Evaluating the Effects of MKAVCFSL Derived from Bighead Carp (Hypophthalmichthys nobilis) Flesh on Antioxidant Activity in Caco-2 Cells In Vitro

(e effect of an antioxidative peptide Met-Lys-Ala-Val-Cys-Phe-Ser-Leu (MKAVCFSL) on oxidative stress in Caco-2 cell lines was investigated. Caco-2 cells exposed to excess oxidative stress could be restored when pretreated with the peptide. Reactive oxygen species (ROS) and malondialdehyde (MDA) within the cells could be scavenged by MKAVCFSL. (e peptide could also enhance the activity of glutathione peroxidase (GPx), glutathione reductase (GR), and superoxide dismutase (SOD), while catalase (CAT) activity did not show a significant difference between treatment and control samples. Meanwhile, it was observed that peptide treatment increased the concentration of glutathione (GSH). Yet the content of glutathione disulfide (GSSG) was hardly affected. (e stability of MKAVCFSL was also assessed and an intact peptide was observed after simulated gastrointestinal digestion. Part of the peptide was hydrolyzed into fragments including MKA, FSL, AVCFSL, and MKAVCF. (is study demonstrated that MKAVCFSL derived from bighead carp hydrolysates could ameliorate oxidative stress to protect the Caco2 cells.


Introduction
Oxidative stress is defined as the potential biological damage caused by free radicals [1]; it demonstrates an excessive production of ROS that cannot be counteracted by the action of antioxidative protection systems of the cells. e imbalance between the oxidant species such as hydrogen peroxide (H 2 O 2 ), superoxide (O 2 -•), singlet oxygen (1/2 O 2 ), and the hydroxyl radical (•OH) was mainly produced from the mitochondrion. e oxidant species may trigger specific factors responsible for oxidative damage in the cells including cellular proteins, membrane lipids, nucleic acids, and eventually cell death. is damage has a correlation with several pathologies like cardiovascular, cancer, diabetes, and inflammatory diseases [2].
All cells possess elaborate antioxidant systems constitutive of interacting various weight components. Among them, SOD, GPx, and CAT play a significant role in scavenging ROS. Increased oxidative stress associated with the disease is often related to a depletion in enzymatic and nonenzymatic antioxidants [3], which reduced the ability to protect against excess ROS exposure. erefore, extra antioxidants are supposed to be utilized as direct scavengers of free radicals, as inhibitors of lipid peroxidation, and so on [4]. erefore, researchers' interest in the identification, characterization, and application of antioxidants has increased to protect organisms from oxidative stress. Natural sources have been specifically considered because they appear to be safer for consumers than synthetic antioxidants, such as butylated hydroxytoluene (BHT), which have shown to be carcinogenic [5]. Food-derived antioxidant peptides have been purified from many protein hydrolysates such as sardinelle (Sardinella aurita) [6], chickpea (Cicer arietinum L.) protein [7], blue mussel (Mytilus edulis) protein [8], and barley glutelin [9]. Yet, antioxidant peptide identified from freshwater fish was limited.
In our previous study, we identified an antioxidant peptide MKAVCFSL from flesh hydrolysate of bighead carp (Hypophthalmichthys nobilis), which had effective DPPH radical scavenging activity, ferrous ions (Fe 2+ ) chelating activity, and reducing power [10]. However, there is a lack of research on the evaluation of antioxidant activity at the cellular level. In this study, the protective role of this antioxidant peptide against H 2 O 2 -induced oxidative stress was further evaluated in Caco-2 cell lines since the intestinal epithelium is the interface between the organism and its luminal environment, which is prone to suffer oxidative stress [11]. Contents of cellular ROS and MDA were assessed to study the antioxidant capacity at the cellular level. e enzyme activities, including SOD, GPx, GR, and CAT, and cellular antioxidant GSH were investigated to evaluate whether this peptide also initiated the cellular antioxidant system to protect Caco-2 cells against H 2 O 2 -mediated cell death. Besides, simulated gastrointestinal digestion was performed to evaluate the stability of the peptide before in vivo experiments.

Simulated Gastrointestinal Digestion.
In vitro pepsinpancreatin-simulated digestion was performed according to the method described by Samaranayaka with slight modifications [12]. e peptide MKAVCFSL (50 μg/mL) was initially dissolved in distilled water, and the pH was adjusted to 2.0 using 5 M HCl. Pepsin was mixed with the peptide in a ratio of 1 : 100 w/w of the substrate, and the mixture was incubated at 37°C for 1 h. After that, the pH was adjusted to 5.3 using saturated NaHCO 2 solution and was further augmented to 7.5 with 5 M NaOH. Subsequently, pancreatin (enzyme : substrate ratio, 1 : 50 w/w) was added to the mixture, which was incubated again at the temperature of 37°C for 2 h. Aliquots of the digested samples were collected at 1 h, 2 h, and 3 h. e enzymatic reaction was terminated by immerging the samples in a 95°C water bath for 10 min and cooling the samples to room temperature on ice. e digested samples were used to evaluate the stability utilizing LC-MS/MS analysis.

Determination of Amino Acid Sequence by LC-MS/MS.
After simulated gastrointestinal digestion, the molecular mass and peptide sequencing analysis was performed using a ermo Q-Exactive high-resolution mass spectrometer ( ermo Scientific, Waltham, MA, USA) in positive ion mode with a capillary temperature of 320°C and a 2 kV spray voltage applied to the emitter. e following electrospray ionization (ESI) conditions were utilized for MS analysis: a high resolution (70,000 FWHM) MS full scan (m/z 300-2000 Da) was performed to select the 10 most intense ions prior to MS/MS analysis using 10 eV energy collisional dissociation.

Induction of Oxidative Stress.
e Caco-2 cells' cultivation was conducted at the concentration of 3.2 × 10 5 cells/ mL in 24-well culture plates for 3 days and the fresh medium was replaced daily. Prior to oxidative induction, Caco-2 cells were incubated with antioxidant peptide for 24 h and then washed twice with phosphate-buffered saline (PBS), pH 7.2. After that, the cells were incubated for 2 h with 1 mM H 2 O 2 to induce oxidative stress. e cells were treated as follows: medium treatment without H 2 O 2 and peptide (control 1: C1), H 2 O 2 treated only (control 2: C2), peptide treated only (sample 1: S1), and medium treatment with H 2 O 2 and peptide (sample 2: S2). e cells were then lysed with cell lysis buffer (Beyotime, Jiangsu, China) and centrifuged at 10,000 g and 4°C for 10 min. e supernatant was collected and stored at −80°C until further experiment. e cell lysate protein concentration was measured using the Bicinchoninic Acid (BCA) Protein Assay Kit (Solarbio, Beijing, China).

Cytotoxicity
Assay. Cell Counting Kit-8 (CCK-8) from Solarbio (Beijing, China) was used to measure the cytotoxicity of MKAVCFSL in Caco-2 cells. e peptide was dissolved in DMEM and then diluted in a culture medium and added to the cultures 24 h after cell seeding. Cells were treated as the method of 2.2, after which 10 µL of CCK-8 was added to each well, and the plates were further incubated for 4 h. ereafter, the absorbance was read using a microplate reader (Bio-Tek, Synergy HT, USA) with 450 nm. e viability of cells was expressed as a percentage of the viability of cells in C1 treatment.

Determination of Intracellular ROS.
e intracellular ROS contents inside the Caco-2 cells were measured according to the instruction of the ROS Assay Kit. Cells were seeded in 96-well plates at a concentration of 2.5 × 10 5 cells/ mL. Cultured cells were treated as the method of 2.4. After incubation, 10 μM DCFH-DA was added to the wells and then incubated at 37°C and 5% CO 2 for 20 min, and cells were then washed with DMEM without FBS for three times. Fluorescence readings were taken at excitation and emission wavelengths of 488 and 525 nm using a Multi-Detection Microplate Reader (Bio-Tek, Synergy HT, USA), respectively. Cellular ROS levels were expressed as fluorescence intensity.

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Measurement of Lipid Peroxidation.
e cellular MDA content was evaluated to investigate the level of lipid peroxidation under oxidative stress according to the method of the MDA Assay Kit (Solarbio, Beijing). 100 µL cell lysate supernatant was mixed with 200 μL MDA working solution and heated at 100°C for 30 min. After cooling the mixture to room temperature, samples were centrifuged at 10,000 g for 10 min. e absorbance at 532 nm and 600 nm was measured in a 96-well plate, respectively. e results of the MDA assay were expressed as nanomoles of MDA per milligram of protein.

Measurement of GSH and GSSG Concentration.
Cellular GSH and GSSG were evaluated according to the direction of GSH and GSSG Assay Kit. Cell lysate supernatants were obtained by being rapidly freezethawed twice with liquid nitrogen and 37°C water baths and then centrifuged at 10,000 g for 10 min at 4°C. 10 μL total GSH and GSSG standards were mixed with 150 μL of 100 mM PBS containing 4 mM EDTA, 0.2 mM NADPH, 0.5 mM DTNB, and 100 units/mL glutathione reductase in a 96-well plate, respectively. Sample supernatants and supernatants which eliminated reduced GSH were mixed with the same PBS as well. e mixture was incubated for 5 min at room temperature and 0.5 mg/mL NADPH was added to each corresponding well. e absorbance was read at 412 nm. e concentration of total glutathione and GSSG in the cell lysate was calculated using a standard curve and GSH concentration was calculated from total glutathione contents deducted GSSG contents.

Glutathione Peroxidase (GPx) Activity.
GPx was assessed according to the procedure of the Cellular Glutathione Peroxidase Assay Kit. Briefly, 10 μL cell lysate was mixed with 175 μL 100 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA and 11 μL of GR solution which contains 30 mM NADPH, 84 mM GSH, and 1 μL 5 U/μL GR. 4 μL 15 mM H2O2 was added to the solution mixture. e absorbance was monitored at 340 nm every 2 min for 10 min. e activity of GPx within the cell was calculated using a standard curve and expressed as milliunit of GPx per milligram of protein. One unit of activity was expressed as the conversion of 1 mM/min NADPH to NADP+.

Glutathione Reductase (GR)
Activity. GR activity was determined using the method described by the Glutathione Reductase Assay Kit. In brief, 20 μL cell lysate was added to 100 mM PBS (0.85 mL, pH 7.5) containing 1 mM EDTA and 2.32 mM GSSG. NADPH (2 mM, 10 μL) was mixed with the solution and the absorbance of 340 nm was read every 1 min for 5 min at 25°C using a Multi-Detection Microplate Reader (Bio-Tek, Synergy HT, USA). Changes in the rate of absorbance were converted into units of GR per milligram of protein using a molar extinction coefficient of 6.22 mM −1 cm −1 .
One unit of activity was defined as the reduction of 1 μmol/min GSSG.

Superoxide Dismutase
Assay. SOD activity was investigated utilizing a Total Superoxide Dismutase Assay Kit according to the manufacture's instruction. e water-soluble tetrazolium-8 (WST-8) method, which is more stable and sensitive, is currently used in the determination of SOD activity. Absorbance was determined at 450 nm. Results were expressed as units milligram of protein where protein content was determined by the BCA method. One unit of SOD is defined as the quantity of the enzyme in 20 μL of the sample solution that inhibits the reduction reaction of WST-8 with superoxide anion by 50%.

Catalase
Assay. In order to measure the level of catalase in Caco-2 cell lysate, the Catalase Activity Assay Colorimetric Kit was used according to the manufacturer's instructions. Absorbance was measured at 520 nm and results were expressed as units milligram of protein. One unit of catalase is defined as the amount of the enzyme that can catalyze 1 μmol H 2 O 2 within 1 minute under the condition of pH7.

Statistical Analysis.
Statistical analysis was performed with SPSS software, version 20 (SPSS Inc., Chicago, IL, USA), using one-way ANOVA followed by Duncan's multiple range test. Differences were considered at a significance level of p < 0.05. ese results suggested MKAVCFSL was highly likely to successfully pass through the digestive tract and reach intestine to exert antioxidant activity pepsinpancreatin.

Analysis of Effects of MKAVCFSL on Cellular Viability, ROS, and MDA.
Our previous study has confirmed that the MKAVCFSL peptide showed effective antioxidant activity such as DPPH scavenging activity, reducing power, and ferrous ions (Fe 2+ ) chelating activity [10]. erefore, we highlighted if the antioxidant peptide could express good antioxidant activity at the cellular level. In our current study,  products was observed (from (0.40 ± 0.08) nmol/mg protein to (1.15 ± 0.08) nmol/mg protein) compared with the C1 sample. However, peptide significantly inhibited the formation of MDA to a concentration of (0.77 ± 0.06) nmol/ mg protein in stressed cells (S2 sample) when compared with C2 sample. ese results were in accordance with those observed with peptides isolated from Hoki fish on human embryonic lung fibroblasts [13] and silver carp peptides in human intestinal epithelial Caco-2 cells [14]. Besides antioxidant peptides, the antioxidant effect of other food compositions was also highlighted such as ascorbic acid, tocopherols, arotenoids, and other varieties of polyphenols and flavonoids [15]. However, the study on foodderived antioxidant peptides that was evaluated at the cellular level was still limited. e results for the protective effect of MKAVCFSL in Caco-2 cells completed the noncellular antioxidant activity that we reported previously.

Analysis of Antioxidant Enzymes Activity Pretreated with MKAVCFSL.
To investigate if the antioxidant peptide could also affect the internal antioxidant system of Caco-2 cells, various antioxidant enzymes including SOD, CAT, GPx, and GR as well as the endogenous antioxidant GSH were determined. SOD could contribute to converting superoxide to hydrogen peroxide, which is then decomposed by CAT or GPx into H 2 O [16]. Deleterious actions of H 2 O 2 on Caco-2 cells were observed since SOD activity was inhibited for about 2-folds compared with C1. e reduction in SOD activity is deemed to be caused by an interaction between H 2 O 2 and a copper moiety within the SOD molecule. Hydroxyl and copper-bound radicals could be produced during the process, which oxidized histidine residues resulting in the denaturation of the SOD protein [17,18]. When preincubated with peptide for 24 h, SOD activity showed no significant difference in S2 compared with C2 treatment, which suggested that peptide had the ability to protect the SOD from the H 2 O 2 toxicity. It is deduced that the SOD-protecting effects of MKAVCFSL may be due to their possible chelation of surplus copper ions in the SOD complex preventing the metal from reacting with H 2 O 2 . H 2 O 2 stimulation, on the contrary, increased the catalase activities from (0.33 ± 0.06) units min −1 mg −1 protein to (0.55 ± 0.04) units min −1 mg −1 protein compared to the untreated baseline cells (Figure 4(b)). Catalase, as a primary defense enzyme against oxidative stress from exogenous, is a porphyrincontaining enzyme which catalyzes the decomposition of hydrogen peroxide to water and oxygen [19]. erefore, it is reasonable that the activity of catalase increased to scavenging excessive H 2 O 2 . On the contrary, Shi et al. [20] have reported that CAT activity decreased after treated by 1 mM H 2 O 2 because it is a high dose for CAT to completely decompose H 2 O 2 into H 2 O [21]. e addition of the peptide had no significant effect (p < 0.05) on the CATactivity of normal cells and H 2 O 2 -challenged cells.
GPx also plays a critical role in protecting the cells from oxidative stress damage induced by a high concentration of [21,24]. GPx utilizes GSH to degrade H 2 O 2 into H 2 O and converts GSH into GSSG. GR catalyzes the decrease of GSSG to the GSH in its sulfhydryl form. erefore, GSH can regenerate through the cyclic action between GPx and GR [25]. In normal cells without H 2 O 2 stimulation, peptides did not enhance GPx and GR activity significantly (Figure 4). After the addition of MKAVCFSL, the activity of GPx, GR in the cells H 2 O 2 treatment were increased with presented in Figures 4(c) and 4(d). is indicated that the peptide could not enhance the GPx and GR activity directly. e protective effect was involved in the removal of oxidative factors that occurred in the cellular metabolic environment. Mammalian GPx1-4 are selenoproteins with selenocysteine (Sec) in the catalytic center, of which GPx2 is mainly expressed in the intestinal epithelium [26]. e presence of Sec as the catalytic moiety of GPx was suggested to guarantee a fast reaction with the hydroperoxide and fast reducibility by GSH [27]. e selenol (-SeH) in SecGPxs reacts in form of a selenolate with H 2 O 2 to selenenic acid (-SeOH) which is reduced back to -SeH by two GSH forming GSSG and H 2 O [28,29]. us, MKAVCFSL might protect Sec in GPx from being attacked by H 2 O 2 . GR contains highly conserved domains, one of which can bind reduced nicotinamide adenine dinucleotide phosphate (NADPH) sustaining reduced GSH in cells.
e antioxidant activity of MKAVCFSL may decrease the NAPDH converting into NADP + contributing to the GR activity. Some studies have also verified the impact of oligopeptide and polyphenols on antioxidant enzyme activities in cells. It is demonstrated that the egg-derived peptide elevated GR, GST, and CAT enzymatic activity in human intestinal cells [30]. Other researchers have demonstrated phenolic compounds from olive oil significantly enhanced GR and GP activities in macrophages and verified that the impact of flavonoids on antioxidant protein expression was genespecific in Caco-2 cells [31,32].

Analysis of Cellular Antioxidants GSH.
GSH is a tripeptide consisting of glutamine, cysteine, and glycine and is a key component in the nonenzymatic antioxidant mechanism of cells [33]. e present study has found that GSH level was significantly decreased in the cells treated with 1 mM H 2 O 2 only ( Figure 5) compared to untreated cells. However, pretreatment of peptide supplementation for 24 h before the addition of H 2 O 2 restored GSH levels significantly. Meanwhile, MKAVCFSL alone could also elevate the GSH concentration in the cells. While GSSG, the oxidized state of glutathione, was not significantly influenced by the peptide without H 2 O 2 stimulation. H 2 O 2 alone augmented the concentration of GSSG from (3.06 ± 0.41) μM to (4.92 ± 0.63) μM compared to the C1 sample. Nevertheless, decreased GSSG concentration was observed in the S2 sample. Increased GSH concentration was unexpectedly in S1 since the activity of GPx and GR was not significantly different from untreated samples (C1). We inferred that the peptides may have a positive effect on GSH synthesis. Increased GSSG in C2 samples was also interesting because GPx and GR activity were both inhibited by excessive H 2 O 2 .

Journal of Food Quality
It could be speculated that the GPx showed effectively antioxidant activity to dispose oxidant stress initially. us, plenty of GSH was converted into GSSH. Besides, the toxicity of H 2 O 2 decreased GR activity, which also prohibited the GSSH from reducing back to GSH, thus bringing about the accumulation of GSSH. e decreased GSSH in S2 was in accordance with the restored activity of GR, which could catalyze GSSG to form GSH at high efficiency to promote GSH level in the cells. MKAVCFSL had the potential for normal activity and functionality in the H 2 O 2 model system to allow cells to maintain a balanced redox.
ese results validate the MKAVCFSL could protect the cells from oxidative stress damage.
Generally, MKAVCFSL enhanced GPx activity for subsequent lipid hydroperoxide and ROS induced detoxification using GSH and increased GR activity to regenerate GSH from GSSG. It also contributed to SOD activity without the addition of H 2 O 2 , but CAT was not influenced. e synergistic actions of cellular antioxidants, antioxidant enzymes, and the peptide efficiently ameliorated the damage of oxidative stress. e protective effect of MKAVCFSL against H 2 O 2 -induced injury may be due to two reasons correlated to its amino acids composition. On one hand, a peptide having radical scavenging activity can suppress free radicalmediated oxidation [34]. As a previous study earlier, MKAVCFSL contains more aromatic and hydrophobic amino acids and exhibited higher radical scavenging activity, which may be responsible for its protection [20]. In particular, phenylalanine and methionine residues in peptides and proteins were regarded as free radical scavengers in vivo [35]. On the other, some studies have exhibited that some peptides can prevent oxidative stress through reducing antioxidant enzyme capacity [30,34], which concurred with our results. Indeed, the cellular regulation of the antioxidant system is complicated and involves intricate signaling networks which is not fully understood. Besides, different signaling networks enhance the complexity and increase difficulties to understand redox control and related processes. Although the novel peptide was proved to have effective antioxidant activity, it is necessary to conduct a profound investigation on the relationship between oxidative stress and the antioxidant activity in vivo.

Conclusion
is study demonstrated that MKAVCFSL derived from bighead carp protein hydrolysates can remain stable through simulated gastrointestinal digestion, although part of the peptides were degraded into smaller fragments. e peptide could effectively scavenge ROS and MDA within the Caco-2 cells. Meanwhile, after being pretreated with the peptide, upregulation of the activity of several cellular antioxidant enzymes was observed including GPx, GR, and SOD in H 2 O 2 -induced Caco-2 cells. However, CAT was not influenced significantly. In addition, the concentration of endogenous antioxidant GSH was also increased, led by MKAVCFSL. In summary, this octapeptide displayed protective effects in Caco-2 cells and may be a potential protective agent or food additives against oxidative stress.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.