Antioxidant Mechanisms of the Oligopeptides (FWKVV and FMPLH) from Muscle Hydrolysate of Miiuy Croaker against Oxidative Damage of HUVECs

In this work, the antioxidant mechanisms of bioactive oligopeptides (FWKVV and FMPLH) from protein hydrolysate of miiuy croaker muscle against H2O2-damaged human umbilical vein endothelial cells (HUVECs) were researched systemically. The finding demonstrated that the HUVEC viability treated with ten antioxidant peptides (M1 to M10) at 100.0 μM for 24 h was not significantly affected compared with that of the normal group (P < 0.05). Furthermore, FWKVV and FMPLH at 100.0 μM could very significantly enhance the viabilities (75.89 ± 1.79% and 70.03 ± 4.37%) of oxidative-damaged HUVECs by H2O2 compared with those of the model group (51.66 ± 2.48%) (P < 0.001). The results indicated that FWKVV and FMPLH played their protective functions through increasing the levels of antioxidant enzymes including superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and decreasing the levels of reactive oxygen species (ROS), malondialdehyde (MDA), and nitric oxide (NO) in oxidative-damaged HUVECs in a dose-dependent manner. In addition, the comet assay revealed that FWKVV and FMPLH could dose-dependently protect deoxyribonucleic acid (DNA) from oxidative damage in the HUVEC model. These results suggested that antioxidant pentapeptides (FWKVV and FMPLH) could serve as potential antioxidant additives applied in the food products, pharmaceuticals, and health supplements.


Introduction
Bioactive peptides (BPs) produced from food resources are short amino acid sequences with 2 to 20 residues, which are inactive in the sequence of the original proteins [1][2][3]. Generally, BPs are liberated by protease hydrolysis during either food processing or gastrointestinal digestion in the body [4,5]. Based on the types of amino acids, sequences, and spatial structures, BPs can effectively perform multiple functions, such as anticancer, antioxidant, angiotensin-converting enzyme (ACE) inhibition, immunomodulatory, antithrombotic hypocholesterolemic, antibacterial, hypotensive, and hypolipidemic activities [2,6,7], and their beneficial func-tions, especially on high nutritional value, health promotion, and chronic disease adjuvant treatment and prevention, are being increasingly recognized [4,8].
Antioxidant peptides (APs) are one of the most popular types of BPs and have been isolated from animals, plants, and microorganism [9][10][11]. Liu et al. prepared the antioxidant peptide fraction from the processing of by-product of hazelnut, being composed of DWDPK, ETTL, ADGF, AGGF, AWDPE, and SGAF, which showed high antioxidant and protective ability against angiotensin (Ang) II-caused oxidant damage by upregulating the levels of superoxide dismutase (SOD) and heme oxygenase-1 (HO-1) and downregulating the level of xanthine oxidase-1 (XO-1) to control the generation of reactive oxygen species (ROS) in human umbilical vein endothelial cells (HUVECs) [12]. An antioxidant peptide YD1 from Bacillus amyloliquefaciens with average molecular weight (MW) of ∼1.0 kDa could strongly reduce the levels of ROS and nitric oxide (NO) and improve the levels of antioxidant enzymes by heightening the transcriptional and translational ability of NF-E2-related factor-2 (Nrf-2) in murine macrophages (RAW 264.7) [13]. EVSGPGLSPN from walnut showed high protection on PC12 cells against H 2 O 2 -caused neurotoxicity by controlling the nuclear factor kappa-B (NF-κB)/caspase pathways and improving the activity of antioxidant enzymes, including glutathione peroxidase (GSH-Px), SOD, and catalase (CAT) [14].
At present, protein hydrolysates and BPs have been prepared from the processing of by-products of multifarious edible marine organisms, such as mollusks, fishes, and seaweeds, and many of them showed significant activities [2,[15][16][17]. Pan et al. found that four oligopeptides (VPR, IEPH, LEEEE, and IEEEQ) from protein hydrolysate of red stingray cartilages exhibited strong lipid peroxidation inhibition activity, Fe 2+ -chelating ability, reducing power, and radical scavenging activity [7]. Research by Oh et al. indicated that blue mussel-α-chymotrypsin hydrolysate (BMCH) showed high radical scavenging activity and inhibitory activity on Cu 2+mediated low-density lipoprotein (LDL) oxidation. Moreover, BMCH could improve the viability of HUVECs and lower ROS generation through downregulating gene expression of p53 and caspase-3, as well as decreasing the ratio of Bcell lymphoma 2-(bcl-2-) associated X (bax)/bcl-2 [18]. In addition, YPPAK, PIIVYWK, TTANIEDRR, and FSVVPSPK from blue mussel hydrolysate exhibited strong radical scavenging activity, and PIIVYWK and FSVVPSPK could activate of HO-1 gene expression to play their hepatoprotective effects [15,19]. Chen and Hou reported that skin gelatin hydrolysate of Pacific cod could protect skin injury caused by ultraviolet (UV) radiation because the gelatin hydrolysate could restrain the decrease in endogenous antioxidases and hold back the expression of proinflammatory cytokines and NF-κB [20]. Those studies confirm that APs could serve as antioxidant agents to inhibit the influences associated with oxidative stress in food and living organisms through enhancing the levels of endogenous antioxidant enzymes to prevent or repair the process of oxidation resulting from ROS [9,15]. Furthermore, these APs showed excellent properties including being easy to absorb, having low MW, and being less toxic or having less side effect and become high-quality ingredients for healthy products and food processing/preservation industries [21,22].

2.2.
Culture and the Viability Assay of HUVECs. The culture method of HUVECs and the cell viability assay were carried out in accordance with the previous method of Cai et al. [23] and Lim et al. [24]. The MTT assay was replicated for three times and applied to determine the viability of HUVECs. In brief, the basal HUVECs were incubated in a 96-well plate for 24 h. After that, the basal HUVECs were cultured in the sample solutions with the concentration of 100 μM. After 12 h, the wells were rinsed with PBS two times and the MTT was added to the wells with the final concentration of 0.5 mg/mL. After being incubated for 4 h, the active cells formed formazan crystals, which were added into 150 μL of DMSO and well combined. Finally, the cell viability was determined according to the absorbance at 570 nm of the blank control group (A control ) and the sample group (A sample ) by the following formula:  [23]. In addition, the levels of antioxidases (SOD and GSH-Px) and oxidation-related indicators (NO and MDA) were measured using assay kits in accordance with the manufacturer's protocols (Nanjing Jiancheng Bioengineering Institute Co., Ltd., China), and the BCA method was employed to determine the protein concentrations for normalizing the levels of antioxidases (SOD and GSH-Px). The level of ROS was indicated as % of blank control values. The levels of antioxidases (SOD and GSH-Px) were indicated as U/mg prot (units of enzymatic activity/mg protein).  [25]. Glutathione (GSH) served as the positive control in the experiment. The DNA comet assay was carried out in accordance with the method described by Cai et al. [23] and applied to analyze the protective ability of FWKVV (M3) and FMPLH (M5) on DNA damage in the oxidative damage model of HUVECs.
2.7. Statistical Analysis. All the assays were performed more than three times, and the resulting data were indicated as means ± standard deviation (SD). The mean value of each treatment was analyzed using the ANOVA test (SPSS Statistics 22.0 software). Duncan's multiple range tests were applied to analyze the significant differences of different groups (P < 0:05, P < 0:01, and P < 0:001).

Results and Discussion
3.1. Influence of APs (M1-M10) on the Viability of HUVEC. HUVECs are separated from the umbilical cord vein and play a major role as a model system for the analysis of the regulation of endothelial cells and the effect of antioxidants on endothelial cells and protection from oxidative damage by peroxide, measured by cell viability and its effect on apoptosis [5,12]. Figure 1 demonstrates the influence of APs (M1-M10) on cell viability. At the concentration of 100 μM, the viability of HUVECs incubated with IRWWW (M10) for 24 h was 93:59 ± 6:41%, which was inferior to those of the blank control and other peptide groups. On the contrary, the viability of cells incubated with YFLWP (M6) was 109:39 ± 9:39%, which was superior to those of the blank control and other peptide groups. However, there was no significant difference between the control group and peptide-treated groups under the same conditions (P < 0:05). Cell viability is a determination of living or dead cells, based on a total cell sample. Viability analysis has a tremendous number of applications, especially calculating the effect of antitumor drug candidates on cancer cells and the cytotoxic activity of chemical compound on normal cells [26][27][28]. Therefore, ten isolated APs (M1-M10) from miiuy croaker revealed the possibility for exploiting nontumor healthy products because they had no significant influence on the conventional proliferation of HUVECs.
In the experiment, H 2 O 2 was used to produce hydroxyl radicals ( • OH) and oxygen radicals, which further induce oxidative stress in cells [27]. Herein, the oxidative damage model built with H 2 O 2 was usually applied to evaluate the antioxidant activity and investigate their molecular mechanisms concerned on the pathogenesis of ROS-caused oxidative injury. Figure 2 shows that the viability of HUVECs decreased with the increase in H 2 O 2 concentrations when the H 2 O 2 concentrations ranged from 100 μM to 350 μM. In addition, it was found that the viability of HUVECs reduced to 51:66 ± 2:48% at the H 2 O 2 concentration of 200 μM, and there was significant difference with the other tested groups (P < 0:05). Then, 200 μM was recognized as the optimal concentration of H 2 O 2 for establishing the oxidative damage model of HUVECs.

Protective Effects of the APs (M1-M10) on the Oxidative Injury of HUVEC by H 2 O 2 .
The protective ability of the 10 APs (M1-M10) on the oxidative injury model of HUVEC caused by H 2 O 2 is presented in Figure 3. The results indicated that the cell viabilities of M1-M10-treated groups ranged from 55:90 ± 2:95% to 75:89 ± 1:79%, which were superior to that of the oxidative damage model group (51:66 ± 2:48%). However, the cell viabilities of M1-M10treated groups were lower than that (82:90 ± 3:05%) of the positive control group (NAC). In addition, the HUVEC viability of the FWKVV-(M3) and FMPLH-(M5) treated group was 75:89 ± 1:79% and 70:03 ± 4:37% at the concentration of 100.0 μM, which was very significantly (P < 0:001) superior to that of the oxidative damage model group       Figure 6. The data demonstrated that there was an extremely (P < 0:001) significant difference between the blank control group and the oxidative damage model group (231:7 ± 12:8% of blank control). The finding proved that the induced oxidative damage caused by H 2 O 2 resulted in a significant increase in ROS levels in the model group. In peptide groups, the intracellular ROS levels were significantly decreased by FWKVV (M3) and FMPLH (M5) pretreatment in a concentrationdependent manner. Moreover, FWKVV (M3) showed stronger activity of ROS scavenging than FMPLH (M5) did. At the concentrations of 10 μM, 50 μM, and 100 μM, the intracellular ROS levels of the FWKVV-(M3) incubated group were 178:2 ± 11:7%, 152:3 ± 8:9%, and 133:4 ± 9:6% of the blank control group. The data indicated that FWKVV (M3) and FMPLH (M5) could play an important role in helping HUVECs from oxidative stress damage through decreasing the levels of ROS.

Effects of FWKVV (M3) and FMPLH (M5) on the Levels of Antioxidases (SOD and GSH-Px) and Oxidation-Related
Indicators (NO and MDA) in the Oxidative Damage Model of HUVECs. The endogenous antioxidant enzymes, such as SOD, GSH-Px, CAT, and GSH-Rx, and nonenzymatic antioxidants form the body's endogenous defense mechanisms to protect vital biomolecules and ultimately body tissues against oxidative damage in cells through catalyzing reactions to neutralize ROS [29]. Under oxidative stress, uncontrolled generation of ROS will be beyond the normal processing capacity of this endogenous defense mechanism, and additional antioxidants are needed to help control oxidative stress damage [2,30]. Hence, the influences of FWKVV (M3) and FMPLH (M5) on the levels of antioxidases (SOD and GSH-Px) and oxidation-related indicators (NO and MDA) in HUVECs were determined and are shown in Figure 7 for explaining their protective action on HUVECs against H 2 O 2 -induced oxidative damage. Previous literatures indicated that some protein hydrolysates and BPs displayed intracellular antioxidant functions through controlling the levels and gene expression of antioxidant enzyme in cells and organisms [2]. Homayouni-Tabrizi et al. reported that YLEELHRLNAGY from camel milk protein hydrolysate could significantly increase the gene expression of SOD and CAT in treated HepG2 cells [31]. Antioxidant peptides derived from Pinctada fucata protein played a protective role in the ultraviolet-induced photoaging of mouse skins by significantly controlling the speed of lipid peroxidation and the reduction of the activity of SOD, GSH-Px, hydroxyproline, and CAT [32]. IYVVDLR and IYVFVR from the hydrolysate of soybean protein were able to protect Caco-2 cells against H 2 O 2 -induced oxidative damage via significantly downregulating intracellular ROS generation and lipid peroxidation, statistically upregulating total reduced glutathione (GSH) synthesis, enhancing activities of CAT and GSH-Px, and suppressing ROS-mediated inflammatory responses via inhibiting interleukin-8 secretion (P < 0:05) [33]. In an in vivo experiment, bovine hair hydrolysates (BHP) (with a major sequence of CERPTCCEHS) [34] and oyster meat hydrolysates by Alcalase (OMA) [35] showed a strong capability to decrease the MDA level and enhance the levels of endogenous cellular antioxidases, including SOD, CAT, and GSH-Px. Those findings in the experiment suggested that FWKVV (M3) and FMPLH (M5) had similar cytoprotective functions with those reported antioxidant peptides because they all could reinforce endogenous antioxidant defense systems to help cells against the H 2 O 2 damage. In the experiment, the pBR322 DNA was cleaved by the hydroxyl radicals generated from the decomposition of H 2 O 2 mediated by Fe 2+ , which changed the SC form into the OC form (Figure 8(h)). In addition, the trace LIN form of pBR322 DNA was discovered as shown in Figure 8

Protective Capacities on DNA against H 2 O 2 -Induced
Injury in the HUVEC Model. Due to the imbalance between the oxidants and antioxidants, redundant ROS is generated and then attacks DNA in cells, which further leads to adverse physiological and biochemical reactions. At present, several methods are applied to estimate the DNA damage in cells, and the comet assay is believed to be a fairly simple, sensitive, and versatile way in single cells to quantitatively and qualitatively evaluate the effects of DNA damage and DNA repair [37]. In the assay, an electrical current is applied to a slide featuring the cell in a gel. The broken strands of DNA will get away from their initial position towards the positive charge and leave a comet trail, which will be easily observable through fluorescence [38]. The comet trail can give out many quantitative data on the degree of DNA damage, which can serve to evaluate the protective effects of antioxidant compounds on DNA.
In the blank control group, the comet with a bright head and almost without comet tail (Figure 9(a)) indicated that the DNA in HUVEC was in a normal state, but the comet with a long and large tail in the oxidative damage model group (Figure 9 (Figure 9(c)).
The images in Figure 9 on the comet tail length or comet area could display visually the experimental results. Moreover, the comet assay had employed some indexes including torque class indicator (Olive tail moment, OTM), head DNA (HDNA), and tail DNA (TDNA) for being more conducive to accurate analysis of the results (Tables 1 and 2). These indexes demonstrated that the HDNA was extremely prominently lower than that of the blank control group (P < 0:001); the tail moment (TM), tail length (TL), OTM, and TDNA in the oxidative damage model group were extremely prominently higher than those in the blank control group (P < 0:001), and the comet length (CL) in the oxidative damage model group was significantly higher than that of the blank control group (P < 0:05). With the increase in the concentration of FWKVV (M3) and FMPLH (M5) from 50.0 μM to 200.0 μM, the TDNA, CL, TL, TM, and OTM decreased gradually, but the HDNA of the comet increased step by step. Furthermore, all the measured indicators of FWKVV (M3) and FMPLH (M5) groups at the concentrations of 100 and 200 μM, except the CL of the FMPLH (M5) group at 100 μM, showed extremely significant differences with those of the model groups (P < 0:001). At the concentration of 50 μM, the HDNA, TL, and TM of FWKVV (M3) and FMPLH (M5) groups showed very significant (P < 0:01) differences with those of the model groups, respectively; the TDNA and OTM of FWKVV (M3) and FMPLH (M5) groups showed very significant differences with those of the model groups, respectively (P < 0:01); the OTM of FWKVV (M3) and FMPLH (M5) groups showed significant (P < 0:05) differences with that of the model groups, respectively, but the CL of FWKVV (M3) and FMPLH (M5) groups showed no significant differences with that of the oxidative damage model groups (P > 0:05). In consequence, the results of the comet assay demonstrated that FWKVV (M3) and FMPLH (M5) showed significantly protective capacity on DNA in the oxidative damage model groups induced by H 2 O 2 . In addition, FWKVV (M3) showed stronger protective effect on DNA in the H 2 O 2 -induced HUVEC injury model than FMPLH (M5) did at the same concentrations. Attack of DNA by ROS produced under conditions of oxidative stress can induce strand breaks, DNA-DNA and DNA-protein crosslinking, and formation of at least 20 modified bases adducts, which play a key role in aging, cancer, arteriosclerosis, neurodegenerative diseases, and diabetes [39,40]. The antioxidant hydrolysate and BPs are stage by stage accepted as food components applied in functional food and nutraceuticals to effectively adjust and control the oxidative injury to protect DNA, lipid, and protein in the human body [41][42][43]. Hoki frame protein hydrolysate (APHPH) could decrease t-butyl hydroperoxide-caused cytotoxicity on human embryonic lung fibroblasts and observably protect DNA against the free radical-induced injury [44]. Similarly, protein hydrolysates prepared from Nile tilapia [45] and shrimp shell [46] could effectively inhibit the DNA scission caused by H 2 O 2 and peroxyl radical in a dose-dependent manner. YGDEY could protect HepG2 cells against alcohol-caused oxidative damage by controlling oxidative stress including reducing the degree of DNA damage. The antioxidant mechanism might be bound up with the Akt/NF-κB/MAPK signal transduction pathways [47]. Sheih reported that VECYG (VG5) presented the protective ability on DNA against oxidative injury, so it further reinforced the capacity of the APs to protect hydroxyl radical-caused injury [48]. WAFAPA and MYPGLA prepared from the protein hydrolysate of the blue-spotted stingray were superior to carnosine in their capacity to control lipid oxidation caused by H 2 O 2 . In addition, WAFAPA and MYPGLA could help plasmid DNA against oxidative injury caused by Fenton's reagent [21]. In our previous report, FWKVV (M3) and FMPLH (M5) have been found to have strong lipid peroxidation inhibition, reducing power, and radical scavenging activities [3], and the current study finding demonstrated that FWKVV (M3) and FMPLH (M5) could weaken the oxidative injury of HUVECs caused by H 2 O 2 through enhancing the contents of endogenous antioxidases (SOD and GSH-Px), lowering the contents of oxidation-related indicators (NO and MDA), and helping DNA from oxidative injury.

Discussion
Oxidative stress contributes to cell pathogenesis [49], ROSmediated damage to DNA [39,40], and an altered mitochondrial function [50]. The oxidative damage to DNA is considered a key factor in vascular disorder [51,52]  10 Oxidative Medicine and Cellular Longevity Excessive oxides deplete tissues of GSH and impair antioxidant defense systems in humans [53,54]. How to increase the level of GSH becomes important. In this experiment, we observed that the levels of GSH in HUVECs had a marked increase in the groups with the treatment with M3 or M5. The results for the potential mechanism of the promoting production of GSH by M3 or M5 might contribute to a broader biological effect of a protective nature with regard to the general detoxification of environmental agents. No evidence was found that GSH was able to pass through cell membranes freely; thus, M3 or M5 must modulate GSH levels by stimulating cells to synthesize GSH intracellularly.
The results indicate that M3 or M5 does protect cells from oxidative stress damage, and the protection includes its capacity to stimulate GSH synthesis.
Cells are in a stable state known as redox homeostasis under normal physiological conditions. Redox homeostasis is maintained by the balance between continuous ROS generation and several mechanisms involved in antioxidant activity [30,55]. An overwhelming production of ROS leads to a prooxidant state also known as oxidative stress, which is a leading factor in the pathogenesis of vascular disorder complications [2,56,57]. NOX is a major contributor to ROS generation in endothelial cells [58]. Discrete compartmental redox signaling is demonstrated by Nrf2-dependent activation of ARE. Nrf2 is a redox-sensitive transcription factor that is activated by an oxidative signal in the cytoplasm. The Nrf2 pathway is regarded as the most important in the cell to protect against oxidative stress [40,59]. Upon extracellular stimulation, the nucleus translocation of Nrf2 induces the transcription of several antioxidant genes, such as NADPH quinineoxidoreductase-1 (NQO-1), heme oxygenase 1 (HO-1), and SOD [60][61][62]. SOD and GSH-Px are two important antioxidant enzymes that remove toxic free radicals [29,47]. Our study revealed that intracellular ROS were significantly increased when HUVECs were cultured in 200 μM H 2 O 2 media. Treatment with M3 or M5 reduced H 2 O 2 -induced ROS production. The present study also showed that SOD and GSH-Px activity markedly decreased in HUVECs and increased the levels of the key cytotoxic lipid peroxide MDA and NO production when they were exposed to 200 μM H 2 O 2 for 24 h, which was blocked by M3 or M5 cotreatments. Major intracellular antioxidant defenses are the GSH pool and ROS-scavenging enzymes such as GSH-Px and SOD. Increased levels or activation of these endogenous antioxidants or enzymes has been shown to protect cells against oxidative damage. In this study, a marked decrease indeed occurred in the level of GSH after the treatment with H 2 O 2 ; as a result, the decreased levels of GSH might be attributed to the less decreased activity of GSH-Px. The imbalance of the H 2 O 2 -induced antioxidant status in HUVECs, such as the changes in the activity of antioxidant enzymes and the depletion of GSH, might be major causes of cell injury. Nevertheless, the cytoprotective effects of M3 or M5 may be mediated, in part, by activation of Nrf2, a redox-regulated transcription factor that binds to the antioxidant response element (ARE). These data suggest that H 2 O 2 -induced ROS production exceeds the natural antioxidant capacity and leads to oxidative stress and that M3 or M5 may exert its protective effect on HUVECs by reducing ROS accumulation and increasing antioxidant enzyme activity.
Finally, in this study, we demonstrated that FWKVV (M3) and FMPLH (M5) could significantly protect cells against H 2 O 2 -induced oxidative stress, DNA damage, and cell apoptosis in cultured HUVECs. Treatment with M3 or M5 reduced the loss of cell viability, improved the antioxidant capacity, and reduced the oxidative damage in HUVECs. Further, we hypothesized that the protective effect of M3 or M5 appears to be mediated by the regulation of the Nrf2 signaling pathway and the maintenance of cellular redox homeostasis. This finding further supports the concept that M3 or M5 may have potential as a vascular protective drug.

Conclusion
In this work, the protective activity and antioxidation mechanisms of FWKVV (M3) and FMPLH (M5) on HUVECs against H 2 O 2 -caused oxidative injury were researched carefully. When incubated for 24 h, no significant difference was found on the viability of HUVECs between the blank control group and the AP (M1-M10) groups at 100 μM (P < 0:05). Moreover, FWKVV (M3) AND FMPLH (M5) could significantly protect HUVECs from H 2 O 2 -induced oxidative damage by enhancing the contents of endogenous antioxidases, lowering the levels of oxidative products, and helping DNA from oxidative injury. These results suggested that FWKVV (M3) and FMPLH (M5) could serve as great potential antioxidants in the health care products. In addition, the molecular mechanism and in vivo antioxidant experiments of FWKVV (M3) and FMPLH (M5) will also be carried out in our laboratory.

Data Availability
The datasets are available from the corresponding author on reasonable request.

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Oxidative Medicine and Cellular Longevity