Actin).

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MATERIALS & METHODS

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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solvability and better solubility comparing with soybean proteins.

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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



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

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

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

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

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

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

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

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

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

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

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

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

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

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2 mL 1x PBS.

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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software (Media Cybernetics, Rockville, MD, USA)

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

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

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

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

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

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

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

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

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

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

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previously described.29

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

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

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

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significant at p ≤0.05.

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RESULTS AND DISCUSSION

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

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

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

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

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



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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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by gel filtration chromatography (GFC).

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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



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

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

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

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

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

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

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

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

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

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

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

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

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protein-derived peptides.

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

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

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

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

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

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

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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


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(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


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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


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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

TITLE Small molecular weight soybean protein-derived peptides

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