Potential skin protective effects after UVB irradiation afforded by an antioxidant peptide from Odorrana andersonii

Biomedicine & Pharmacotherapy 120 (2019) 109535

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Potential skin protective effects after UVB irradiation afforded by an antioxidant peptide from Odorrana andersonii

T

Saige Yina,1, Ying Wangb,1, Naixin Liua, Meifeng Yanga, Yan Hua, Xiaojie Lic, Yang Fuc, ⁎ ⁎ ⁎ Mingying Luoa, , Jun Suna, , Xinwang Yanga, a

Department of Anatomy and Histology & Embryology, Faculty of Basic Medical Science, Kunming Medical University, Kunming, 650500, Yunnan, China Key Laboratory of Chemistry in Ethnic Medicine Resource, State Ethnic Affairs Commission & Ministry of Education, School of Ethnomedicine and Ethnopharmacy, Yunnan Minzu University, Kunming, 650500, Yunnan, China c Department of Biochemistry and Molecular Biology, Faculty of Basic Medical Science, Kunming Medical University, Kunming, 650500, Yunnan, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Amphibian peptides UV irradiation Photodamage Odorrana andersonii Antioxidant repair peptide

With increasing demand, the development of new natural antioxidants has become a primary direction of scientific research. We previously identified a short gene-encoded peptide (OA-VI12) from Odorrana andersonii frog skin secretions that exerted direct scavenging capacity against free radicals, suggesting a possible function in protecting skin against photodamage caused by their high-altitude habitat. In the current research, we examined the effects of OA-VI12 on both UVB-irradiation and hydrogen peroxide-induced oxidative stress models established with human immortalized keratinocytes. In addition, we identified the differentially expressed genes (DEGs) in the oxidative stress and OA-VI12 groups and further performed transcriptome as well as functional and pathway enrichment analyses. Results showed that OA-VI12 protected cell viability, promoted the release of catalase, and decreased the levels of lactate dehydrogenase and reactive oxygen species. Moreover, the peptide promoted the production of superoxide dismutase and glutathione, alleviated epidermis and dermis thickness, and decreased the production of light spots and collagen fibers in skin from the photo-injured mouse model. Kyoto Encyclopedia of Genes and Genomes analysis showed mitogen-activated protein kinase (MAPK) to be the most abundant signaling pathway. Gene Ontology (GO) analysis indicated that the top 55 significantly enriched GO terms mainly involved cellular processes, parts, and binding. These results revealed the beneficial role of the small molecule gene-encoding antioxidant peptide (OA-VI12) and its potential application as a protective agent against photodamage.

1. Introduction Among the factors related to skin aging injury, genetic and physiological factors only account for about 3%, with environmental factors accounting for 97% [1]. In particular, solar irradiation is one of the most important causes of photo-aging damage. There are three kinds of ultraviolet (UV) rays in sunlight: i.e., long-wave UVA (315–400 nm), medium-wave UVB (280–315 nm), and short-wave UVC (100–280 nm). Almost all UVC is absorbed by the ozone layer and the biological activity of UVA is much lower than that of UVB; thus, UVB is considered the main cause of photo-aging injury [2–4]. When the intensity of UV irradiation is more than 3.7 mJ/cm2 per day, the redox homeostasis of

skin can be destroyed, followed by lipid peroxidation of cell membranes, release of lactate dehydrogenase (LDH), and reduction in the levels of enzymatic antioxidants such as superoxide dismutase (SOD) and catalase (CAT) and antioxidant peptides such as reduced glutathione (GSH) [5–7]. When skin is exposed to excessive UV radiation, there is an associated accumulation of reactive oxygen species (ROS) and imbalance in the oxidation and antioxidant system, resulting in oxidative stress. Moreover, ROS-mediated oxidative stress can cause lipid oxidation and DNA damage. These oxidative injuries contribute to skin aging and photodamage, including erythema generation and melanin deposition, and can eventually lead to skin defects [8,9]. Long-term irradiation has also influenced the evolution of a unique

Abbreviations: DEGs, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology; MAPK, mitogen-activated protein kinase; UV, ultraviolet; LDH, lactate dehydrogenase; ROS, reactive oxygen species; SOD, superoxide dismutase; CAT, catalase; GSH, glutathione; VC, vitamin C; H2O2, hydrogen peroxide; DMEM/F12, Dulbecco’s modified Eagle’s/F12 medium; FBS, fetal bovine serum; PBS, phosphate buffer solution; FDR, false discovery rate ⁎ Corresponding authors. E-mail addresses: [email protected] (M. Luo), [email protected] (J. Sun), [email protected] (X. Yang). 1 Authors contributed equally to this work. https://doi.org/10.1016/j.biopha.2019.109535 Received 1 July 2019; Received in revised form 28 September 2019; Accepted 2 October 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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positive charge produced by tleap in peptide treatment. To ensure structure and correct position of hydrogen atoms, the gradient descent and conjugate gradient methods were used to minimize the energy of the initial structure before folding simulation. The whole simulation system was heated to 300 K (room temperature) in the NVT (canonical) ensemble, and the balance of the NPT (isothermal-isobaric) ensemble was operated under standard atmospheric pressure using the Langevin dynamic algorithm. After balancing, molecular dynamic simulation of 10,000 ps (smallest unit of time in molecular dynamics simulation) was carried out at 300 K. Finally, the structure with the lowest energy (folding state) was used as the structure of OA-VI12 [21].

and effective antioxidant peptide skin defense system in amphibians in order to cope with oxidative stress. Skin antioxidant defense can be divided into three categories: 1) High molecular weight gene-coding enzymes composed of SOD; 2) Non-genetically coded low molecular weight antioxidants composed of vitamin C (VC) or endogenous antioxidants such as reduced glutathione and ubiquitin-10; and 3) Small molecular peptides encoded by genes. These antioxidant skin defense systems can rapidly produce antioxidant peptides that scavenge free radicals to reduce oxidative damage to the skin [10,11]. Free radicals can cause lipid peroxidation, abnormal accumulation of metabolites, and ultimately oxidative stress in cells and tissues. About 80% of free radicals are thought to be caused by UVA and UVB irradiation. Therefore, antioxidants often maintain balance by scavenging free radicals and resisting UVB light damage to protect the body from oxidative stress-induced diseases [12,13]. Local application of antioxidants has become one of the most important measures to prevent skin photo-aging injury. Antioxidants from various sources show different effects in their protection of cells and tissues against free radicals [14]. Non-genetically coded low molecular weight antioxidants, such as VC, play an important role in protecting the internal structure and water content of cells [15]; high molecular weight gene-coding enzymes, such as SOD, protect the defense system of mitochondria and tissues [16]; however, small molecular peptides with abundant gene-coding sources are considered to have broad prospects for development because of their low energy consumption, high speed, and high efficiency; to date, however, our knowledge on these peptides remains limited [11]. At present, the known small molecular gene-coding antioxidant peptides, which include AOP-P1 from O. andersonii and RL from Odorrana livida, are limited relative to the richness of their sources [17,18]. Therefore, the development of small molecule gene-coding peptides has become an area of intense research in recent years. We previously identified a natural polypeptide (OA-VI12) with a molecular mass of 1298.65 Da and sequence of 'VIPFLACRPLGL', which was extracted from skin secretions of O. andersonii and which showed potent free radical scavenging ability [19]. We speculated that this antioxidant peptide may protect against UV-induced oxidative stress and may play a direct role in protection against skin photodamage. Thus, in the present study, we validated the peptide at the cellular and animal level and found that OA-VI12 played a beneficial role in protecting cells from oxidative stress induced by UV irradiation and hydrogen peroxide (H2O2) stimulation. More importantly, OA-VI12 also played a role in protecting skin from UV irradiation-induced photodamage. In addition, we also applied RNA sequence (transcriptome) analysis [20] to accurately measure transcriptional levels and subtypes and thus analyze the differentially expressed genes (DEGs) in the oxidative damage group induced by H2O2 and in the OA-VI12 treatment group. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of DEGs were carried out to determine the possible antioxidant mechanism of OA-VI12. This study further demonstrated that OA-VI12, an antioxidant peptide derived from O. andersonii, could repair UVB-induced skin photodamage, which should contribute to future research on oxidative damage caused by UVB irradiation to the skin.

2.2. Cell culture Cell culture was performed as per previous research [22]. Human keratinocytes (HaCaT cells) (KCB 200442 YJ) were commercially provided by Conservation Genetics, Kunming Cell Bank of the Kunming Institute of Zoology, Chinese Academy of Sciences. The cells were cultured in Dulbecco’s modified Eagle’s/F12 medium (DMEM/F12, BI, Israel) supplemented with 1% antibiotics (100 unit/mL penicillin, 100 unit/mL streptomycin) and 10% fetal bovine serum (FBS, BI, Israel) at 37 °C in a humidified incubator with 5% CO2. 2.3. Effects of OA-VI12 on viability of HaCaT cells irradiated by UVB The HaCaT cells were thrice washed with phosphate buffer solution (PBS) to detach dead cells and then divided into three groups (UVB irradiation model, blank, and sample groups). The cells were then seeded in 96-well plates (3.0 × 103 cells/well) containing 90 μL of DMEM/F12 (serum free). After exposure to a 9 W/01 UVB lamp (Philips, Holland) (30 mJ/cm2), cells in the sample group were pretreated with different concentrations of OA-VI12 (0.5, 1, 5, and 10 μM) or 10 μM VC (Sigma, St. Louis, MO, USA) at 37 °C for 2 h. After this, 20 μL of MTS (Promega, Madison, WI, USA) was added to the incubated cells for another 2–4 h to test the effects of OA-VI12 or VC on the viability of HaCaT cells. The plates were read on a plate reader at 490 nm. 2.4. Effects of OA-VI12 on viability of HaCaT cells treated with H2O2 Cells were grouped and seeded on plates as per the above methods. The cells were then treated with 200 μM of H2O2 (Sigma, St. Louis, MO, USA) for 2 h after pretreatment with different concentrations of OAVI12 or VC at 37 °C for 2 h. The MTS assay for detecting cell viability was the same as above. 2.5. Effects of OA-VI12 on ROS levels in HaCaT cells treated with H2O2 or UVB irradiation Cells were divided into four groups (H2O2 treatment, UVB irradiated, blank, and sample groups) and seeded in 6-well plates (1.2 × 106 cells/well). The cells were then treated with H2O2 for 2 h or irradiated by UVB after pretreatment with different concentrations of OA-VI12 or VC at 37 °C for 2 h. After 2 h, cells were resuspended in warmed PBS containing 1 μM of H2DCFDA fluorescent probe (Invitrogen, Thermo, USA) at 37 °C for 45 min. The cells were then thrice washed with PBS, digested with 0.25% trypsin (BI, Israel) for 4 min, centrifuged at 1 000g for 5 min at room temperature, and then suspended with 1 mL of cold PBS. The production of ROS in cells was detected by flow cytometry (CyFlow Space, PARTEC, Germany).

2. Material and methods 2.1. Peptide synthesis and prediction of advanced structure The mature OA-VI12 peptide, ‘VIPFLACRPLGL’, with a purity > 95% was commercially synthesized and provided by Wuhan Bioyeargene Biotechnology Co., Ltd. (China). The structure of OA-VI12 was predicted by PEP-FOLD3 online service, with the highest score then chosen as the initial structure for further optimization with the Sander module in Amber 16. The ff14SB force field was used to simulate the folding of OA-VI12 and chloride ions were added to neutralize the

2.6. Effects of OA-VI12 on CAT and LDH levels in HaCaT cells treated with H2O2 or UVB irradiation HaCaT cells were thrice washed with PBS, then digested with 0.25% trypsin for 4 min, centrifuged at 1 000g for 5 min at 37 °C, suspended in 2

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2.10. Analysis of quantification and differential expression of transcripts

0.5 mL of cold PBS, stewed in an ice box, and then disrupted by an ultrasonic cell pulverizer (VCX750, Xinchen, Nanjing, China). Disruptive mixtures of cells were centrifuged at 12 000 g for 15 min at 4 °C, with the supernatants then collected to determine the levels of CAT using commercial kits. The supernatants of HaCaT cells treated with H2O2 or UVB irradiation were collected to determine the levels of LDH using commercial kits (i.e., CAT and Lactate Dehydrogenase assay kits, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), which were operated according to the standard protocols provided by the manufacturer.

Bowtie 2 was used to align clean reads to reference gene sequences, then RSEM (RNA-Seq by Expectation-Maximization) was used to calculate gene expression levels in each sample [24,25]. Each read was independently and evenly sampled from RNA sequencing samples. Under this assumption, the number of reads in the transcriptome followed a binomial distribution. Using the above model, DEGs were highlighted based on MA-plots to test the hypotheses for each gene on the MA map and to calculate the p-value according to normal distribution [26]. The p-values of difference tests were corrected by the multiple hypothesis test, and the p-value threshold was determined by controlling the false discovery rate (FDR). In order to improve the accuracy of DEGs, significant differentially expressed genes were defined by more than twice the difference, Q-value and FDR ≤ 0.001.

2.7. Animal model of UVB-irradiated dorsal skin photodamage Female Kunming mice (18–22 g) from the same generation were obtained from the Laboratory Animal Department of Kunming Medical University. All animal experiments and care and handling procedures were conducted in accordance with the requirements of the Ethics Committee of Kunming Medical University (KMMU20180012). Mice were kept in individual ventilated cages (Fengshi, China) in the laboratory animal room of Kunming Medical University. The experimental mice were provided with food and free drinking water in an airconditioned room at a temperature of 22 ± 2 °C and light-dark cycle of 12/12 h. The mice underwent 7 d of acclimation before the experiment, after which they were randomly divided into four groups (six in each group): i.e., control, model (UVB), sample (UVB + OA-VI12), and positive control (UVB + VC). Mice were anesthetized with 1% sodium pentobarbital (0.1 mL/20 g body weight) by intraperitoneal injection (i.p.), with the dorsal skin of mice then depilated and exposed to UVB irradiation (UVB lamp, TL20W/12, Philips, Holland) at 150 mJ/cm2 per day for the first 7 d and 300 mJ/cm2 every other day for the following two weeks [17]. The intensity of irradiation was monitored with a UV radiometer (TM-213, Tenmars, Taiwan, China). OA-VI12 (10 μM) or VC (10 μM) (both liquids) were topically administered once daily on the irradiated mouse skin sections. After the final UVB irradiation, the mice were sacrificed, and the dorsal hairless skin was removed for the following procedures. Images of photodamaged skin were taken on the final day. In view of the individual differences in mice, the most representative pictures are shown in Fig. 5. All study protocols and procedures were approved by the Ethics Committee of Kunming Medical University and were conducted in accordance with the guidelines for Animal Care and Use at Kunming Medical University.

2.11. Bioinformatic analysis and protein interaction Pathway significance enrichment analysis was determined using the KEGG Pathway database (http://www.genome.jp/kegg/) [27], with the hypergeometric test used to identify pathways that were significantly enriched in candidate genes compared with the whole genome background. Pathways with a Q-value ≤0.05 were defined as pathways significantly enriched in DEGs. Significant pathway enrichment can identify the major biochemical metabolic pathways and signal transduction pathways in which candidate genes participate. GO functional significance enrichment analysis highlights GO functional terms that are significantly enriched in candidate genes compared with all genetic backgrounds of the species, thus indicating the biological functions of candidate genes. All candidate genes were first mapped to each term in the GO database (http://www.geneontology.org/), with the number of genes in each term then calculated, and the hypergeometric test applied to identify GO entries significantly enriched in candidate genes compared with all genetic backgrounds of the species. STRING version 11.0 (https://string-db.org/) was employed to explore the protein interaction networks in the model and OA-VI12 treatment groups. 2.12. Western blot analysis Cells (normal group, H2O2 stimulation group, OA-VI12 treatment group) were treated with lysate (RIPA and PMSF, Meilun Biotechnology, Dalian, China; phosphatase inhibitors, Roche, Shanghai, China) on ice for 30 min. After being fully lysed, cells were scraped and collected in 1.5-mL centrifugal tubes, then centrifugated at 12,000g for 5 min at 4 °C. The supernatant was collected and protein content was detected using a BCA protein analysis kit (Meilun, Dalian, China). To verify the activation of the MAPK signaling pathway, sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis were performed for proteins in HaCaT cells. Each sample protein was decomposed by 12% SDS-PAGE, transferred to polyvinylidene fluoride membranes and sealed with 5% skim milk for 2 h, then incubated with primary antibody (GAPDH, p-P38, p-Erk1/2, Cell Signaling, Danvers, MA, USA) at 4 °C overnight and secondary antibody (anti-mouse, antirabbit, Cell Signaling, Danvers, MA, USA) for 1 h. After exposure, the membranes were washed with membrane regeneration solution for 30 min (Solarbio, Beijing, China), then sealed with 5% skim milk, incubated overnight with primary antibody (P38, ERK1/2, Cell Signaling, Danvers, MA, USA) at 4 °C, and detected by enhanced chemiluminescence after incubation with secondary antibody for 1 h. Specific bands were detected, analyzed, and quantified by Image J software.

2.8. Hematoxylin and eosin (H&E) and Masson’s trichrome staining of UVB-irradiated mouse skin The mouse skin tissue samples were fixed in 4% paraformaldehyde for 24 h, then dehydrated and hyalinized as per previous study [23]. For histological analysis, tissue samples were sectioned into 6-μm thick slices and stained with H&E or Masson’s trichrome reagents (Solarbio, Beijing, China). Image J software (National Institutes of Health, Bethesda, USA) was used to estimate the thickness of the epidermal and dermal layers. 2.9. Measurement of SOD and GSH levels in UVB-irradiated mouse skin Mouse skin tissue samples (0.1 g) were homogenized for 3 min in 1 mL of cold PBS, then centrifuged at 10,000g for 15 min at 4 °C. Supernatants were collected for measurement of SOD (SOD assay kit (WST-1 method), Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and GSH (GSH assay kit, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) levels. All experimental procedures were operated in accordance with the standard protocols provided by the company.

2.13. Statistical analysis Data were depicted as means ± SD. Statistical analysis was performed using GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA, USA). Pairwise comparison was conducted using one-way ANOVA. A p3

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Fig. 1. Chemical structure and prediction of the advanced structure of OA-VI12. A. Structure was manually produced by ChemDraw software, with peptide bonds labeled in blue; B. Structure consists of a α-helix and side-chain residues (N: blue; O: red; S: yellow) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

3.3. OA-VI12 showed antioxidant activities in HaCaT cells challenged by UVB and H2O2

value of < 0.05 was considered statistically significant. 3. Results

To test the antioxidant activities, recognized as an index of oxidative damage in vivo, the levels of CAT, LDH, and ROS were tested [5]. As shown in Fig. 3, the intracellular ROS fluorescence intensity greatly increased with both UVB irradiation and H2O2 challenge in comparison to the control group. OA-VI12 exposure significantly reduced ROS release after UVB irradiation and H2O2 stimulation. The effect of OA-VI12 on HaCaT cells was the same as that of VC. Both UVB exposure and H2O2 challenge against HaCaT cells induced significant increase in LDH activity but decrease in CAT activity. After UVB irradiation, LDH activity increased (117.63 ± 8.52%) and CAT activity decreased (77.31 ± 4.34%); however, pretreatment with OA-VI12 (10 μM) resulted in a significant 100.68 ± 10.9% decrease in LDH activity and 54.49 ± 3.15% increase in CAT activity (Fig. 4A, C). Simultaneously, the activity of LDH was markedly increased but the activity of CAT was reduced in HaCaT cells stimulated with H2O2. In the group pretreated with OA-VI12, LDH activity decreased by 73.52 ± 16.86% and CAT activity increased by 23.91 ± 3.78% (Fig. 4B, D).

3.1. Chemical structure and prediction of advanced structure of OA-VI12 The molecular formula of OA-VI12 is ‘VIPFLACRPLGL’ and its chemical formula is C62H103N15O13S (Fig. 1A). The predicted advanced structure, as displayed using two maps from the front and side views, was mainly composed of a α-helix and side-chain residue (Fig. 1B). 3.2. Pretreatment with OA-VI12 sustained HaCaT cell viability against UVB irradiation and H2O2 treatment As illustrated in Fig. 2, OA-VI12 (0.5–10 μM) showed no obvious influence on the viability of HaCaT cells. The viability of HaCaT cells decreased significantly when exposed to UVB irradiation or H2O2; however, pretreatment with OA-VI12 protected the HaCaT cells from the decrease in viability induced by both UVB irradiation and H2O2 (Fig. 2A, B). In the OA-VI12 pretreatment group (10 μM), cell viability was the same as that in the VC group (positive control), indicating that OA-VI12 had a significant protective effect on cell viability. 4

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Fig. 2. Effects of OA-VI12 on viability of HaCaT cells challenged by UVB or H2O2. A. OAVI12 showed significant protective activity against UVB irradiation in HaCaT cells. B. OAVI2 showed significant protective activity against oxidative damage in HaCaT cells treated with H2O2. HaCaT cells (3.0 × 103) were irradiated with 30 mJ/ cm2 using a UVB lamp or exposed to 200 μM H2O2 for 2 h, with pretreatment of different concentrations of OAVI12 (0.5, 1, 5, 10 μM) or VC (10 μM, positive control) for 2 h, or directly pretreated with OAVI12 or VC without or with UVB or H2O2 treatment. Cell viability was measured using MTS assay. All data are means ( ± SD) of three independent experiments performed in triplicate. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ‘#’ represents value compared with previous group, # p < 0.05; ## p < 0.01. The data from control group was normalized as 100% and data were normalized accordingly.

Fig. 3. Effects of OA-VI12 on ROS levels in HaCaT cells challenged with UVB irradiation or H2O2. A. OA-VI12 significantly reduced ROS release in UVB-irradiated HaCaT cells; B. Histogram of mean fluorescence intensity after UVB irradiation; C. OA-VI2 significantly reduced ROS release in HaCaT cells treated with H2O2; D. Histogram of mean fluorescence intensity after H2O2 stimulation. Cells (1.2 × 106) were pretreated with OA-VI12 (10 μM) or VC (10 μM, positive control) for 2 h before UVB irradiation (A) or H2O2 stimulation (B); after 2 h, the cells were maintained in DMEM/F12 medium with 1 μM H2DCFDA fluorescent probe at 37 °C for 45 min. Production of ROS in cells was detected by flow cytometry. Data are mean values of three independent experiments. * p < 0.05; ** p < 0.01. One control group was considered as 100% and data were normalized accordingly. 5

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Fig. 4. Effects of OA-VI12 on LDH and CAT activities in HaCaT cells challenged with UVB irradiation or H2O2. A, B. LDH activities; C, D. CAT activities. HaCaT cells were pretreated with OA-VI12 (10 μM) or VC (10 μM, positive control) for 2 h before UVB irradiation (A, C) or H2O2 stimulation (B, D). All data are means ( ± SD) of three independent experiments. Significant differences are indicated by asterisks: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. One control group was considered as 100% and data were normalized accordingly.

3.5. OA-VI12 reduced endogenous oxidative stress in UVB-irradiated mouse skin

3.4. OA-VI12 prevented UVB-induced photodamage to skin in vivo Overexposure to UVB irradiation can damage the integrity of skin, producing erythema and increasing epithelial and dermal thickness [8]. The skin-damage mouse model induced by UV irradiation was used to evaluate the photoprotective effects of OA-VI12. Following UVB irradiation for 21 d, the morphology of the mouse dorsal skin changed, with skin damage, erythema, and epidermal thickening observed (Fig. 5A, B). However, as displayed in Fig. 5C and D, OA-VI12 effectively inhibited the above phenomena induced by light damage, indicating that OA-VI12 and VC had comparative beneficial activities. The topical administration of OA-VI12 and VC caused no adverse effects on mouse body weight, general health, or behavior (data not shown). Compared with normal skin, the thickness of the epidermis significantly increased by 160.81 ± 6.93 μm after UVB irradiation, whereas OA-VI12 reduced the thickness to 62.14 ± 3.39 μm (Fig. 6A, C). Dermal thickness increased by 355.92 ± 8.28 μm after UVB irradiation; however, OA-VI12 exposure reduced the thickness to 266.67 ± 6.36 μm (Fig. 6B, D). Masson’s trichrome staining showed that UVB irradiation reduced the content of collagen fibers, which was inhibited by both OA-VI12 and VC treatment (Fig. 6B). Results also demonstrated the same effects of anti-photodamage between VC and OA-VI12.

Changes in the levels of endogenous antioxidant enzyme SOD and antioxidant peptide GSH can be used as an index of oxidative damage [6]. As illustrated in Fig. 7, both SOD activity and GSH levels were significantly decreased by 46.90 ± 0.539% and 90.36 ± 4.58%, respectively, in UVB-irradiated mouse skins. Conversely, exposure to antioxidant OA-VI12 increased the levels by 79.39 ± 1.96% and 67.42 ± 10.11%, respectively (Fig. 7A, B), indicating that OA-VI12 and VC exerted equivalent effects on SOD and GSH reduction.

3.6. Identification of DEGs in H2O2-induced and OA-VI12-treated HaCaT cells Genes with more than twice the difference and a significance level of FDR ≤ 0.001 were considered to be significantly different. In the control-vs-model (H2O2 treated) group, 3 757 genes were significantly changed. In the model-vs-treatment (OA-VI12 treated) group, 1 532 genes were significantly changed. Among them, 467 genes were common to both (Fig. 8A).

Fig. 5. Protective effects of OA-VI12 on dorsal UVB-irradiated mouse skin. Mice were divided into four groups (six in each group): A. Control group without UVB irradiation; B. Model group with UVB exposure alone; C. UVB-irradiative +10 μM OA-VI12 group; D. UVB-irradiative +10 μM VC group. Mice were irradiated with UVB (150 mJ/cm2) for 7 d, then exposed to 300 mJ/cm2 every other day for two weeks. Images of photodamaged mouse skin were taken on final day. Macroscopic changes were observed, and UVB-induced erythema and eschars are indicated with arrows.

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Fig. 6. Effects of OA-VI12 on collagen degradation and epidermis and dermis thickness in UVB-irradiated dorsal skin of mice. A. H&E staining; B. Masson’s trichrome staining; C. Histogram of epidermal thickness with H&E staining; D. Histogram of dermal thickness with Masson’s trichrome staining. After final UVB irradiation, dorsal skin was collected immediately and sectioned into 6-μm thick slices for H&E and Masson’s trichrome staining. The scale bar indicated 200 μm. ** Significant difference at p < 0.01; ***p < 0.001; **** p < 0.0001. All bars represent means ± SD from three different sections for each experiment.

The top 55 significantly enriched GO terms are shown in Fig. 9. Biological process analysis showed that the largest groups of DEGs were related to cellular processes, metabolic processes, and biological regulation. Similarly, cellular component and molecular function analyses showed that the largest groupings were related to cellular parts and binding.

3.7. KEGG pathway and GO enrichment analysis The top 20 significantly enriched KEGG pathways with the smallest Q-value are shown in Fig. 8B. The most significantly enriched pathways included the MAPK signaling pathway, Salmonella infection, and pathways in cancer, with 18, 25, and nine enriched DEGs, respectively. We identified 102 genes involved in classification of transport and catabolism, 66 involved in signal transduction, 47 involved in cancers, 26 involved in global and overview maps, and 37 involved in the immune system.

3.8. Protein interaction analysis of differentially expressed proteins (DEPs) STRING analysis was conducted to explore the interactions among Fig. 7. Effects of OA-VI12 on SOD activity and GSH level in UVB-irradiated mouse skin. A. SOD activity after UVB irradiation; B. GSH level after UVB irradiation. Skin tissue (0.1 g) was homogenized in 1 mL of cold PBS and centrifuged at 10 000 g for 15 min at 4 °C to obtain supernatants, after which SOD activity and GSH level were measured by a microplate reader. All data are means ( ± SD) of three independent experiments. ** Significant difference at p < 0.01; *** p < 0.001; **** p < 0.0001. One control group was considered as 100% and data were normalized accordingly.

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Fig. 8. Differentially expressed genes (DEGs) and KEGG analysis. A. DEG map between control group, model group (200 μM H2O2 treatment), and treatment group (10 μM OA-VI12 treatment). Significant differentially expressed genes were defined by more than twice the difference, Q-value and FDR ≤ 0.001. B. KEGG analysis of significantly enriched pathways in OA-VI12 treated group.

physiological challenge to amphibians [28,29,43]. Given the complex functions of amphibian skin, the need to maintain skin integrity in aquatic and terrestrial environments, and the vulnerability of the cuticle, amphibians (compared with other vertebrates) must effectively protect their skin from oxidative stress [11,44]. Past studies have found that skin secretions from O. andersonii frogs, which live at high altitudes, have more complex peptides and stronger antioxidant capacity than skin secretions from low-altitude O. wuchuanensis, indicating the strong adaptability of O. andersonii to harsh environments under massive UV irradiation [11]. Moreover, the gene-encoded peptide OA-VI12, previously identified from skin secretions of O. andersonii with 12 amino acids and a tertiary structure of α-helix and side-chain residues (Fig. 1), has strong direct free radical, DPPH, and iron ion scavenging abilities, suggesting it might also be able to resist oxidative damage [19]. According to the growth characteristics of O. andersonii, we speculated that such peptides may protect against oxidative and direct light damage caused by strong UV irradiation. To verify whether the antioxidant peptide OA-VI12 plays such a role, we conducted cellular and animal level experiments. Firstly, the protective effects of OA-VI12 on direct light damage and oxidative stress were verified in HaCaT cells. HaCaT cells are human

identified DEPs (Fig. 10). DEGs with more than twice the difference was selected for further analysis, with most found to be closely related to inflammatory pathways. The proteins that interacted with each other in the model group and OA-VI12 treatment group are shown in Fig. 10A and B, respectively. 3.9. Influence on MAPK signaling pathway To determine the possible mechanisms involved in the effect of OAVI12 on H2O2-stimulated oxidative stress, we studied the phosphorylation of ERK1/2 and P38 in HaCaT cells. As shown in Fig. 11, oxidative stress activated the MAPK signaling pathway and significantly phosphorylated the proteins ERK1/2 (0.4038 ± 0.0117) and P38 (0.401 ± 0.1206). Furthermore, phosphorylation was inhibited by OAVI12 (ERK1/2: 0.2787 ± 0.0902; P38: 0.2231 ± 0.0617) (Fig. 11B, C). 4. Discussion Amphibians provide a rich diversity of small molecule gene-coding peptides. In addition, high-altitude environments pose a serious 8

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Fig. 9. GO enrichment analysis of significantly enriched pathways in OA-VI12 treated group. Top 55 significantly enriched GO terms were mainly involved in cellular processes, cell parts, and cellular binding.

antioxidant enzymes such as CAT in the body [7]. Results showed that OA-VI12 and VC effectively reduced the increase in LDH and consumption of CAT (Fig. 4) caused by UVB irradiation and H2O2 stimulation. In conclusion, OA-VI12 demonstrated a significant protective capacity against oxidative damage and direct light damage. The protective effects of OA-VI12 were further studied in the light damage mouse skin model. UVB irradiation can cause skin erythema formation, melanin deposition, and damage, and can significantly increase the thickness of skin dermis and epidermis [33]. This process is not only the manifestation of skin damage caused by UVB, but also from skin self-protection against UVB damage. As seen in Figs. 5 and 6, UVB irradiation resulted in thicker skin, erythema, and skin damage, whereas OA-VI12 and VC treatment significantly inhibited these changes. It should be noted that we used OA-VI12 after each UVB irradiation for treatment. Thus, the 24-h stability of OA-VI12 at 37 °C was tested and was found to be 99.28% (data not shown). This means that when the mice were irradiated by UVB in following day, OA-VI12 may have also acted as a sunscreen. This suggests that OA-VI12 may not only

epidermal cells widely used in light-induced injury experiments in vitro [30]. As seen in Fig. 2, OA-VI12 had no effect on the viability of normal cells. When cells were irradiated by UVB or stimulated by H2O2, the viability of cells was significantly reduced. However, cell viability was restored after OA-VI12 or VC pretreatment. The effect of OA-VI12 was almost parallel to that of VC at concentrations of 10 μM, indicating that OA-VI12 was able to resist direct light damage and oxidative stress (Fig. 2). When cells are injured by UVB or experience oxidative damage, ROS are released in large quantities, and the imbalance between excessive ROS and antioxidant defense eventually lead to oxidative stress [31]. Here, the fluorescent probe H2DCFDA results confirmed the excessive production of ROS caused by UVB and oxidative stress. We found that pretreatment with antioxidant OA-VI12 or VC significantly inhibited the production of ROS and reduced damage to cells (Fig. 3). Excessive UV irradiation can destroy the lipid layer structure of cell membranes, resulting in increased cell membrane permeability, leading to intracellular LDH in the cell culture medium [32]. These increased oxidases can destroy the balance of oxidation and further deplete 9

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Fig. 10. Protein interaction analysis of differentially expressed proteins by STRING. A. Protein which differentially expressed more than twice in the 200 μM H2O2 treated group and control group. B. Protein which differentially expressed more than twice in the 200 μM H2O2 treated group and 10 μM OA-VI12 treated group.

[36]. These changes increase the thickness of the skin epidermis and dermis. When OA-VI12 was applied on the skin surface, it might penetrat the skin, inhibited the secretion of pro-inflammatory and proproliferative factors in keratinocytes, reduced the thickness of the epidermis and dermis, and promoted the production of antioxidant enzymes to resist light damage [37]. However, it is unknown OA-VI12 acts on the epidermis or dermis, and therefore more targeted research is required to explore the specific mechanism. To elucidate the underlying mechanisms of OA-VI12 against oxidative stress damage, transcriptome sequencing was used to analyze the DEGs between the oxidative damage and OA-VI12 treatment groups. We identified 5 289 significantly expressed DEGs (2 730 up-regulated and 2 559 down-regulated) (Fig. 8A). There are three main factors related to UV light damage to the skin. The first is the cross-linking of cystine residues of extracellular matrix protein, which is also the main cause of skin aging: Cystine can resist the influence of inflammatory factors on the body, protect the redox ability of cells, and maintain the oxidation balance of the body [38]. As shown in Fig. 8B, the main enriched KEGG pathway related to DEGs was the MAPK signaling

be a therapeutic drug for UVB irradiation but may also exhibit the dual effects of prevention and treatment. Previous research has reported that antioxidant enzymes and peptides in the oxidative system are depleted in a dose-dependent manner when the skin is damaged by direct light [34]. As the main free radical scavenger in the body, SOD blocks the toxicity of free radicals and the degradation of nitric oxide [35]. Furthermore, GSH participates in the detoxification process of many poisons and is also an important weapon against free radicals [8]. Therefore, it is important for the body to maintain high SOD and GSH activities. As shown in Fig. 7, the use of antioxidant OA-VI12 helped reduce the consumption of UVB antioxidant enzymes and peptides to help skin against light damage. These data suggest that the protective effects of OA-VI12 on skin photodamage and oxidative stress may be related to changes in endogenous antioxidant enzymes and peptides, and these changes may be closely related to antioxidant signaling pathways in vivo. In addition, the skin damage caused by light injury is multifaceted, not only related to changes in antioxidant balance in vivo, but also to hyperkeratosis of the epidermis, matrix thickening, and vascular expansion of the dermis

Fig. 11. Effects of OA-VI12 administration on H2O2-induced phosphorylation of MAPK signaling pathway. A. HaCaT cells were either pretreated or not with 10 μM OA-VI12 for 2 h before addition of 200 μM H2O2. After 30 min of incubation at 37 °C, total proteins were prepared and detected by Western blotting. Antibodies (p-P38, total-P38, p-ERK, total ERK, GAPDH) were used to detect protein expression. GAPDH was internal control. p, phosphorylated. B, C. Degrees of phosphorylation of ERK and P38 are indicated by column diagrams, respectively. * p < 0.05; ** p < 0.01. Protein bands were from three independent experiments.

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pathway. Among the DEGs, many inflammatory factors, including IL-6, IL-1, protein kinase C (PKC), and p21 activated kinase 1/2 (PAK1/2), were down-regulated to varying degrees after OA-VI12 treatment. The second factor is the formation of thymine dimer of DNA, which can trigger the activation of the DNA damage repair pathway [39]: the GADD45 (growth arrest and DNA damage gene 45) family plays a key role in the DNA repair pathway as a protein group involved in cell-cycle regulation and apoptosis. Furthermore, expression of the GADD45α gene can also down-regulate the activation of the MAPK signaling pathway and further protect DNA in repaired skin. As shown in Fig. 10, the level of ATF3 (transcription factor 3), a transcription regulator of GADD45α in oxidative stress and a gatekeeper for protecting gene integrity during UV-induced DNA damage [40], was significantly altered when treated with H2O2 stimulation and OA-VI12 treatment. In addition, KEGG results (Fig. 8B) showed that the MAPK signaling pathway was activated, so it may be reasonable to suggest that OA-VI12 plays an anti-UVB role by protecting DNA from damage induced by UV irradiation [41]. The third factor is that UV radiation can cause the self-excitation of some kinases and eventually lead to skin cancer [42]. As shown in Figs. 8B and 10, when oxidative stress was imposed, many kinases, such as PKC and PAK1/2, were activated, which will influence various inflammatory signaling pathways, including the MAPK and IL-17 signaling pathways, and ultimately affect the normal existence of the cell signaling network and eventually lead to cell death. Based on these results and analysis, it is reasonable to speculate that OA-VI12 may play an antioxidant role by regulating inflammatory factors and the MAPK signaling pathway. In addition, GO analysis showed that most of these proteins were closely related to cellular processes, metabolic processes, cell parts, and cellular binding, thus potentially revealing the molecular mechanism of OA-VI12 (Fig. 9). In addition, the activation of the MAPK signaling pathway by H2O2 was validated in Fig. 11, with OA-VI12 shown to markedly inhibit this phosphorylation effect. Based on the above, it is possible to conclude that the gene-encoded antioxidant peptide OA-VI12 extracted from skin secretions of O. andersonii showed significant resistance to oxidative damage and photodamage. Compared with traditional antioxidants such as VC, OA-VI12 shows some obvious advantages, including much easier synthesis and simpler storage conditions. This study also provides a basis for the development and application of novel gene-coding small molecule antioxidants.

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Author contributions M.L., J.S., and X.Y. acquired funding and designed the research. S.Y. and Y.W. performed most of the experiments. M.Y., Y.F., and N.L. assisted in the experiments. Y.H. and X.L. analyzed the data. All authors contributed substantially to this research and reviewed the manuscript. Declaration of Competing Interest The authors declare that there are no conflicts of interest regarding the publication of this paper. Acknowledgments This work was supported by the Chinese National Natural Science Foundation (81760648, 31670776, 31460571, and 81260178), Yunnan Applied Basic Research Project Foundation (2017FB035), and Yunnan Applied Basic Research Project-Kunming Medical University Union Foundation (2018FE001 (-161)). References [1] F. Liebel, S. Kaur, E. Ruvolo, N. Kollias, M.D. Southall, Irradiation of skin with visible light induces reactive oxygen species and matrix-degrading enzymes, J.

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