2017 Volume 40 Issue 7 Pages 984-991
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 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 of γ-glutamylcysteine synthetase (γ-GCS), which constitutes the rate-limiting enzyme of GSH synthesis, and of another two enzymes, hemeoxygenase-1 (HO-1) and reduced nicotinamide adenine dinucleotide (phosphate): quinone oxidoreductase 1 (NQO1). This induction was preceded by the accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2) 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.
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), reduced nicotinamide adenine dinucleotide (phosphate): quinone oxidoreductase 1 (NQO1), catalase, and GSH S-transferase, is mainly regulated by the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2).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.
Defatted RB was a gift from Satake Corporation (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, U.S.A.). 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 Industries, Ltd. (Osaka, Japan).
Preparation of RBPHRBPH 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 2000×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 2000×g for 30 min. The supernatant (RBPH) was divided into aliquots and freeze-dried. The RBPH was dissolved in 25 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (Hepes)–NaOH pH 7.4 before further analysis. RB protein without hydrolysis was prepared by a similar method to RBPH, except for the absence of Umamizyme G.
Cell CulturesHepG2 and COS7 cells were purchased from the RIKEN Cell Bank (Tsukuba, Japan), and maintained in Eagle’s minimum essential medium (MEM) and Dulbecco’s modified Eagle’s medium, respectively. Both media were supplemented with 10% fetal bovine serum (FBS), 100 µg/mL streptomycin, 100 units/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 LevelsCells 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 phosphate buffered saline (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 AnalysisTotal 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, Shiga, 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, U.S.A.). 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: γ-GCSh forward primer, 5′-TGC TGT CTC CAG GTG ACA TTC-3′ and reverse primer, 5′-CCC AGC GAC AAT CAA TGT CT-3′)21); γ-GCSl forward primer 5′-TCC AGT TCC TGC ACA TCT ACC A-3′ and reverse primer, 5′-TCA TCG CCC CAC TTG AGA A-3′); HO-1 forward primer, 5′-GCA ACC CGA CAG CAT GC-3′ and reverse primer, 5′-TGC GGT GCA GCT CTT CTG-3′22); NQO1 (forward primer, 5′-CAT GAA TGT CAT TCT CTG GCC A-3′ and reverse primer, 5′-CTG GAG TGT GCC CAA TGC TA-3′); Nrf-2 forward primer, 5′-TGC TTT ATA GCG TGC AAA CCT CGC-3′ and reverse primer, 5′-ATC CAT GTC CCT TGA CAG CAC AGA-3′23); β-actin forward primer, 5′-CCT GGC ACC CAG CAC AAT-3′ and reverse primer, 5′-GCC GAT CCA CAC GGA GTA CT-3′.
Western BlottingTreated 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, U.S.A.). Cells were incubated in the lysis buffer for 30 min on ice. After centrifugation at 13000×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 ethylenediaminetetraacetic acid (EDTA), 5 mM dithiothreitol (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 13000×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 13000 g, at 4°C.
Samples of cell lysates and nuclear extracts containing 20 µg of protein were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride (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, U.S.A.), 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, U.S.A.) in a LAS-1000 imager (Fuji, Tokyo, Japan). Band intensities were analyzed using ImageJ software (Public Domain).
Lactate Dehydrogenase (LDH) Cytotoxicity AssayThe 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 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:
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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 (Figs. 2A, B). 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).
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±S.D. (n=3). * p<0.05, ** p<0.01 vs. control group.
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±S.D. (n=3). * p<0.05, *** p<0.001 vs. control group.
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 (Figs. 3A, B). 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 (Figs. 3C, D). 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)
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±S.D. (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±S.D. (n=3).
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).
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±S.D. (n=3). * p<0.05, ** p<0.01 vs. control group.
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.
(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±S.D. (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±S.D. (n=3). * p<0.05 and ** p<0.01 vs. control group.
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 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 (Figs. 6A, B). 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).
(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±S.D. * p<0.05 and ** p<0.01 vs. the group that was not pre-treated with RB protein hydrolysate.
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 superoxide dismutase, 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-butylhydroquinone,31) the nuclear import of Nrf2 started as early as 1 h and Nrf2 was present in the nucleus between 1 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.
We would like to acknowledge the contribution from 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.
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.
