Jiying Huo, Yuzhng Ming, Huifng Li, Anjun Li, Jiwn Zho,Mingqun Hung*, Wizhng Sun, Jihong Wu*, Jinglin Zhng

a Key Laboratory of Brewing Molecular Engineering of China Light Industry, Beijing Technology and Business University, Beijing 100048, China

b School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China

c Inner Mongolia Taibusiqi Grassland King Wine Co., Ltd., Inner Mongolia 027000, China

d Anhui Gujing Distillery Co., Ltd., Anhui 236000, China

e Shandong Bandaojing Co., Ltd., Shandong 256300, China

ABSTRACT

Antioxidant peptides have been widely reported. However, only a few reports have been published examining the antioxidant peptides derived from Chinese baijiu. In this study, 6 novel peptides derived from Chinese baijiu were identified successfully using high-performance liquid chromatography-quadrupoletime-of-flight mass spectrometry (HPLC-QTOF-MS) with a concentration of 0.835–24.540 μg/L. The underlying molecular mechanisms were investigated, and their cytoprotective effects were examined against 2,2’-azobis (2-methylpropanimidamidine) dihydrochloride (AAPH)-induced oxidative stress in HepG2 cells. The results showed that these peptides exerted protective effects by suppressing reactive oxygen species (ROS) generation, preventing malondialdehyde (MDA) formation, and upregulating cellular antioxidant enzyme activities (SOD, CAT, and GSH-Px) in a dose-dependent manner. Further experiments proved that these peptides exerted antioxidant effects via Nrf2/ARE-mediated signaling pathway by promoting Nrf2 nuclear translocation, inhibiting ubiquitination, and enhancing transcription capacity of Nrf2 in HepG2 cells. These findings provide the molecular basis for the effects of antioxidant peptides derived from Chinese baijiu, which is important for a deeper understanding of the relationship between human health and moderate drinking.

Keywords:

Chinese baijiu

High-performance liquid chromatography

quadrupole-time-of- flight mass spectrometry

Antioxidant peptides

HepG2 cells

Nrf2 signaling pathway

1. Introduction

Oxidative stress, caused by an increase in the levels of reactive oxygen species (ROS) or a decrease in antioxidant enzyme activities [1],can induce cell damage and apoptosis due to elevated oxidation and modification of intracellular lipids, proteins, and DNA, which increases the risk of DNA mutation and tumorigenesis [2]. As a key regulator of antioxidant signaling, nuclear factor E2 related factor 2(Nrf2) typically binds to kelch-like ECH-associated protein 1(Keap1) under homeostatic conditions to form a complex. This complex is located in the cytoplasm and is rapidly degraded by the ubiquitin enzyme to ensure that the balanced state of the normal redox system in the human body [3]. The Nrf2/Keap1 complex is degraded in two ways via Keap1 self-conformation change and Nrf2 self-phosphorylation, when cells are stimulated by oxidants or electrophilic agents. Subsequently, Nrf2 enters the nucleus to recognize and bind to the antioxidant response element (ARE), which activates the gene expression of phase II detoxifying/antioxidant enzymes (SOD, CAT, and GSH-Px) to protect cells from oxidative stress [4,5]. Therefore, the activation of the Nrf2 signaling pathway is a major antioxidant mechanism to explore. At present, many antioxidants regulate oxidative stress by activating the Nrf2 signaling pathway via the activation of phase II detoxification/antioxidant enzymes [6-8].

Peptides, specific small fragments of inactive proteins, are released by enzymatic hydrolysis, either during gastrointestinal digestion or food processing (e.g., fermentation) [9]. Typically, they contain 2–20 amino acids and exhibit biological activities depending on their chain length, amino acid composition, and sequence [10]. As a type of natural antioxidants, peptides have garnered interest due to their potential beneficial effects on human health. Accumulated evidence has shown that peptides isolated from many types of food proteins,such as soybean protein [11], dairy products [12], seafoods [13],and egg white protein [14], not only eliminate free radicals, but also inhibit oxidative stress by increasing the activities of antioxidant enzymes in cells. Typically, the antioxidation of bioactive peptides is based on their free radical scavenging abilities and strong chelating abilities for transition metals. Recently, it has been proposed that the antioxidant action of peptides may be defined as the ability of specific molecules to increase the amount of antioxidant enzymes in the process mediated by the Nrf2-Keap1 system [15,16].

Chinese baijiu, with a 2 000-year history since the Western Han dynasty, is produced from grains, such as sorghum, corn, rice, wheat, peas,millet, and highland barley, with Jiuqu as fermentation starters by solid state brewing technology. This includes two important processes [18]:solid-state fermentation and solid-state distillation process [17].Accumulating evidence has demonstrated various beneficial antioxidant compounds in Chinese baijiu, such as tetramethyl pyrazine [18],vanillin [19], terpenes [20], 4-methylguaiacol [8], 4-ethylguaiacol [8],and bioactive peptides [2]. Most of these compounds showed antioxidant activities in chemical assays under in vitro conditions and inhibited 2,2’-azobis (2-methylpropanimidamidine) dihydrochloride(AAPH)-induced oxidative stress in HepG2 cells. However, to the best of our knowledge, few reports have been published examining the molecular mechanism of the antioxidant activity of peptides present in Chinese baijiu.

Therefore, in this study, 6 novel peptides were identified using high-performance liquid chromatography-quadrupole-time-offlight mass spectrometry (HPLC-QTOF-MS), and their protective effects were investigated against AAPH-induced oxidative stress in HepG2 cells. Several markers of oxidative damage, including malondialdehyde (MDA), ROS, and antioxidant enzymes (CAT,GSH-Px, and SOD), were also evaluated. Furthermore, the gene and protein expression of Nrf2 and Keap1, Nucleo-cytoplasmic separation, IP Nrf2-ubiquitination, and dual luciferase reporter assay were examined to validate whether these peptides modulated the antioxidant enzymes via the Nrf2 signaling pathway in HepG2 cells.

2. Material and methods

2.1 Chemicals

Acetonitrile (HPLC grade, purity > 99%) was purchased from Thermo Fisher Scientific, Inc. (Beijing, China). Formic acid (HPLC grade, purity > 99%) was purchased from Dikema Technology Co.,Ltd. (Beijing, China). The synthetic peptides, with 99% purity, were purchased from GL Biochem Ltd. (Shanghai, China). 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), fetal bovine serum (FBS), AAPH, and antibiotics were obtained from Sigma-Aldrich (St. Louis, USA). MDA, SOD, CAT, GSH-Px, and bicinchoninic acid (BCA) protein assay kits were purchased from Nanjing Jiancheng Institute of Biotechnology (Jiangsu, China). ROS assay kit was purchased from Beyotime Institute of Biotechnology(Shanghai, China). Cell-counting kit (CCK-8) was obtained from Dojindo (Kumamoto, Japan). Dulbecco’s Modified Eagle Medium(DMEM) was purchased from Corning Corporation (USA). Trizol reagent was from Life Technologies (USA). Anti-CAT (GTX110704)was purchased from GenTex Inc. (USA). Anti-SOD (Ab13533),anti-GSH-Px (Ab22604), anti-Nrf2 (Ab31163), and anti-Keap1(Ab218815) were purchased from Abcam (Cambridge, UK). Antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH, AB-P-R001)was from JK GREEN Company (Beijing, China). All other chemicals and reagents with analytical grade used in this study were purchased from Sinopharm Group Chemical Reagents Co. Ltd. (Beijing, China).

2.2 Chinese baijiu samples

Bandaojing, Caoyuanwang, and Gujinggong baijiu (base liquor,65%, 58%, and 70% alcohol by volume, respectively, 3 parallel samples) were purchased from Shandong Bandaojing Co., Ltd.(Shandong Province, China), Inner Mongolia Taibusiqi Grassland King Wine Co., Ltd. (Inner Mongolia, China), and Anhui Gujing Distillery Co., Ltd. (Anhui Province, China). The samples had been aged for 3 months and stored at 4 °C prior to analysis. The display of the brand names was not for advertising purpose.

2.3 Isolation of peptides from Chinese baijiu

According to a previous report [21], all Chinese baijiu samples were concentrated from 5.0 L to 50 mL at 55 °C and -0.1 MPa with a rotary evaporator (RE-52AA, Shanghai, China). The residues were extracted thrice with 50 mL redistilled ethyl acetate, and the aqueous phases were collected and lyophilized using a vacuum freeze drier (FD-1B-80, Nanjing, China).Finally, the freeze-dried samples were rehydrated with 1.0 mL of Milli-Q water before HPLC-Q-TOF-MS analysis.

2.4 Qualitative analysis of peptides from Chinese baijiu by HPLC-QTOF-MS

The peptides isolated as described above were identified using HPLC-ESI-QTOF-MS with a 1290 HPLC coupled with an Agilent 6530 QTOF-MS equipped with an electrospray ionization source (ESI)[21]. There were 6 peptides were identified, which were Asp-Cys-Asn (DCN), Lys-Val-Val-Ala (KVVA), Val-Cys-Trp-Asn (VCWN),Trp-ILe-Lys-Lys (WIKK), Lys-Tyr (KY), and Ala-Cys-Phe (ACF).A total of 10.0 μL of the aforementioned rehydrated solution was directly injected into the HPLC-QTOF-MS, and separated on a ZORBAX SB-C18reversed-phase column (150 mm × 4.6 mm, 5 μm,Agilent, Inc., USA) with the following solvent system: (A) 0.10% formic acid in water and (B) 0.10% formic acid in acetonitrile, with a linear gradient of B in A, 8% infirst 3 min, 8%–80% in 3–15 min,80%–92% in 15–20 min, 92%–8% in 20–25 min, andfinally 8% in 25–30 min at 0.50 mL/min. The elution peaks were monitored at 214 nm using a diode array detector (DAD), and the spectra were recorded over the mass/charge (m/z) range of 100–1 000. Nitrogen was used as the nebulizing and drying gas. The peptide sequences were confirmed using MS/MS spectra combined with ade novosequencing algorithm. The optimized conditions of the MS and MS/MS modes are shown in Table S1.

2.5 Cell culture

HepG2 cells were obtained from Chinese Center for Disease Control and Prevention. The cells were incubated with DMEM containing 2.5% fetal bovine serum (FBS) and 50 μg/mL antibiotics(gentamicin, penicillin, and streptomycin) in a humidified incubator with 5% CO2and 95% air at 37 °C. To avoid interference from FBS,the plates were replaced with FBS-free medium before the next tests. Logarithmic growth phase cells were used for the follow-up experiments.

2.6 Cytotoxicity evaluation of peptides and their effects preventing ROS generation

To determine the suitable concentrations of the peptides for subsequent experiments, the cytotoxicity of the peptides was evaluated using the CCK-8 assay [22]. Effects of the samples with different concentrations, which were 0.125, 0.250, 0.500, 1.000,2.000, 4.000, 8.000, and 12.000 mg/mL for DCN, 0.01, 0.10,0.50, 1.00, 2.00, 4.00, and 8.00 mg/mL for VCWN, KVVA, and WIKK, 0.05, 0.10, 0.50, 1.00, 2.00, 5.00, 8.00, and 10.00 mg/mL for ACF and KY, respectively, on cytotoxicity were measured.Cellular oxidative stress attributed to AAPH-induced ROS levels was measured using dichloro fluorescin diacetate (DCFH-DA) assay according to a previously reported method with some modifications in the sample preparation protocol [23]. Briefly, the cells were treated with 100 μL of serum-free DMEM containing samples with different concentrations as mentioned in section 3.2 (sample groups)and without samples (AAPH group and control group) before being incubated with 10 μmol/L of DCFH-DA in serum-free DMEM solution. Trolox was used as a standard antioxidant.

2.7 Determination of MDA levels and antioxidant enzyme activities

HepG2 cells were inoculated at a concentration of 1 × 105cells/mL into a Costa 24-well plate, and 1.0 mL of the cells were adherently cultured in an incubator containing 5% CO2and 95% air at 37 °C for 24 h. After being washed thrice with PBS, the cultured cells were treated with 600 μL of serum-free DMEM with different concentration of samples as mentioned in section 3.2 (sample groups), or without samples (the AAPH group and control group). These mixtures were incubated for 2 h in the cell culture incubator. After that, the cells were washed thrice with PBS. The cells of sample groups and the AAPH group were treated with 600 μL of AAPH at a concentration of 200 μmol/L to induce oxidative stress, and the control group was only added an equal volume of serum-free DMEM. All groups were then incubated in a cell culture incubator for 3 h. Cells of different groups were washed thrice with PBS, and then lysed with RIPA lysis buffer (Cwbio, Beijing, China) containing 1.0 mmol/L phenylmethane sulfonyl fluoride (PMSF) at 4 °C for 10 min. The lysed cells were collected and centrifuged at 10 000 ×gfor 5 min to obtain the supernatant. Finally, a commercial enzyme assay kit was used to determine the contents of trace MDA in the supernatant along with the GSH-Px, SOD, and CAT activities. The cytosolic protein concentrations were quantified using a commercially available BCA kit with bovine serum albumin (BSA) as the standard. Trolox was used as the standard antioxidant.

2.8 Real-time PCR

Real-time PCR was performed based on the method reported by a previous study [3] to further investigate the gene expression of antioxidant enzymes. After 24 h of incubation, the cultured cells were collected and washed thrice with cold PBS. Total RNA was extracted with Trizol reagent according to the manufacturer’s instructions. Subsequently, these RNA samples were treated with DNase and reverse-transcribed into cDNA using the M-MLV Reverse Transcriptase Kit, following the manufacturer’s instructions.The mRNA expression levels of CAT, SOD, GSH-Px, Nrf2, and Keap1 were analyzed with real-time PCR using SYBR Green I gene expression assays. The CFX96 Fluorescence Quantitative PCR detection system (Bio-Rad) was used for real-time PCR detection. Real-time PCR analysis was performed with the following amplification profile: 1 cycle at 95 °C for 10 min, 40 cycles at 95 °C for 10 s and 60 °C for 1.0 min. The primer sequences (forward and reverse) used for real-time PCR are listed in Table 1. The expression levels of mRNA were calculated using the 2?ΔΔCtmethod with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene.

2.9 Protein determination by Western blotting

Each tube cell sample was treated with 500 μL pre-cooled RIPA protein extraction reagent, and the cells were fully suspended and incubated on ice for 30 min. After the cultured cells were centrifuged at 8 000 ×gand 4 °C for 15 min, the supernatants were collected for testing. The proteins in the supernatants were quantified using a commercial BCA kit according to the manufacturer’s instruction.The OD was determined using a microplate reader by measuring the absorbance at 570 nm wavelength, followed by calculating the concentrations. Finally, the protein expression was detected by a previously published method described in the Support information [22].The Western blot results were quantified using the Quantity One software. GAPDH was used as an internal standard reference.

2.10 Nuclear and cytoplasmic protein extraction

Nuclear and cytoplasmic proteins were obtained by treating the cells with the NE-PER Nuclear and Cytoplasmic Extraction Reagents(Thermo fisher scientific, USA) according to the manufacturer’s instructions. After cell fractionation, the samples were separated by SDS-PAGE and immunoblotted with indicated primary antibodies according to the Western bolt method in section 2.9.

2.11 Immunoprecipitations (IP) and immunoblot (IB)analyses

Immunoprecipitation analysis. Cells with indicated treatments were lysed in lysis buffer containing protease inhibitors and phosphatase inhibitors (Thermofisher scientific, USA) on ice for 30 min followed by centrifuging at 10 000 × g for 15 min at 4 °C. Subsequently,the supernatant was collected and the protein concentration was measured by BCA kit (Thermofisher scientific, USA).

Immunoblot analysis. Equal amounts of the whole cell lysates were incubated with anti-Nrf2 antibody overnight at 4 °C.Subsequently, the supernatant was incubated with Protein A/G beads for 1 h at 25 °C. The recovered immuno-complexes were washed four times with cold DPBS (Gibco) containing protease inhibitors. The protein A/G beads were resuspended with 2 × loading buffer followed by being separated on SDS-PAGE gel and immunoblotted with indicated antibodies. Samples were also separated by SDS-PAGE and immunoblotted with indicated antibodies followed by secondary antibody incubation (Jackson Labs) and ECL (Millipore) treatment to detect the endogenous indicator.

2.12 Dual-Luciferase reporter gene assay

293T cells were seeded in 6-well plates and incubated for 18 h. The cells were transfected with pGL3-ARE-luciferase plasmid with pRT-TK plasmid as the internal control. After 12 h post-transfection, the cells were then exposed to KY together with or without AAPH or ML383 for 24 h.Subsequently, the cells were collected for dual-Glo Luciferase detection according to the manufacturer’s instructions (Promega).

2.13 Statistical analysis

All experiments were repeated four times, and the results were expressed as the mean ± standard deviation (SD). All data were analyzed by one-way analysis of variance (ANOVA) using SPSS (version 16.0,IBM, Inc., NY, USA). Significant differences (P < 0.05) were determined using the least significant difference (LSD) range test.

3. Results and discussion

3.1 Sequence of peptides and their concentration in Chinese baijiu

HPLC-ESI-QTOF-MS and de novo sequencing algorithms were used to identify the peptide sequences. Peptides are typically protonated with ESI-MS/MS. They are primarily degraded by disrupting the amide bonds with 10–200 eV of collision energy;however, the chemical bonds of the side chains are difficult to disrupt with such low energy. Therefore, b and y ions act primarily as the secondary fragment ions [21,24].

In the present study, 6 novel peptides were identified from 3 types of Chinese baijiu samples (1 from Bandaojing, 3 from Caoyuanwang,and 2 from Gujinggong). The MS/MS spectrum of a charged ion at m/z 351.094 4 (from Baodaojing) with a retention time of 5.901 min is shown in Fig. 1A (only y and b ions are shown). The ion fragment m/z 236.056 3 was regarded as the y2 ion, which proved to be Cys-Asn, and m/z 219.045 0 was regarded as the b2 ion, which stood for Asp-Cys. The y1 ion (m/z 133.060 8) was the typical fragment[Asn+H]+, and the b1 ion (m/z 116.034 2) was speculated to be Asp.According to these data, the sequence of the peptide was determined to be Asp-Cys-Asn (DCN).

Fig. 1 MS/MS spectra and structures of the peptides from baijiu. (A) DCN, (B) KVVA, (C) VCWN, (D) WIKK, (E) KY, and (F) ACF.

Similarly, the other 5 peptides from Caoyuanwang and Gujinggong baijiu were identified as Lys-Val-Val-Ala (KVVA,m/z = 416.286 6), Val-Cys-Trp-Asn (VCWN, m/z = 521.218 4), Trp-Ile-Lys-Lys (WIKK, m/z = 574.382 4), Lys-Tyr (KY, m/z = 310.169 6),and Ala-Cys-Phe (ACF, m/z = 340.126 6); the b and y ions are shown in Figs. 1B-1F, respectively.

The concentrations of peptides were quantitated by the internal standard method. Pro-His-Pro (PHP, Fig. S1) was used as an internal standard. The internal standard curves of the 6 peptides are shown in Table 2. As a result, the concentration of peptides was determined to be 0.835–24.540 μg/L, which was similar to the reported peptide from Chinese baijiu [21,24]. The peptides may be derived from brewing materials, fermented grains (Jiupei). Some peptides with antioxidant and ACE inhibitory activities have already been isolated from Jiupei [25,26].Moreover, the total content of peptides from Jiupei was approximately 57.682 mg/g of Jiupei[26], which was higher than the content of peptides in Chinese baijiu. This evidence confirmed that only a small portion of peptides entered the liquid along with water and alcohol or through entrainment of mist and foam during the process of Chinese baijiu distillation [27].

Fig. 1 (Continued)

Table 2The concentrations of 6 peptides extracted from baijiu.

3.2 Effect of peptides on cell viability

After the designed treatment, the effects of the peptides on the toxicity of HepG2 cells were monitored (Fig. 2). For the peptide DCN, when the sample concentration was within 4.00 mg/mL, the cell viability did not change with the increase of the sample concentration.However, when the sample concentration reached 4.00 mg/mL, the cell viability significantly decreased (P< 0.05), indicating that HepG2 cells had been damaged under this concentration, but the survival rate was still more than 80%, which was regarded as the normal rate for cells under this condition (Fig. 2A). In order to investigate the effect of different concentrations on the antioxidant capacity of cells,3 concentrations (1.00, 2.00, and 4.00 mg/mL) of DCN were selected for further study. The cells, treated with KVVA, VCWN, and WIKK with concentrations of 1.00 mg/mL or less still survived normally.However, when the concentrations of these peptides exceeded 1.00 mg/mL, the cell survival rates were less than 80% (Figs. 2B-2D).Therefore, three concentrations of 0.25, 0.50, and 1.00 mg/mL were selected for the peptides KVVA, VCWN, and WIKK for further research. Similarly, cell viability decreased in a dose-dependent manner with increasing concentrations of KY and ACF. However,they sharply decreased when the concentrations of samples were 5.00 mg/mL, indicating severe damage to the HepG2 cells. Hence, the appropriate concentrations for both KY and ACF were 2.00 mg/mL.These concentrations (0.50, 1.00, and 2.00 mg/mL) were selected for both KY and ACF (Figs. 2E-2F).

Fig. 2 Cell viabilities of HepG2 cells treated with various concentrations of peptides. (A) DCN, (B) KVVA, (C) VCWN, (D) WIKK, (E) KY, and (F) ACF. Data were shown as the mean ± SD from four independent experiments. Different letters represented the significant difference at P < 0.05.

3.3 Effect of peptides preventing ROS generation and MDA levels in HepG2 cells

As reported previously [27], the antioxidants function is achieved by directly acting as free radical scavengers providing hydrogen or electrons (non-enzymatic antioxidant system), or by improving antioxidant systems increasing gene and protein expression in twophase detoxification enzymes or antioxidant enzymes (enzymatic antioxidant system). In the present study, both antioxidative mechanisms were evaluated, including non-enzymatic and enzymatic antioxidant systems.

DCFH-DA present in the ROS kit used in the experiment is a fluorescein-labeled dye that is non-fluorescent. When it crosses the plasma membrane and reaches the cytoplasm, it is hydrolyzed by intracellular esterase to produce DCFH without fluorescence.DCFH is then oxidized to DCF, a highly fluorescent compound, by intracellular ROS. Hence, the ROS levels in cells are demonstrated by fluorescence intensity.

The effects of the peptides preventing ROS generation in HepG2 cells are shown in Fig. 3A. Trolox was used as the standard.Compared to the AAPH group, different concentrations of peptides significantly (P < 0.05) reduced DCF fluorescence intensities. Thisfinding proved that these six peptides exerted antioxidative effects by reducing intracellular ROS generation in a dose-dependent manner.Specifically, DCN demonstrated the strongest ROS-scavenging ability. Even at a low concentration (1.00 mg/mL), the relative DCF fluorescence intensity was 0.53-fold of the control group. Compared to the standard antioxidant Trolox at the same concentration(1.00 mg/mL, 0.91-fold of the control group), the intracellular ROS level was still significantly reduced (P < 0.05). KY also showed relatively strong ROS scavenging ability, because when KY was administered at high, medium, and low concentrations, the relative DCF fluorescence intensities were 0.56-, 0.67-, and 0.70-fold of that in the control group, respectively. In cells treated with medium and low concentrations (0.50 and 0.25 mg/mL) of WIKK,the relative DCF fluorescence intensities were 1.05 and 1.08-fold of the control group, respectively, which were significantly higher(P < 0.05) than that demonstrated by treatment with Trolox (1.0 mg/mL,0.91-fold). This showed that the ROS scavenging ability of WIKK was significantly (P < 0.05) lower than that of Trolox at both concentrations. However, the relative DCF fluorescence intensity was 0.79-fold of the control group at high concentration (1.00 mg/mL),indicating that the ROS scavenging ability was significantly (P < 0.05)higher than Trolox. However, for KVVA, VCWN, and ACF, when administered at high, medium and low concentrations, the relative DCF fluorescence intensities were significantly lower than that of the AAPH group but higher than that of the Trolox group (P < 0.05).This showed that KVVA, VCWN, and ACF exerted ROS scavenging effects, however, their capacities were lower than Trolox.

Excessive ROS levels can cause lipid peroxidation and directly increase MDA levels [28]. MDA is a tricarbonic compound formed by the cleavage of peroxidic PUFAs (mainly arachidonic acid) and is considered one of the main products of lipid peroxidation [10,29].Moreover, excessive amounts of MDA negatively impact tumor formation, cell metabolism disorders, and cell membrane dysfunction.Therefore, the amount of MDA directly reflects the degree of peroxidation in the body and indirectly re flects the degree of cell damage.

The effect of the peptides on MDA content in HepG2 cells is shown in Fig. 3B. Compared to the control group, after treatment with AAPH (200 μmol/L) for 3 h, MDA content in HepG2 cells increased significantly (P < 0.05). However, the MDA levels of the cells treated with peptides at all tested concentrations were significantly lower than those in AAPH-treated cells (P < 0.05). Specifically, the MDA levels in cells treated with the different concentrations of DCN(4.00, 2.00, 1.00 mg/mL) were (0.280 ± 0.001), (0.55 ± 0.01) and(1.01 ± 0.10) nmol/mg of protein, respectively. Compared to that in the Trolox group ((0.330 ± 0.036) nmol/mg protein), the MDA levels in the cells treated with a high concentration of DCN was significantly lower (P < 0.05). However, the MDA level in the cells treated with a low concentration of DCN was significantly higher (P < 0.05). These results indicated that DCN had better antioxidant activity than the standard antioxidant Trolox at high concentration; however, it showed lower antioxidant activity at low concentration. The contents of MDA in WIKK treatment cells with high, medium and low concentrations were (0.21 ± 0.02), (0.40 ± 0.08), and (0.46 ± 0.03) nmol/mg of protein, respectively. The MDA levels in cells treated with a high concentration of WIKK were significantly lower than that in the cells treated with Trolox ((0.330 ± 0.036) nmol/mg protein) (P < 0.05). No significant difference was observed between the low concentration and Trolox treatment groups (P > 0.05). Additionally, the KY treatment group showed similar results to the WIKK-treated group.Moreover, the MDA levels in HepG2 cells treated with KVVA,VCWN, and ACF were significantly higher than those in the Trolox group but significantly lower than those in the AAPH group (P < 0.05).These results were in accordance with the ROS scavenging capacities of the three peptides.

Fig. 3 Various in fluence of peptides on HepG2 cells. (A) Relative ROS generation, (B) relative MDA contents, (C) relative SOD activities, (D) relative CAT activities, and (E) relative GSH-Px activities. Data were shown as the mean ± SD from 4 independent experiments. Different letters represented the significant difference at P < 0.05.

Based on the aforementioned results, these six peptides derived from Chinese baijiu prevented AAPH-induced MDA accumulation by decreasing ROS generation to protect HepG2 cells from AAPH-induced oxidative damage, in accordance with previous reports [30,31]. Among them, when administered at the same concentration (1.00 mg/mL), KY showed stronger antioxidant activity. A study conducted by Zheng et al. [32] demonstrated that the hydroxyl (-OH) on the side chain group of Tyr is a good electron/proton donor, and peptides with Tyr residues show strong free radical scavenging abilities. Therefore, the strong antioxidant capacity of KY may be attribute to the existence of Tyr residue, which was the only different amino acid residue among these peptides.

3.4 Effects of peptides on the activities of GSH-Px, SOD, and CAT

In order to determine whether the enzymatic antioxidative system played a role in HepG2 cells, the activities of antioxidant enzymes, including SOD, CAT, and GSH-Px were examined,which inhibit the peroxidation reaction by reducing free radicals in the human body [33].

The effects of the peptides on SOD activity in HepG2 cells are shown in Fig. 3C. Compared to the control group, AAPH treatment for 3 h significantly inhibited SOD activity (P < 0.05) by 47.33%, while the SOD activity of the cells treated with peptides was significantly enhanced. This indicated that AAPH increased the oxidative stress in HepG2 cells. However, the peptides protected the cells from AAPH-induced oxidative damage, especially KY. Collectively, the results indicated that DCN and KY protected the cells in a dose-dependent manner. However, for VCWN, KVVA, and WIKK, the concentration dependence was not apparent from the results. Specifically,compared to the AAPH group, the SOD activity in the cells treated with KY at high, medium, and low concentrations were elevated by 47%, 36%, and 36%, respectively. The activities were significantly(P < 0.05) higher than those in the Trolox group. In addition, the SOD activity in DCN-treated cells at a high concentration (4.00 mg/mL)increased by 19.69%, while there was no significant difference in SOD activity between the cells treated with medium concentration (2.00 mg/mL)of DCN and the AAPH group. In addition, there was no significant difference among the SOD activities (P > 0.05) in the cells treated with KVVA and ACF at high and medium concentrations (1.00 and 2.00 mg/mL,0.50 and 1.00 mg/mL), which were (11.44 ± 0.22) and(0.910 ± 0.012) U/mg protein, (11.30 ± 0.31) and (0.900 ± 0.009) U/mg protein,respectively. However, the activities were significantly(P < 0.05) lower than the SOD activity observed in the Trolox group.Similarly, there was no significant difference (P > 0.05) among the cells treated with high, medium, and low concentrations of VCWN and WIKK (1.00, 0.50, and 0.25 mg/mL), indicating that the activities of SOD of these four peptides had no dependence on their concentrations.

As shown in Fig. 3D, the group treated with AAPH alone showed very low CAT activity, which was 0.45-fold that of the control group, indicating that AAPH increased oxidative stress in HepG2 cells.However, after treatment with Trolox and different concentrations of peptides, CAT activity increased significantly, thereby proving that Trolox and peptides played a role in antioxidation. Specifically, compared to that observed in the AAPH group ((0.99 ± 0.06) U/mg protein),the activities of CAT in the DCN treatment group were increased by 575.00%, 124.31%, and 40.53% at high, medium, and low concentrations (4.00, 2.00 and 1.00 mg/mL), respectively.The CAT activities were increased by 245.68%, 214.81%, and 200.54% at different concentrations (2.00, 1.00, and 0.50 mg/mL)of KY, respectively, compared to that observed in the AAPH group. In addition, the CAT activities observed at high and medium concentrations of VCWN and KVVA, high concentration of WIKK and three concentrations of ACF, were significantly higher (P < 0.05)than that observed in the AAPH group ((0.99 ± 0.06) U/mg protein).There was no significant difference between the cells treated with VCWN at low concentration and the AAPH group (P > 0.05), while the CAT activities in cells treated with low concentrations of KVVA,medium and low concentrations of WIKK were significantly lower(P < 0.05) than that in the AAPH group. It could be inferred that the cells might suffer from serious oxidative damage; however, the damage was not increased under the aforementioned conditions. The CAT activities in cells treated at high concentrations of DCN, VCWN,and KVVA and those treated with three different concentrations of KY were significantly higher than that observed in the Trolox group (P < 0.05). As for the cells treated with the high concentration(1.00 mg/mL) of VCWN and KVVA, the CAT activities were (2.72 ± 0.15)and (2.72 ± 0.08) U/mg protein, respectively, which were also significantly higher (P < 0.05) than that observed in the Trolox group((2.19 ± 0.04) U/mg protein). Moreover, the CAT activities in cells treated with the medium concentration (0.50 mg/mL) of VCWN and KVVA and the high concentration (1.00 mg/mL) of WIKK were(2.11 ± 0.04), (2.25 ± 0.09), and (1.97 ± 0.14) U/mg protein,respectively. There was no significant difference among the three groups and the Trolox group (P > 0.05). These results proved that the CAT activity in KY-treated cells was higher than that in cells treated with Trolox and the otherfive peptides, indicating that KY showed better antioxidant capacities among all peptides.

The effects of the peptides on the activities of GSH-Px in HepG2 cells are shown in Fig. 3E. After treatment with AAPH for 3 h, GSH-Px activity in HepG2 cells was inhibited significantly (P < 0.05) by 64.28% compared to that observed in the control group, proving that AAPH increased oxidative stress in HepG2 cells. Compared to the AAPH group, GSH-Px activities in the cells of the sample group increased by 207.17%, 182.13%, and 50.05%, respectively, after treatment with DCN at different concentrations (4.00, 2.00, and 1.00 mg/mL).This proved that DCN inhibited AAPH-induced oxidative stress and protected the cells from oxidative damage to a certain degree.Moreover, compared to the AAPH group ((14.38 ± 0.93) U/mg protein), GSH-Px activities in peptide treatment groups were not all significantly higher (P < 0.05). Specifically, the group treated with KVVA at a high concentration (1.00 mg/mL), and the groups treated with VCWN, WIKK, and KY at all concentrations (low, medium,and high) showed significantly higher GSH-Px activities (P < 0.05).In addition, there was no significant difference in GSH-Px activities between the cells treated with the highest concentration of WIKK(1.00 mg/mL) ((44.37 ± 1.96) U/mg protein) and those treated with Trolox ((45.61 ± 1.68) U/mg protein) (P > 0.05). However, GSHPx activities in the rest of the sample groups were significantly lower(P < 0.05) than that in the Trolox group. In summary, most of the peptides could protect the cells from oxidative damage induced by AAPH only at higher concentrations, and their antioxidant capacities were not as strong as that of Trolox when evaluated based on the GSH-Px activity.

Based on the above results, with respect to the activities of SOD,CAT, and GSH-Px, these six peptides could inhibit AAPH-induced oxidative stress in HepG2 cells via enzymatic antioxidant systems.This was consistent with the study reported by Jiang et al. [26], who found that the peptides from Jiupei exhibited strong inhibition of AAPH-induced oxidative stress via increasing the activities of CAT,GSH-Px, and SOD in a concentration-dependent manner. Moreover,KY with Tyr residue showed a more pronounced protective effect on AAPH-induced oxidative stress in HepG2 cells, in accordance with a previous report [32].

3.5 Relative mRNA and protein expression of antioxidant enzymes, Nrf2, and Keap1

Nrf2 is recognized as the most critical transcription factor in the antioxidant defense system [8]. Activation of Nrf2 and its downstream phase II detoxifying/antioxidant enzymes (CAT,SOD, and GSH-Px) has therapeutic potential in preventing diseases associated with oxidative stress and inflammation [34]. These 6 peptides demonstrated antioxidant abilities by regulating the activities of antioxidant enzymes and thus were further analyzed in this study to verify whether they inhibited AAPH-induced oxidative stress via the Nrf2 signaling pathway. For this purpose, real-time PCR and Western blotting were used to detect the gene and protein expression of SOD,CAT, GSH-Px, Nrf2, and Keap1, respectively.

As shown in Figs. 4A-4C, compared to the control group, the relative mRNA levels of CAT, SOD, and GSH-Px in cells treated with AAPH decreased by 83.75%, 91.09%, and 82.01%, respectively.However, KY treatment at high concentrations increased the relative mRNA levels of CAT, SOD, and GSH-Px by 14.2-, 8.21-, and 6.90-fold,respectively, in comparison to the control group. Even KY treatment at low concentrations increased the relative mRNA levels of CAT,SOD, and GSH-Px by 10.86-, 8.22-, and 6.96-fold, respectively.Similarly, the relative mRNA levels of CAT, SOD, and GSH-Px in the cells treated with different concentrations of WIKK increased by 3.62–6.18, 6.15–10.15, and 4.36–10.14 folds, respectively. Moreover,the rest of the peptides slightly elevated the mRNA expression of antioxidant enzymes in a dose-dependent manner. Treatment with different concentrations of DCN, VCWN, KVVA, and ACF upregulated the mRNA levels of CAT by 2.75–5.50, 2.50–5.59, 2.88–4.35,and 3.43–4.44 folds, respectively. The mRNA levels of SOD in the cells treated with different concentrations of DCN, VCWN, KVVA,and ACF were increased by 1.56–2.82, 0.43–1.54, 0.48–1.46, and 0.07–0.34 folds, respectively. Similarly, the mRNA levels of GSH-Px in cells treated with different concentrations of DCN, VCWN, KVVA,and ACF were elevated by 0.61–2.89, 0.81–1.53, 0.00–2.20, and 0.00–0.54 folds, respectively. Notably, the ability of ACF to regulate the gene expression of antioxidant enzymes (SOD, CAT, and GSH-Px) was lower than that of the otherfive peptides at the same concentration(1.00 mg/mL), which was consistent with the results of a previous study conducted examining the activities of antioxidant enzymes.

Based on the above results, the AAPH treatment group showed the lowest mRNA levels of CAT, SOD, and GSH-Px, indicating that AAPH increased oxidative stress in HepG2 cells. However, different concentrations of peptides significantly (P < 0.05) upregulated the gene expression of these antioxidant enzymes in a dosedependent manner, which was similar to the antioxidant peptides(LEEQQQTEDEQQDQL, YLEELHRLNAGY and RGLHPVPQ)extracted from camel milk protein hydrolysate [35]. Particularly,compared to other peptides, KY demonstrated higher expression of genes encoding antioxidant enzymes, indicating that it possessed a stronger ability to inhibit the oxidative stress induced by AAPH, which was consistent with the previous results outlined in sections 3.3 and 3.4.

The peptide treatment also altered the mRNA expression of Nrf2 and Keap1 (Fig. 4D and 4E). The lowest relative mRNA level of Nrf2 (0.258 ± 0.020) and the highest relative mRNA level of Keap1 (7.54 ± 0.25) were observed in the AAPH group without the peptide treatment. The gene expression of Nrf2 was upregulated,whereas Keap1 gene expression was downregulated in the sample groups treated with a range of concentrations of peptides. This result demonstrated that these 6 peptides extracted from Chinese baijiu led to dissociation of the Nrf2/Keap1 complex, similar to a previous report [35]. It was noteworthy that the gene expression levels of Nrf2 in the KY treatment group were 1.75–2.19 folds than that observed in the control group, which was comparable to the activity of the antioxidant peptide (FOs) derived from wheat bran [36].Moreover, the other 5 peptides also significantly upregulated the gene expressions of Nrf2, from 0.38-fold by KVVA (0.25 mg/mL) to 5.23-fold by DCN (4.00 mg/mL). However, the gene expressions of Keap1 were decreased by 27% (low-level of VCWN, 0.25 mg/mL) to 59% (highlevel of KVVA, 1.00 mg/mL) in comparison with that observed in the AAPH treatment group. Antioxidant peptides might decrease the affinity of Nrf2 and Keap1, leading to the aforementioned results [3].

The above results proved that the 6 peptides decreased AAPH-induced oxidative stress in HepG2 cells by activating Nrf2 and its related genes. Therefore, in order to identify whether the protein expression of antioxidant enzymes (CAT, SOD, and GSH-Px), Nrf2,and Keap1 were also modulated by peptides, Western blotting analysis was further performed (Fig. 4F). It was found that AAPH treatment alone downregulated the protein expression levels of CAT, SOD,and GSH-Px, similar to their relative mRNA levels and activities in comparison with those observed in the control groups, indicating that the activities of the antioxidant enzymes were decreased by AAPH-induced ROS levels. However, the protein expression levels of antioxidant enzymes in the cells treated with peptides were higher than those observed in the AAPH group, suggesting that the mRNA and protein expression levels of CAT, SOD, and GSH-Px were increased simultaneously due to the peptide treatment, consistent with the results of a previous study [37].

Meanwhile, in contrast to those of the control group, the levels of Nrf2 protein decreased and Keap1 protein levels increased in HepG2 cells after AAPH treatment (Fig. 4F, the densitometric analysis are shown in Fig. S2). This finding demonstrated that these peptides decreased the affinity of Nrf2 to Keap1, similar to the previous results reported by Balogun [38]. The 6 peptides might stimulate Nrf2-mediated ARE activation by enhancing Nrf2 expression and reducing Keap1 protein levels at the same time, in other words, by increasing the ratio of Nrf2/Keap1 [39].

Furthermore, as a new-type and selective Nrf2 inhibitor, ML385 was used in the present study to further prove that Nrf2 plays an important role in the antioxidation of these peptides. 3 peptides (KY(1.0 mg/mL), VCWN (0.5 mg/mL), and ACF (1.0 mg/mL)) were randomly selected for the following experiments. The results showed that 10 μmol/L ML385 as Nrf2 inhibitor did not affect cell viability(Fig. S3). Moreover, these three peptides pretreatment activated Nrf2 signaling to enhanced the expression of antioxidant enzymes(SOD, GSH-Px, and CAT) significantly compared with the normal and oxidative stress groups (Fig. 5). However, the expression levels of antioxidant enzymes were significantly downregulated in HepG2 cells treated with 10 μmol/L ML385, proving that these peptides protected HepG2 cells against AAPH-induced oxidative stress in an Nrf2-dependent manner.

Fig. 4 Effect of peptides on the relative mRNA expression levels of (A) CAT, (B) SOD, (C) GSH-Px, (D) Nrf2, (E) Keap1, (F) protein expression visualized by Western blot. Data were shown as the mean ± SD from 4 independent experiments. Different letters represented the significant difference at P < 0.05.

3.6 The peptides activated Nrf2 pathway through promoting Nrf2 nuclear translocation, inhibiting ubiquitination, and enhancing transcription capacity of Nrf2 in HepG2 cells

It is well known that Nrf2 migrates to the nucleus, where,after binding to the ARE, it activates massive gene expression of antioxidant enzyme to exert an antioxidant effect [40]. To elucidate the molecular mechanism by which peptides are able to protect HepG2 cells against oxidative stress injury, the expression of Nrf2 was evaluated by Western blot analysis. In particular, the involvement of the Keap1-Nrf2 pathway was explored by estimating the translocation to the nucleus of the transcription factor Nrf2 (Fig. 6A).The results of nuclear cytoplasmic separation showed that the expression of Nrf2 in cytoplasm and nucleus both decreased under AAPH treatment, indicating that AAPH induced oxidative stress in HepG2 cells. Compared to the AAPH group, Nrf2 expression in cytoplasm and nucleus were significantly up-regulated in 3 peptides(KY, VCWN, ACF) treatment groups as reported in Fig. 6A. In addition, peptides-induced inhibition of Nrf2 ubiquitination was observed (Fig. 6B). Additionally, dual luciferase reporter assay was conducted to evaluate the transcription capacity of Nrf2 through exploring its binding to ARE. As shown in Fig. 6C, the peptide KY could promote the binding of Nrf2 to plasmid promoter and increase luciferase expression. However, ML385 inhibited this reaction. This result further confirmed that the peptide induced the antioxidant response in an Nrf2-dependent manner. These data demonstrated that these peptides activated the Nrf2/ARE signaling pathway in HepG2 cells through promoting Nrf2 nuclear translocation, inhibiting ubiquitination and enhancing transcription capacity of Nrf2 in HepG2 cells. As reported by Tonolo et al. [40], the peptides present in milk proteins in particular the peptide K-8-K, activate the Keap1-Nrf2 system by allowing the translocation of the transcription factor Nrf2 from the cytosol to nucleus so as to trigger the overexpression of the antioxidant enzymes.

Fig. 5 Effect of peptides on the relative protein expression of SOD, GSH-Px, and CAT with or without Nrf2 inhibitor (ML385). Data were shown as the mean ± SD from 4 independent experiments. Different letters represented the significant difference at P < 0.05.

Fig. 6 The peptides activates Nrf2 pathway through promoting Nrf2 nuclear translocation, inhibiting ubiquitination, and enhancing transcription capacity of Nrf2 in HepG2 cells. (A) Nucleo-cytoplasmic separation. (B) Effect of the peptides on Nrf2 ubiquitination. (C) ARE-luciferase activity induced by peptide KY(1.0 mg/mL). Data were shown as the mean ± SD from 4 independent experiments. Different letters represented the significant difference at P < 0.05.

Fig. 6 (Continued)

In summary, the modulation of CAT, SOD, GSH-Px, and Nrf2/ARE signaling was tested in the present study. The results showed that,these peptides led to the dissociation of the Nrf2-Keap1 complex,promoted Nrf2 nuclear translocation, inhibited ubiquitination, and enhanced transcription capacity of Nrf2 in HepG2 cells to activate Nrf2 pathway, which promote the transcriptional activation of phase II antioxidant/detoxifying enzymes by binding to ARE.

4. Conclusions

In this study, six novel antioxidant peptides, including DCN,KVVA, VCWN, WIKK, ACF, and KY extracted from three flavor styles of Chinese baijiu (Bandaojing, Caoyuanwang, and Gujinggong), were successfully identified by HPLC-QTOF-MS with concentrations of 0.835–24.540 μg/L. The results of this study demonstrated these peptides protected HepG2 cells against AAPH-induced oxidative stress by suppressing ROS generation, preventing MDA formation, and upregulating cellular antioxidant enzyme activities, including SOD, CAT, and GSH-Px, in a dose-dependent manner. Among them, KY showed more effective inhibition of AAPH-induced oxidative stress in HepG2 cells at a high concentration due to the presence of Tyr since the hydroxyl group in its residue is a good electron/proton donor. Further experiments proved that these peptides exerted antioxidant effects by activating the Nrf2/ARE-mediated signaling pathway. Thesefindings not only provide the molecular basis for the antioxidant activities of peptides derived from baijiu but also lay the foundation for further design and production of functional baijiu.

Con flicts of interest

The authors declare no con flict of interest.

Acknowledgements

This work was supported by National Key Research &Development Program of China (2017YFC1600401-3) and National Natural Science Foundation of China (31871749 and 31701567).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://doi.org/10.1016/j.fshw.2022.06.010.