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Biological and Pharmaceutical Bulletin Advance Publication by J-STAGE DOI:10.1248/bpb.b16-00971

Ⓒ 2017 The Pharmaceutical Society of Japan

Advance Publication April 6, 2017

Biol. Pharm. Bull.

Regular Article

Anti-oxidative activity of hydrolysate from rice bran protein in HepG2 cells

Chie Moritania, Kayoko Kawakamia, Akiko Fujitab, Koji Kawakamib, Tadashi Hatanakac and

Seiji Tsuboi*, a

aDepartment of Biochemistry, School of Pharmacy, Shujitsu University; 1-6-1 Nishigawara,

Okayama 703-8516, Japan, bSatake Corporation, 2-30 Saijo Nishihonmachi; Higashi-

Hiroshima-shi, Hiroshima, 739-8602, Japan and cOkayama Prefectural Technology Center

for Agriculture, Forestry, and Fisheries, Research Institute for Biological Sciences (RIBS);

7549-1 Yoshikawa, Kibi-chuo, Okayama 716-1241, Japan

*To whom correspondence should be addressed.

Seiji Tsuboi: Department of Biochemistry, School of Pharmacy, Shujitsu University, 1-6-1

Nishigawara, Okayama 703-8516, Japan

E-mail address: [email protected]

Biological and Pharmaceutical Bulletin Advance Publication

SUMMARY

Glutathione (GSH) is an ubiquitous thiol-containing tripeptide, which plays important

roles in cellular protection from oxidative stress. In our search for a dietary source that can

increase glutathione (GSH) levels, we discovered that a 24 h treatment of HepG2 cells with

rice bran protein hydrolysate (RBPH), prepared by Umamizyme G-catalyzed hydrolysis,

increased the GSH content in a dose-dependent manner. RBPH elevated the expression levels

ofγ-glutamylcysteine synthetase (γ-GCS), which constitutes the rate-limiting enzyme of

GSH synthesis, and of another two enzymes, hemeoxygenase-1 (HO-1) and NAD(P)H

quinone oxidoreductase1 (NQO1). This induction was preceded by the accumulation of Nrf2

(nuclear factor erythroid 2-related factor 2) inside the nucleus, which is a key transcription

factor for the expression of the γ-GCS, HO-1, and NQO1. Pre-treatment of cells with RBPH

produced a significant protective effect against cytotoxicity caused by H2O2 or ethanol. These

results indicate that RBPH exerts a protective effect against oxidative stress by modulating

GSH levels and anti-oxidative enzyme expression via the Nrf2 pathway.

KEYWORDS: rice bran; anti-oxidative; glutathione; nuclear factor erythroid 2-related factor

2


INTRODUCTION

Rice is the most important cereal food in Japan. Rice bran (RB), which constitutes

approximately 10% of the grain, is a major by-product of rice milling. RB is rich in protein,

lipids, dietary fibers, and vitamins.1, 2) Recently, RB was recognized as a functional ingredient,

containing such antioxidants as tocopherols, tocotorienols, and γ-oryzanol.3, 4) Moreover,

enzymatically produced RB protein hydrolysates (RBPHs) were found to possess various

biological functions with potential medical applications. In a previous study, we

demonstrated that RBPH produced with Umamizyme G, a commercial protease from

Aspergillus oryzae, from defatted RB protein exhibited the inhibitory activity of

dipeptidylpetidase-IV (DPP-IV) that is a key regulator involved in the prevention and

treatment of type 2 diabetes. 5) RBPHs produced with other peptidases were shown to have an

anti-proliferative effect on cancer cells,6) and the ability to reduce micellar cholesterol

levels.7)

Excess generation of reactive oxygen species (ROS) leads to oxidative stress, a condition

characterized by ROS attacks on proteins, lipids, and DNA, leading to cell-function disorders.

Oxidative stress is thought to be involved in the pathogenesis of various diseases, e.g.

cancer,8) diabetes,9) cardiovascular diseases,10) and neurodegenerative disorders.11) Thus,

maintenance or restoration of the mammalian cell balance between ROS generation and

detoxification through the action of anti-oxidative molecules and enzymes that decrease

oxidative stress, may be important in the prevention of these pathological conditions.12)

Glutathione (GSH), a ubiquitous thiol-tripeptide, is a major cellular anti-oxidative

molecule, as it has the ability, by itself or in combination with GSH peroxidase, to scavenge

H2O2, other peroxides and free radicals. GSH is biosynthesized from glutamate, cysteine, and

glycine through a two-step ATP-dependent reaction. The first rate-limiting step is catalyzed

by the enzyme γ-glutamylcysteine synthetase (γ-GCS), which is a dimer consisting of a heavy


chain (γ-GCSh) and a light chain (γ-GCSl).13) The expression of γ-GCS and various other

antioxidant and phase-2 enzymes, such as hemeoxygenase 1 (HO-1), NAD(P)H quinone

oxidoreductase 1 (NQO1), catalase, and GSH S-transferase, is mainly regulated by the

transcription factor Nrf2 (Nuclear factor erythroid 2-related factor 2).14, 15) Under normal

oxidation conditions, Nrf2 is located in the cytoplasm, bound to the Kelch-like ECH

associated protein 1 (Keap1), which inhibits Nrf2 translocation to the nucleus. In response to

oxidative stress, Nrf2 is released from Keap1, translocates to the nucleus, and activates the

expression of the aforementioned genes, exerting an anti-oxidative cytoprotective effect.16)

Therefore, activation of Nrf2 contributes to the regulation of GSH levels and the maintenance

of normal redox status in cells.

Various hydrolysates derived from dietary proteins have been shown to exert biological

functions, including anti-oxidative, anti-hypertensive, anti-diabetic, and immuno-modulating

activities.17, 18) After studying the biological functions of RBPH, we discovered that it can

increase GSH levels in HepG2 cells, and exerts a protective effect against cytotoxicity

induced by oxidative stress, through the induction of the Nrf2 pathway.

MATERIALS AND METHODS

Materials Defatted RB was a gift from SATAKE Co. Ltd. (Higashi-Hiroshima, Japan).

Umamizyme G was obtained from AMANO Enzyme Co. Ltd. (Nagoya, Japan), soybean

protein (FUJIPRO E) from FUJI OIL Co. Ltd. (Osaka, Japan), and collagen peptide from

Nippi (Tokyo, Japan). The protein assay kit was purchased from Bio-Rad Laboratories Inc.

(Hercules, CA, USA). 7-Benzo-2-oxa-1,3-diazole-4-sulfonic acid (SBD-F) and 4-

(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) were obtained from Dojindo Labs

(Kumamoto, Japan), while 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) was bought from

Wako Pure Chemical (Osaka, Japan).


Preparation of RBPH RBPH was prepared as described previously.5) In brief, defatted RB

was solubilized in distilled water, whose pH had been adjusted to 12.5 using NaOH, by

stirring for 2 h at 45 °C. After centrifugation at 2,000 × g for 15 min, the supernatant was

collected, and the pH was adjusted to 4.0 with 1 M HCl. After a new centrifugation, the solid

residue (RB proteins) was dried in a vacuum oven, overnight at 40 °C. The obtained proteins

were hydrolyzed with a 1% (w/w) solution of Umamizyme G, overnight at 45 °C. After a 30

min incubation at 80 °C for protease inactivation, the hydrolysate was centrifuged at 2,000 ×

g for 30 min. The supernatant (RBPH) was divided into aliquots and freeze-dried. The RBPH

was dissolved in 25 mM Hepes-NaOH before further analysis. RB protein without hydrolysis

was prepared by a similar method to RBPH, except for the absence of Umamizyme G.

Cell cultures HepG2 and COS7 cells were purchased from the RIKEN Cell Bank (Tsukuba,

Japan), and maintained in MEM and DMEM, respectively. Both media were supplemented

with 10% fetal bovine serum (FBS), 100 µg/ml streptomycin, 100 µg/ml penicillin, and 0.56

µg/ml amphotericin B. The cells were cultured at 37 °C in a humidified atmosphere

containing 5 % CO2.

Measurement of intracellular GSH levels Cells were seeded into 6-well plates at a

concentration of 1.5·105 cells / well. After 48 h, the culture medium was replaced with

medium containing RBPH. After incubating for the indicated periods of time, cells were

rinsed twice with PBS and collected. Following homogenization and deproteinization, the

obtained supernatants were used for measuring the GSH content. The concentrations of GSH

and its oxidized form, glutathione disulfide (GSSG), were determined simultaneously by

HPLC-fluorescence detection, after labeling with ABD-F and SBD-F, respectively.19) The

total GSH content was determined by the enzymatic recycling method using GSH reductase


and DTNB.20)

Real-time PCR analysis Total RNA from RBPH-treated cells was extracted with the

RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s

instructions. First strand cDNA was synthesized from 1 µg of total RNA using the

PrimeScript RT-PCR kit (Takara, Tokyo, Japan). Real-time PCR was performed using SYBR

Premix EX Taq (Takara), and fluorescence was quantified with the ABI PRISM 7700

sequence detection system (Thermo Fisher Scientific, Waltham, MA, USA). The cDNA

levels of the house keeping gene, β -actin, were used as an endogenous control. The

sequences of the primers used in the PCR were as follows: β-GCSh forward primer, 5′-

TGCTGTCTCCAGGTGACATTC-3′ and reverse primer, 5′-cccagcgacaatcaatgtct-3′)21);

β-GCSl forward primer 5′-TCCAGTTCCTGCACATCTACCA-3′ and reverse primer,

5 ′ -TCATCGCCCCACTTGAGAA-3 ′ ); HO-1 forward primer, 5 ′ -

GCAACCCGACAGCATGC-3′ and reverse primer, 5′-TGCGGTGCAGCTCTTCTG-3′

22); NQO1 (forward primer, 5′-CATGAATGTCATTCTCTGGCCA-3′ and reverse primer,

5 ′ -CTGGAGTGTGCCCAATGCTA-3 ′ ); Nrf-2 forward primer, 5 ′ -

TGCTTTATAGCGTGCAAACCTCGC-3 ′ and reverse primer, 5 ′ -

ATCCATGTCCCTTGACAGCACAGA-3 ′ 23); β -actin forward primer, 5 ′ -

CCTGGCACCCAGCACAAT-3 ′ and reverse primer, 5 ′ -

GCCGATCCACACGGAGTACT-3′.

Western blotting Treated cells were washed twice with PBS and harvested using a cell

scraper. Harvested cells were lysed using the RIPA Lysis Buffer System (Santa Cruz


Biotechnology, Dallas, TX, USA). Cells were incubated in the lysis buffer for 30 min on ice.

After centrifugation at 13,000 × g for 15 min at 4 °C, the supernatants were collected as cell

lysates.

Nuclear extracts were prepared as described.24) Briefly, harvested cells were suspended in

200 µl of extraction buffer containing 10 mM HEPES, pH 7.5, 150 mM NaCl, 0.6 % Nonidet

P-40, 1 mM EDTA, 5 mM DTT, supplemented with a proteinase-inhibitor cocktail (Roche

Applied Science, Penzberg, Germany) just before use. After a 20-min incubation on ice,

nuclei were pelleted by centrifugation at 13,000 × g for 15 min at 4 °C. The nuclear pellet

was extracted with a solution containing 10 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM

MgCl2, 0.2 mM EDTA, 5 mM DTT, supplemented with a proteinase-inhibitor cocktail.

Nuclear fractions were collected after a 15 min centrifugation at 13,000 g, at 4 °C.

Samples of cell lysates and nuclear extracts containing 20 µg of protein were separated by

SDS-polyacrylamide gel electrophoresis and transferred onto a PVDF membrane (Bio-Rad).

The membrane was incubated with primary antibodies against γ -GCSh (Santa Cruz

Biotechnology), γ-GCSl, Nrf2 (Santa Cruz Biotechnology), HO-1 (Enzo Life Science),

Lamin B2 (Santa Cruz Biotechnology) or β-actin (Sigma, St. Louis, MO, USA), followed by

incubation with horseradish peroxidase-linked second antibodies. The immune complexes on

the membrane were detected with the Amersham ECL Prime western blotting detection

reagent (GE Healthcare, Chicago, IL, USA) in a LAS-1000 imager (Fuji, Tokyo, Japan).

Band intensities were analyzed using ImageJ software (Public Domain).

Lactate dehydrogenase (LDH) cytotoxicity assay The activity of LDH released from

damaged cells into the medium was measured using the Cytotoxic Detection Kit (Roche

Applied Science). HepG2 cells seeded into 96-well microplates at a concentration of 1.5 · 104


cells / well were treated with RBPH for 24 h. After incubation, the medium was replaced with

FBS-free medium containing H2O2 or ethanol. The culture medium was collected after

incubation periods of 1 h and 24 h for H2O2 and ethanol, respectively, and used to measure

the activity of released LDH (sample). The activity of the total LDH in the culture (LDHhigh

control) was determined by lysing cells in 1 % Triton X-100, while the LDH activity from the

medium of untreated cells was defined as LDHlow control. After the subtraction of background

absorbance from all other values, the cytotoxicity was calculated as follows:

% = – ℎ ℎ – x100

RESULTS

Effect of various protein hydrolysates on the intracellular GSH level We examined the

effect of hydrolysates of various proteins on the intracellular GSH levels of HepG2 cells after

24 h incubations with each hydrolysate, at a concentration of 5 mg/ml. Cultures treated with

RBPH displayed about double the amount of GSH compared to the untreated controls (Fig.

1A). Soybean protein hydrolysate produced by Umamizyme G and collagen peptide did not

cause statistically significant changes in intracellular GSH levels. Treatment of the cells with

RB protein without hydrolysis did not increase the intracellular GSH levels (Fig. 1B). A dose-

response experiment revealed that RBPH elevated the intracellular GSH levels in HepG2 and

COS7 cells in a dose-dependent fashion (Fig. 2A, 2B). On the other hand, the ratio of GSH to

GSSG, which reflects the redox status in the cell, was not significantly affected (Fig. 2C).

The time course analysis indicates that the increase of the intracellular GSH levels was

relatively slow and tended to reach a plateau at 16 h (Fig. 2D).

Effect of RBPH on the expression of γ-GCSh and γ-GCSl In an effort to determine the


mechanism behind RBPH's ability to increase intracellular GSH levels, we determined the

mRNA levels of the γ-GCSh and γ-GCSl genes, that encode the two subunits comprising

the rate-limiting enzyme of GSH synthesis, γ-GCS mRNA levels of both genes began to

increase at 3 h after the addition of RBPH, reaching a statistically significant increase of

about 50% at 8 h (Fig. 3A, 3B). Protein levels of both subunits were found to be decreased at

1 and 3 h after addition, but this reduction had disappeared at the samples collected at 8 h and

the protein levels increased at 24 h though without significance (Fig. 3C, 3D). In addition, the

increase of the intracellular GSH levels was inhibited by about 35% after treatment with 1

mM methionine sulfoximine, which is a known inhibitor of γ-GCS (data not shown).25)

Effect of RBPH on NQO1 and HO-1 expression levels To determine the effect of RBPH

on the expression of anti-oxidant and phase II detoxifying enzymes, the expression of NQO1

and HO-1 was investigated at various times after RBPH addition. At 3 h, mRNA levels of

NQO1 were increased by about 50%, and remained roughly at this level until the end of the

experiment (24 h) (Fig. 4A). RBPH also induced the mRNA levels of HO-1, but more slowly.

The levels had statistically significantly increased by about 100% at 8 h, and kept up to 24 h

(Fig. 4B). The HO-1 protein levels were also increased by 150% at 24 h (Fig. 4C).

Effects of RBPH on Nrf2 Nrf2 is known as a key regulator for anti-oxidant and phase II

detoxifying enzymes such as γ -GCS, HO-1 and NQO1. Since mRNA levels of these

enzymes were induced by RBPH, we decided to examine the expression and nuclear

translocation of Nrf2. mRNA levels of Nrf2 appeared increased about 1.5-fold at 3 h after

RBPH addition. At 8 h they had fallen to about 50 % the initial value, and remained low until

the end of the experiment (Fig. 5A). The Nrf2 protein levels in total cell lysate also reached a


maximum increase (about 9-fold) at 3 h, only to fall afterwards, correlating with mRNA

expression (Fig. 5B). On the other hand, the nuclear Nrf2 protein levels began to increase at 3

h, indicating that RBPH treatment induces the translocation of Nrf2 into the nucleus (Fig. 5C).

The levels of Nrf2 in the cytosolic fraction were generally unchanged, although they

increased slightly after 3 h.

Protective effects of RBPH against cytotoxicity induced by oxidative stress Cells treated

with RBPH, as well as untreated controls, were incubated with H2O2 or ethanol. Afterwards,

cytotoxicity was evaluated by measuring the activity of released LDH in the medium.

Treatment of control cells with 100 μM or 200 μM H2O2 1 h had a cytotoxicity of 10 % and

25 %, respectively. In contrast, at the RBPH-pretreated cells cytotoxicity had been decreased

to less than 5 %, and this reduction was statistically significant fashion. Pretreatment of RB

protein without hydrolysis did not show the cytoprotective effect (Fig. 6A, 6B). Ethanol

treatment was also included in the study, as ROS are known to be produced during ethanol

metabolism, especially through the action of CYP 2E1.26) Pre-treatment of control cultures

with 200 mM or 500 mM of ethanol caused cytotoxicity levels of 20 % and 24 %,

respectively, whereas pre-treatment with RBPH reduced these levels to less than 1% at 200

mM, and to 7% at 500 mM (Fig. 6C).

DISCUSSION

In this study, we demonstrated that RBPH increases intracellular GSH levels in a dose-

and time- dependent manner (Fig. 2), whereas soybean protein hydrolysate or collagen

peptide did not exhibit a similar effect (Fig. 1). The mRNA levels of both γ-GCS subunits

began to increased at 3 h after addition (Fig. 3). In addition to γ-GCS, HO-1 and NQO1


were induced, suggesting that RBPH activated the Nrf2 pathway (Fig. 4). Nrf2 expression

temporarily increased at 3 h after RBPH treatment. Moreover, the increased Nrf2 nuclear

accumulation started at 3 h after the addition of RBPH, which corresponded to the expression

of anti-oxidant enzymes (Fig. 5). As our results indicated that RBPH was able to activate the

Nrf2 pathway, we assumed that it might protect against cell damage caused by oxidative

stress. The hypothesis was verified because pre-treatment of cells with RBPH did offer

significant protection against damage brought about by H2O2 and ethanol (Fig. 6). HO-1

converts heme into biliverdin, releasing free iron and carbon monoxide. Biliverdin is rapidly

metabolized to the antioxidant bilirubin.27) NQO1 detoxifies quinones, which protects the cell

against oxidative stress, and reduces the antioxidants vitamin E and coenzyme Q to their

active form.28) In addition to the induction of HO-1 and NQO1, other anti-oxidant enzymes

that are regulated by Nrf2, including catalase and SOD, could contribute to the protection of

the cell from oxidative stress. Taken together, our results strongly suggest that RBPH may be

able to suppress oxidative stress in cells not only through the up-regulation of GSH

biosynthesis, but also by increasing of expression of other antioxidant enzymes.

Even though our results show that RBPH increase on GSH levels takes place through a

mechanism related to the Nrf2 pathway, this response is relatively slow. Furthermore, the

levels of the γ-GCS protein actually decreased during the first few hours of treatment,

before the induction. In LC2 cells, intracellular GSH levels were found to increase at 6 h after

exposure to 2,3-dimethoxy-1,4-naphthoquinone, which is known to generate ROS,29) whereas

ionizing radiation and TNF-α have been shown to increase intracellular GSH levels with a

peak appearing at 6 or 3 h.30) A 3-h treatment with pyrrolidine dithiocarbamate induces γ-

GCS expression by an Nrf2-associated mechanism.15) After treatment with tert-

butylhydroquinone31), the nuclear import of Nrf2 started as early as 1 h and Nrf2 was present


in the nucleus between 1 h and 4 h. After stimulation with dieckol, a hexamer of

phloroglucinol with known anti-oxidant activity, the nuclear translocation of Nrf2 was

induced at 1 h.32) Changes in GSH levels, γ-GCS levels, and Nrf2 nuclear translocation to

these oxidants and anti-oxidants are faster than those to RBPH. These observations suggest

that activation of the Nrf2 pathway by RBPH is mediated through an additional step: RBPH

might induce a weak oxidative stress in cells, such as an imbalance of the GSH/GSSG ratio,

which subsequently triggered the activation of the Nrf2 pathway. However, the mechanism of

the RBPH-induced decrease of γ -GCS levels is unclear. As it has been reported that

inhibition of NO synthesis leads to a decrease in GSH levels through downregulation of γ-

GCS expression,33) a rational hypothesis would be that RBPH might inhibit NO synthesis.

Several studies have demonstrated the anti-oxidative effects of phytochemicals isolated

from dietary sources.34, 35) Protein hydrolysates derived from soy, egg, milk, whey etc., have

also been reported to exhibit anti-oxidative activity.36) Both, scavenging of ROS and free

radicals as well as sequestering pro-oxidative metals through chelation, have been described

as the primary mechanisms of anti-oxidative activity. To this day, only a few peptides have

been shown to exert anti-oxidative action via increasing the expression levels or the activity

of anti-oxidative enzymes. Egg-derived peptides have been reported to increase the

intracellular GSH levels and upregulate anti-oxidative enzymes in Caco-2 cells37) whereas,

peptides derived from chickpeas have been reported to increase the expression of anti-

oxidative enzymes including γ-GCS, HO-1, NQO1, and Nrf2, in Caco-2 and HT-29 cells.38)

Thus, RBPH is one of the few anti-oxidants that exert their anti-oxidative activity by

regulating the oxidative defense system.

Many studies have shown that oxidative stress is related to the progression or the

aggravation of diseases including cancer, neurodegenerative disorders, and diabetes.8-11)


Supplementation with exogenous GSH has been suggested as a novel treatment for

Parkinson’s disease,39) psychiatric disorders,40) and diabetes.41) In our previous study, the

apparent molecular weight of RBPH was estimated around 300 Da, corresponding with the

molecular weight of di- or tripeptides, and inhibitory peptides of DPP-IV from RBPH were

successfully identified.5) In this case, even though the sequences of the bioactive peptides in

RBPH have not yet been identified, our results suggest that novel anti-oxidative agents

among them may prevent the development and progression of disorders caused by oxidative

stress such as those mentioned above.

In summary, RBPH increased intracellular GSH levels and induced the expression levels

of anti-oxidative enzymes such as γ-GCS, HO-1 and NQO1 through an activation of the

Nrf2 pathway. In addition, RBPH provided the cytoprotective effect against oxidative stress.

Acknowledgements We would like to acknowledge the contribution Mr. Yoshikazu Inoue,

who was one of our colleagues and passed away suddenly in 2015. This project was

financially supported by the Iijima Memorial Foundation for the promotion of food science

and technology.

Conflict of interest Chie Moritani, Kayoko Kawakami and Seiji Tsuboi received a research

grant from Satake Corporation. Akiko Fujita and Koji Kawakami are employees of Satake

Corporation. Tadashi Hatanaka has no conflict of interest.


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Figure legends

Figure 1. Effects of hydrolysates of various proteins on the intracellular GSH levels in

HepG2 cells. Cells were treated with 5 mg/ml of RB protein hydrolysate (RBPH), or soybean

protein hydrolysate (Soybean), or collagen peptide (Collagen) (A), or RB protein without

hydrolysis (RB/−) (B), for 24 h. Values are the means ± SD (n=3). *p < 0.05, **p < 0.01 vs.

control group.

Figure 2. Dose- and time-dependent dependent effects of RB protein hydrolysate on the

intracellular GSH levels. HepG2 (A) or COS7 (B) cells were incubated with the indicated

concentrations of RB protein hydrolysate for 24 h, followed by measurement of GSH levels.

(C) Effects of RB protein hydrolysate on GSH/GSSG ratios. HepG2 cells were treated with

the indicated concentrations of RB protein hydrolysate for 24 h. (D) Time-dependent effect of

RB protein hydrolysate on GSH levels. Cells were treated with 5 mg/ml of RB protein

hydrolysate for the indicated times. Values are the means ± SD (n=3). *p < 0.05, ***p <

0.001 vs. control group.

Figure 3. Effects of RB protein hydrolysate on γ-GCS expression levels. mRNA levels of

heavy (A) and light (B) subunits were estimated by real-time PCR analysis after HepG2 cells

were treated with 5 mg/ml RB protein hydrolysate for the indicated times. Values are the

means ± SD (n=3). *p < 0.05 vs. control. Protein levels of each subunit (C, D) were analyzed

by western blotting using corresponding antibodies. Values are the means ± SD (n=3).

Figure 4. Effects of RB protein hydrolysate on NQO1 and HO-1 expression levels. mRNA

levels of NQO1 (A) and HO-1 (B) were estimated by real-time PCR analysis after HepG2

cells were treated with 5 mg/ml RB protein hydrolysate for the indicated times. (C) HO-1


protein levels were analyzed by western blotting using corresponding antibodies. Values are

the means ± SD (n=3). *p < 0.05, **p < 0.01 vs. control group.

Figure 5. Effects of RB protein hydrolysate on Nrf2 expression. (A) mRNA levels of Nrf2

were estimated by real-time PCR analysis after HepG2 cells were treated with 5 mg/ml RB

protein hydrolysate for the indicated times. Values are the means ± SD (n=3). Nrf2 protein

levels in cell lysate (B) and the cytosolic or nuclear fraction (C) were analyzed by western

blotting using corresponding antibodies. Values are the means ± SD (n=3). *p < 0.05 and **p

< 0.01 vs. control group.

Figure 6. Protective effect of RB protein hydrolysate against cell damage caused by

oxidative stress in HepG2 cells. (A) Cells were treated with 5 mg/mL RB protein with

(RBPH) or without hydrolysis (RB/−) for 24 h and subsequently exposed to 200 µM H2O2 for

1 h (n=4). (B, C) Cells treated with 5 mg/mL RB protein hydrolysate for 24 h, as well as

untreated controls, were exposed to various concentrations of H2O2 for 1 h (n=3-4) and

various concentrations of ethanol for 24 h (n=3-4). The cytotoxicity was determined by

measurement of LDH activity released from damaged cells into the medium. Values are the

means ± SD. *p < 0.05 and **p < 0.01 vs. the group that was not pre-treated with RB protein

hydrolysate.

0

100

200

300

400

500

600To

tal G

SH

(nm

ol/m

g pr

otei

n)*

Control CollagenRBPH Soybean

Figure 1. Effects of hydrolysates of various proteins on the intracellular GSH levels in HepG2 cells.

0

50

100

150

200

Control RBPH RB/-Tot

al G

SH

(nm

ol/m

g pr

otei

n)A

B

**


*

0

50

100

150

200

250

300

0 1.25 2.5 5Tot

al G

SH

(nm

ol/m

g pr

otei

n)

RB protein hydrolysate （mg/mL）

AHepG2

0

50

100

150

200

250

300

Tota

l GSH

(nm

ol/m

g pr

otei

n)

RB protein hydrolysate (mg/mL)0 2.5 5

*

***

BCOS7

0

20

40

60

80

100

0 1.25 2.5 5

GSH

/GSSG

RB protein hydrolysate（mg/mL）

0

20

40

60

80

0 10 20

Tota

lGSH

(nm

ol/m

g pr

otei

n)

Time (h)

*** ***C D

Figure 2. Dose- and time-dependent dependent effects of RB protein hydrolysate on the intracellular GSH levels.


C

0.0

0.5

1.0

1.5

2.0

0 3 8 24

Rel

ativ

e ex

pres

sion

leve

l

Time (h)

*

A γ-GCSh B

0.0

0.5

1.0

1.5

2.0

0 3 8 24Rel

ativ

e ex

pres

sion

leve

lTime (h)

*

γ-GCSl

D

0.0

0.5

1.0

1.5

0 1 3 8 24

γ-G

CSh/

β-ac

tin (

fold

)

Time (h)

0 1 3 8 24 (h)

β-actin

γ-GCSh0 1 3 8 24 (h)

β-actin

γ-GCSl

0.0

0.5

1.0

1.5

2.0

0 1 3 8 24

γ-G

CSl/

β-ac

tin (

fold

)

Time (h)

Figure 3. Effects of RB protein hydrolysate on γ-GCS expression levels.


0 1 3 8 24 (h)

0.0

0.5

1.0

1.5

2.0

2.5

0 3 8 24Rel

ativ

e ex

pres

sion

leve

l

Time (h)

HO-1

** **

BA

0.0

0.5

1.0

1.5

2.0

0 3 8 24

Rel

ativ

e ex

pres

sion

leve

l

Time (h)

NQO1

** *

HO-1

β-actin

C

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0 1 3 8 24

HO

-1/β

-act

in (

fold

)

Time (h)

**

*

**

Figure 4. Effects of RB protein hydrolysate on NQO1 and HO-1 expression levels.


0 1 3 8 24 (h)

0 1 3 8 24 (h)

0.0

0.5

1.0

1.5

2.0

0 3 8 24

Rel

ativ

e ex

pres

sion

leve

l

Time (h)

Nrf2

Nrf2

Lamin B

β-actin

A

C

B

**

**

0

2

4

6

8

10

12

0 1 3 8 24

Tota

l Nrf

2/β-

actin

(fo

ld)

Time (h)

Figure 5. Effects of RB protein hydrolysate on Nrf2 expression.

Nrf2

β-actinCytosol

Nucleus0

2

4

6

8

0 1 3 8 24Rel

ativ

e N

rf2

prot

ein

leve

ls(f

old)

Time (h)

Cytosol

Nucleus*

**

**


0

10

20

30

40

0 50 100 150 200

Cyt

otox

icity

(％）

H₂O₂ (µM)

0%

10%

20%

30%

40%

0 200 400 600

Cyt

otox

icity

(％）

Ethanol (mM)

**

Control

RB protein hydrolysate

Control

RB protein hydrolysate40

30

20

10

0

Cyt

otox

icity

(％

）

A

Figure 6. Protective effect of RB protein hydrolysate against cell damage caused by oxidative stress in HepG2 cells.

0

10

20

30

40

Control RBPH RB/-

Cyt

otox

icity

(％

）

**

C

B

***


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