Skipjack (Katsuwonus pelamis) elastin hydrolysate-derived peptides attenuate UVA irradiation-induced cell damage in human HaCaT keratinocytes

We aimed to identify skipjack elastin hydrolysate-derived peptides, and explore its mechanism of photoprotection against ultraviolet A (UVA) induced skin damage. Two peptides, TGVLTVM (peptide A) and NHIINGW (peptide B), were successfully identified using Sephadex G-15, analyzed and detected using LC-MS/MS fitted with electrospray ionization (ESI-MS/MS), and further synthesized using the Fmoc strategy. A UVA radiation-induced damage cell model of Human keratinocytes cell line (HaCaT) was established and applied. We found that both peptides significantly reduced reactive oxygen species (ROS) generation, reversed mitochondrial membrane potential (MMP) by 12.40%–13.13% and 10.80%–12.90% and reduced cell apoptosis by 3.43%–6.44%, and4.56%–6.03%forpeptideAandB,respectively,comparedtotheUVAmodelgroup. In conclusion, peptides TGVLTVM and NHIINGW protected against UVA-induced photoaging through the attenuation of oxidative stress, thus both peptides might be useful as promising ingredients in sunscreens or antiaging cosmetic products. peptide B decreased by 10.80% ± 0.72% and 12.90% ± 3.35% for 0.5 and 0.05 mM, respectively, compared with the UVA group. Accordingly, the results suggest that peptides A and B may reverse MMP decline.

skin's inherent antioxidant capacity, inducing cell damage, photoaging, and/or skin cancer (Wenk et al., 2001). The overproduction of ROS also drives the pro-apoptotic proteins, Bax and Bak, to translocate into the mitochondrial outer membrane, inducing the release of cytochrome C and subsequently activate caspases and trigger cell apoptosis program (Lopez & Tait, 2015;Tait & Green, 2010), ultimately leading to apoptosis in HaCaT cells (Tada-Oikawa, Oikawa, & Kawanishi, 1998).
Therefore, through the generation of ROS, UVA irradiation has been shown to be a particularly potent inducer of DNA damage, serious oxidative stress and cell death.
Elastin is the core protein (>90%) of elastic fibers and gives elasticity to tissues, such as skin, aorta, ligaments, and lungs (Mithieux & Weiss, 2005). A small amount of elastin in the dermis allows for the elasticity and flexibility of the skin. However, breakdown and loss of elastic fiber function becomes prevalent with aging (Zhang & Duan, 2018). Food-derived peptides have been suggested to induce biological responses including enhanced synthesis of elastin, fibroblast growth, and the extracellular matrix, all of which are beneficial for the skin (Shigemura et al., 2012;Tajima, Wachi, Uemura, & Okamoto, 1997).
hydrophobic protein-derived peptides from natural materials including food have long attracted tremendous research attention due to their antioxidative properties (Hattori, Hirotomo Kumagai, Kumagai, Feng, & Takahashi, 1998). Food-derived elastin peptides such as Pro-Hyp, Pro-Hyp-Gly, Ala-Hyp, and Ala-Hyp-Gly (Shigemura et al., 2012) have been shown to be potentially valuable in skin care, but detail mechanism is largely lacking. In this regard, it is suggested that ingestion of elastin hydrolysate might improve the condition of the skin. Recent consumer preference for marine products, the availability and relatively low cost coupled with its demonstrated presence of elastin peptides and biological potency of skipjack (Shiratsuchi, Nakaba, Shigemura, Yamada, & Sato, 2013) makes it a good candidate for further exploring the mechanism of photoaging. We hypothesized that skipjack elastin hydrolysate small peptides have free radicals scavenging ability. Because UVA-induced cell damage is primarily ascribed to the deleterious effects of ROS, molecules with free radical scavenging properties are particularly promising as radio-protectors. In the present study, we focused on identifying small peptides from the skipjack elastin hydrolysate with various bioactivities, especially in free radicals scavenging and explored the mechanism for their photoprotective effects.

Measurement of oxygen radical absorbance capacity (ORAC)
The ORAC of the synthesized peptides was evaluated according to the procedure described by Prior R.L (Prior et al., 2003). Analyses were conducted by automated plate reader in phosphate buffer (PBS,pH 7.4) at 37 • C. The measurements were made in plates with 96 black flatbottom wells. Trolox was used as the standard solution. Fluorescein (FL) was used as the fluorescent substrate. The reaction was started by thermal decomposition of 2,2-Azobis(2-Amidinopropane) Hydrochloride (AAPH) (Sigma, St. Louis, MO, USA) in 75 mM phosphate buffer (pH 7.4). In each well, 200 uL of FL (78 nM) and 20 uL of sample, blank (PBS), and standard (Trolox) were placed, and then 20 uL of AAPH (119.4 mM) was added. The mixture was preheated at 37 • C for 15 minutes prior to the addition of AAPH. The reaction was activated by the addition of AAPH. Fluorescence was measured at 5-minute intervals for 180 minutes at an excitation of 485 nm and an emission of 538 nm. The measurements were taken in triplicate. The ORAC values were calculated by applying formula (1) and expressed as μmol Trolox equivalents/μmol peptide (μmol TE/μmol peptide).

ORAC
( μmol TE mol peptide In formula (1), C Trolox is the concentration (μM) of Trolox, k is the sample dilution factor, and AUC is the area below the fluorescence decay curve of the sample, blank or Trolox, respectively, which is calculated by applying formula (2) in a Microsoft Excel spreadsheet (Washington, USA).
In formula (2), F 0 is the initial fluorescence and F n is the fluorescence at time n.

Cell culture
Human keratinocyte, HaCaT, cell line was maintained at 37 • C in an incubator with a humidified atmosphere of 5% CO 2 . The cells were cultured in minimum Eagle's medium containing 10% heat-inactivated fetal bovine serum.

UVA irradiation intensity and cell viability
HaCaT cells were seeded into 96-well plates at a density of 3 × 10 3 cells/well and maintained in culture medium for 24 hours. Before exposure to UVA irradiation, the media in each well was replaced by PBS to eliminate possible phototoxic reaction of some components in the culture medium. Cells were then treated with various intensities (2.8, 5.6, and 10 J/cm 2 ) of UVA light after the confluence reached 80%-90%. To prevent the cells from overheating during irradiation, plates were kept on ice during the whole test. Cells without UVA irradiation were set as the negative control. UVA irradiation were supplied by two paralleled UVA lamps with a peak emission frequency of 365 nm at 8000 μW/cm 2 output (emission spectrum: 320-400 nm). Cells were irradiated at a distance of approximately 20 cm. The irradiation intensity was measured by a UVA radiometer, which was put at the same distance from the UVA source as the cells. It took about 3 hours to reach the highest UVA irradiation intensity of 10 J/cm 2 . After UVA irradiation, cells were incubated with fresh complete medium at 37 • C for 24 hours.
The effect of the different UVA irradiation doses on cell viability was monitored using the MTT (3-(4-5-dimethylthiazol-2-11)-2, 5-diphenyl tetrazolium bromide) assay. MTT assay depends on the principle that mitochondrial dehydrogenase reduces tetrazolium salt in viable cells. In brief, 10 μL of 5 mg/mL MTT was added to each well to rich a final concentration of 0.5 mg/mL. After incubation at 37 • C for 4 hours in light-proof conditions, the supernatants were aspirated. Formazan crystals in each well was dissolved in Dimethyl sulfoxide (DMSO, 150 μL) and the plate was further analyzed by Tiangen microplate reader (Beijing, China) at a wavelength of 490 nm.

Cell viability of the peptides under UVA irradiation
The effect of the peptides on the viability of cells under UVA (2.8, 5.6, 10 J/cm 2 ) treatment was monitored by MTT essay. HaCaT cells were seeded into 96-well plates at a density of 3×10 3 cells/well. When the confluence reached 80%-90%, cells were pretreated with culture medium containing predetermined (results not shown) doses (0.5 and 0.05 mM) of the peptides for 14 hours. Cells preincubated with peptides and without UVA irradiation were set as the control. Other cells were treated with 10 J/cm 2 UVA irradiation and then incubated for 24 hours at 37 • C. Finally, MTT essay was performed by the same protocol as mentioned in 2.4.1. Considering that 10 J/cm 2 is the actual daily UVA intensity to the human skin (10J/cm 2 ≈ 30 minutes of sun exposition, at 12 noon in Central Europe) (Mapelli, Calo, & Marabini, 2016), 10 J/cm 2 UVA irradiation was chosen and applied for the subsequent assays.

Measurement of intracellular ROS
Intracellular ROS was assessed after cells were pretreated with the synthesized peptides (0.5 and 0.05 mM) and then exposed to 10 J/cm 2 UVA irradiation. Production of intracellular ROS was quantified according to the method of Wang and Joseph using fluorometric assay with the cell-permeable, nonfluorescent probe 2′7′-dichlorofluorecin diacetate (DCFH-DA) by performing a fluorometric assay (Wang & Joseph, 1999). This probe is a stable compound that readily diffuses into cells and is hydrolyzed by intracellular esterase to yield DCFH, which could be trapped within cells and subsequently changes to

Measurement of mitochondrial membrane potential (MMP)
The mitochondrial membrane potential of cells was assessed using JC-1 staining. The JC-1 fluorescent probe, which enters the mitochondria and changes from red to green as the membrane potential declines, was used to estimate mitochondrial polarity. In brief, cells were seeded at 2.5 × 10 5 cells/well in a 6-well plate and incubated at 37 • C for 12 hours. After growing adhesively, the cells were, respectively, treated with peptides A and B (0.5 and 0.05 mM) for 14 hours before being exposed to 10 J/cm 2 UVA irradiation. After irradiation, JC-1 (10 μM) was added to the cells and incubated at 37 • C for 20 minutes, following the manufacturer's instructions. After that, cells were washed with PBS twice and images were visualized by fluorescence microscope. Besides, cells under the same treatment were collected and detected by flow cytometry. For each tube, 500 μL JC-1 staining buffer was added and then cells were incubated at 37 • C for 30 minutes. Cells were then washed once by adding 1.5 mL of PBS to each tube and resuspended in a final volume of 500 μL PBS for flow cytometry. Flow cytometry was performed using 488 nm excitation, green and red filters for detection.
The ratio of the intensity of green fluorescent monomers to the intensity of JC-1 aggregates reflects the mitochondrial potential. Therefore, mitochondrial membrane potential was expressed as the ratio of red/green fluorescence and the fraction of damaged cells.

Apoptosis analysis by Annexin V-Fluorescein Isothiocyanate (FITC) staining
Staining was performed according to the Annexin V-FITC apoptosisdetection kit protocol to determine whether the synthesized peptides had protective effects on UVA-exposed HaCaT cells by reducing apoptosis. Phosphatidylserine (PS) is translocated from the inner to the outer layer of the plasma membrane in apoptotic cells. Therefore, Annexin V, which is a Ca 2+ -dependent phospholipid-binding protein with high affinity for PS, can be used for identifying apoptotic cells (Mapelli et al., 2016). Briefly, cells were seeded at 2.5 × 10 5 cells/well in a 6-well plate and incubated at 37 • C for 12 hours. After growing adhesively, the cells were respectively treated with peptides A or B (0.5 and 0.05 mM) for 14 hours before exposure to 10 J/cm 2 UVA irradiation. Both floating and attached cells which received UVA irradiation were harvested with 0.25% EDTA-trypsin. Then, the cells were washed twice with PBS and incubated with Annexin V-FITC for 20 minutes at room temperature. Thereafter, propidium iodide was added and then incubated for 10 minutes.

Statistical analysis
All measurements were performed in triplicates. All statistical analysis was carried out using the Origin software version 8.0 (Origin-Lab. Co, USA). Data are presented as means ± SD. Group comparison was analyzed using paired t-tests. Results were classified as significant (p < 0.05), highly significant (p < 0.01), or extremely significant (p < 0.001). The peptides A and B with molecular structure as shown in Figure 1c were confirmed to be the main constituents of the peak sample by both database search and ion comparison.

In vitro antioxidant activity of peptides
The in vitro antioxidant activity, Oxygen Radical Absorbance Capacity (ORAC), of the two peptides was determined by ORAC assay. The results (Figure 1d)

Peptides A and B increases the viability of UVA-induced HaCaT cells
In order to evaluate the cytotoxicity of UVA irradiation, the viability of HaCaT cells exposed to UVA irradiations of 2.8, 5.6, and 10 J/cm 2 were measured by MTT assay. After UVA irradiation, obvious changes in cell morphology were observed under microscope (Figure 2a). Compared with the control group, cells in the UVA (model) group exposed to UVA irradiation detached from the wall, shrank, became round, and ± 7.30% (p < 0.05), 22.92% ± 5.19% (p < 0.001), and 73.33% ± 5.30% (p < 0.01) under 2.8, 5.6, and 10 J/cm 2 UVA, respectively, compared with the normalized UVA group (Figures 2d-f). The results showed that, peptides A and B exhibited no apparent cytotoxicity on HaCaT cells, making them good candidates for further exploration.

ROS scavenging capacity of peptides A and B in UVA-induced HaCaT cells
MTT was performed to analyze the capacity of peptides A and B to scavenge ROS induced by UVA irradiation in HaCaT cells. 10 J/cm 2 UVA irradiation was used because that is the actual daily UVA intensity to the human skin (10 J/cm 2 ≈ 30 minutes of sun exposition, at 12 noon. in Central Europe) (Mapelli et al., 2016). A cell-permeable, nonfluorescent probe 2′7′-dichlorofluorecin diacetate (DCFH-DA) was used to perform a fluorometric assay. This probe is a stable compound that readily diffuses into cells and is hydrolyzed by intracellular esterase to yield DCFH, which could be trapped within cells and subsequently change to highly fluorescent 2′,7′-dichlorofluorescein upon oxidation by intracellular ROS. Therefore, the fluorescence intensity is proportional to the amount of cellular ROS generation (Chen, Zhong, Xu, Chen, & Wang, 2010). As shown in Figure 3a, an increase in fluorescence

F I G U R E 2 Effect of peptides A and B on cell viability. Cell viability was determined by MTT assay and measured by microplate reader. (a)
Morphology of HaCaT cells before (control group) and after UVA irradiation (UVA group). (b) Cell viability of HaCaT cells treated with 0, 2.8, 5.6, and 10 J/cm 2 UVA irradiation, respectively. (c)-(f) Cell viability of HaCaT cells pretreated (14 hours) with peptides A and B under 0, 2.8, 5.6, and 10 J/cm 2 UVA irradiation. *p < .05; **p < .01; ***p < .001 compared with the UVA group intensity was observed in cells irradiated with UVA (10 J/cm 2 ), which was reduced in the presence of peptides A and B. Quantitative analysis of the fluorescence intensity showed that UV irradiation significantly increased (p < .05) intracellular levels of ROS. However, pretreatment with peptides A and B remarkably inhibited the generation of ROS (p < .05) ( Figure 3b). As shown in Figure 3c, the mean fluorescence intensity in cells treated with of 0.5 and 0.05 mM of peptide A, reduced by 56.57% ± 12.62% and 64.86% ± 9.52%, respectively, whiles peptide B treatment (0.5 mM) showed a 40.68% ± 15.67% reduction in mean fluorescence intensity. However, the change induced by peptide B at 0.05 mM did not reach significant levels. Thus peptides A and B effectively reduced intracellular ROS generation and/or accumulation.  (Qian, Jung, & Kim, 2008). Fluorescence images taken by fluorescence microscope were shown in  (Figure 4d). UVA irradiation caused severe cell damage (25.20% ± 2.01%); however by treatment with peptides, the damage was recovered as reflected by the fraction of damaged cells decreasing by 12.40% ± 0.28% (0.5 mM) and 13.13% ± 3.96% (0.05 mM) in peptide A, while that of peptide B decreased by 10.80% ± 0.72% and 12.90% ± 3.35% for 0.5 and 0.05 mM, respectively, compared with the UVA group. Accordingly, the results suggest that peptides A and B may reverse MMP decline.

Peptides A and B ameliorate UVA-induced apoptosis rate in HaCaT cells
To observe the apoptosis rate of UVA-irradiated HaCaT cells, nuclear morphology was evaluated by fluorescence microscopy using Hoechst 33258 staining. As shown in Figure 5a, the control group showed clear images indicating very rare apoptotic cells. However, in the UVA group (10 J/cm 2 UVA), shrunk, bright nuclei, and condensed chromatic cells typical of cell apoptosis were observed. Interestingly, the number of apoptotic nuclei in cells treated with peptides A and B decreased, revealing the protective effect of the two peptides. Phosphatidylserine (PS) is translocated from the inner to the outer layer of the plasma membrane in apoptotic cells. Therefore, Annexin V, which is a Ca 2+dependent phospholipid-binding protein with high affinity for PS was also used for identifying apoptosis cells (Mapelli et al., 2016). As shown in Figures 5b-c, the apoptosis rate in the UVA group was 13.08% ± 0.36%. However, this was significantly reduced by 3.43% ± 0.99% (0.5 mM) and 6.44% ± 0.50% (0.05 mM) for peptide A, and 8.52% ± 0.30% (0.5 mM) and 7.05% ± 0.70% (0.05 mM) for peptide B. Taking together, peptides A and B effectively ameliorates the rate of UVAinduced apoptosis in HaCaT cells.

DISCUSSION
Numerous reports suggest that natural sources of peptides exert significant benefits on human health. As such the search (generation, separation, purification, and identification) for novel peptides from different protein sources have attracted enormous attention over the years.
Elastin peptides in particular have long been known to be potent photoprotective agents (Zhang et al., 2017). Herein two novel peptides, TGVLTVM (peptide A) and NHIINGW (peptide B), with significantly different antioxidant capacities as measured by ORAC were identified.
In the two peptides studied, both had G. In addition, peptide A had 3 (V, L, and M), whiles peptide B contained 2 (W and H) known highly antioxidant amino acids. This higher number of constituent antioxidant amino acids in peptides A B relative to the others may have accounted for the relatively higher ORAC values of the two peptides.
HaCaT cells are immortalized human keratinocytes and have been widely used to study the molecular mechanism and pathophysiology associated with UVA irradiation of the skin which is usually characterized by significant reduction in cell viability with obvious nuclear morphological changes (Choi & Jeon, 2018;Gao, Guo, Chen, Du, & Wang, 2007;Hseu et al., 2018). The results showed that a successful UVAirradiated HaCaT cell model was established, evident in reduced cell viability and increased formation of apoptosis cell bodies.
Exposure to UVA leads to increases in fibroblast damage through different photoaging pathways (Wang et al., 2014). Peptides A and B substantially ameliorated UVA-induced cytotoxicity without exhibiting apparent self-toxicity. It thus suggests that peptides A and B may interfere with UVA-induced cellular photoaging pathways and holds great potential in reversing the decline in cell proliferation that is characteristic of UVA irradiations.  in peptide A could be the most significant driving force of the antioxidant activity (Nimalaratne et al., 2015;Shen, Chahal, Majumder, You, & Wu, 2010). Interestingly, the Food and Drug Administration has stated that ORAC values on their own may not realistically reflect the in vivo situation (Prior, 2015). Furthermore, ORAC is best used to quantify and reflect peroxyl radical scavenging capacity and may not be able to adequately quantify an antioxidant's reducing capacity. As such ORAC values on their own may not be comprehensive enough in determining all aspects of the antioxidation of a substance (Huang et al., 2005).
MMP is a key parameter in assessing cellular energy metabolism.
Research findings suggest that the over production of ROS and Ca 2+ overload are major activators of membrane permeability transition pore (MPTP) and mitochondrial membrane potential depolarization.
Consequently, it has been proven that attenuating oxidative-stressinduced mitochondrial Ca 2+ overload can reduce the loss of mitochondrial membrane potential (MMP) (Wang et al., 2018). Accordingly, the results suggested that peptides A and B may reverse the decline in energy metabolism ostensibly by preventing mitochondrial dysfunction and oxidative stress (Liang & Sundberg, 2011) caused by UVA irradiation in HaCaT cells.
Evidence also indicates that the excessive production of UVA irradiation induced-ROS initiates cell damage leading to decreased MMP, the release of cytochrome C, and eventual cell death (Svobodova, Walterova, & Vostalova, 2006). It was reported (Kim & Yoo, 2016) that propolis reduced UVA-induced mitochondria damage, apoptosis, and cell death through its ROS-scavenging capacity. It is therefore reasonable to infer that the inhibition of cellular ROS overproduction and accumulation by peptides A and B may be responsible for the observed attenuation of mitochondrial damage, reversed MMP decline and consequently reduced cell death and apoptosis.

CONCLUSION
In conclusion, two novel elastin peptides, TGVLTVM (peptide A) and NHIINGW (