Multiple Mechanistic Action of Brevinin-1FL Peptide against Oxidative Stress Effects in an Acute Inflammatory Model of Carrageenan-Induced Damage

Amphibian skin is acknowledged to contain an antioxidant system composed of various gene-encoded antioxidant peptides, which exert significant effects on host defense. Nevertheless, recognition of such peptides is in its infancy so far. Here, we reported the antioxidant properties and underlying mechanism of a new antioxidant peptide, brevinin-1FL, identified from Fejervarya limnocharis frog skin. The cDNA sequence encoding brevinin-1FL was successfully cloned from the total cDNA of F. limnocharis and showed to contain 222 bp. The deduced mature peptide sequence of brevinin-1FL was FWERCSRWLLN. Functional analysis revealed that brevinin-1FL could concentration-dependently scavenge ABTS+, DPPH, NO, and hydroxyl radicals and alleviate iron oxidation. Besides, brevinin-1FL was found to show neuroprotective activity by reducing contents of MDA and ROS plus mitochondrial membrane potential, increasing endogenous antioxidant enzyme activity, and suppressing H2O2-induced death, apoptosis, and cycle arrest in PC12 cells which were associated with its regulation of AKT/MAPK/NF-κB signal pathways. Moreover, brevinin-1FL relieved paw edema, decreased the levels of TNF-α, IL-1β, IL-6, MPO, and malondialdehyde (MDA), and restored catalase (CAT) and superoxide dismutase (SOD) activity plus glutathione (GSH) contents in the mouse injected by carrageenan. Together, these findings indicate that brevinin-1FL as an antioxidant has potent therapeutic potential for the diseases induced by oxidative damage. Meanwhile, this study will help us further comprehend the biological functions of amphibian skin and the mechanism by which antioxidants protect cells from oxidative stress.


Introduction
Free radicals including superoxide anion radical, peroxyl radical, and hydroxyl radical are unstable and indispensable intermediates of aerobic metabolism during respiration in organisms. In general, they are highly reactive with the other groups or substances in the body due to their single and unbalanced electrons. Consequently, free radicals can trigger a cascade of damaging reactions such as lipid peroxidation as well as protein and DNA oxidation, affecting cellular sig-naling and culminating in cell damage and death [1,2]. ROS are effectually obliterated by the antioxidant defense system including nonenzymatic factors and antioxidant enzymes under normal conditions. But, under pathological conditions, the homeostasis between the generation and scavenging of ROS is broken in vivo, which is generally acknowledged to be involved in some health disorders such as diabetes mellitus, cancer, atherosclerosis, aging, and neurodegenerative and inflammatory diseases [2,3]. Thus, effectively scavenging excessive free radicals or preventing their generation has been momentous methods for the prevention and treatment of such diseases. Many synthetic antioxidants, such as vitamin C, butylated hydroxyanisole, and propyl gallate, are used for retarding lipid peroxidation. Yet their potential health hazards and low stability limit their further medical application [4]. Hence, the isolation and identification of natural antioxidants which can scavenge free radicals and protect cells from oxidative damage have been of great interest among researchers [5].
Amphibian has evolved an efficient antioxidant defense system in their skin to antagonize nonbiological injuries from ROS in their environments. Antioxidant peptides are important components of the amphibian skin antioxidant system, and more than 40 peptides with free radial scavenging functions have been identified by purification, proteomic analysis, or cDNA trapping from R. catesbeiana [6,7]. Fejervarya limnocharis is a mediumsized frog (30-60 mm) inhabiting throughout East, Southeast, and South Asia [8,9]. Except that a lectin-like peptide inhibiting HIV-1 entry has been identified by us, there is no any bioactive peptide reported from this species [10]. In this work, we firstly characterized brevinin-1FL from the skin of frog F. limnocharis which showed potent antioxidant activity in vitro. Then, we explored the protective effects of brevinin-1FL against H 2 O 2 -induced ROS generation, oxidative stress, and cytotoxic effect in PC12 cells for evaluating its pharmaceutical potential. Finally, we conducted animal experiment to test its antioxidant and anti-inflammatory activities in vivo. To the best of our knowledge, it is the first report about an antioxidant peptide from F. limnocharis.

Animals and Ethical Statement.
Male and female adult F. limnocharis frogs (n = 3) obtained from the countryside of Guangzhou, Guangdong Province, China (23.12°N, 113.28°E) are not a protected species without the need for specific permissions. After collection, the frogs were humanely euthanized using CO 2 , and the skin was subsequently sheared and stored in liquid nitrogen until use. Kunming mice (4 weeks, 18-20 g) were bought from the Laboratory Animal Center of Southern Medical University (Guangdong, China) and maintained in plastic cages under standard conditions at 25 ± 2°C and 55 ± 10% humidity, with free access to food and water on a 12 h light/dark rhythm. The Animal Care and Use Ethics Committee of Southern Medical University (no. L2018254) authorized all protocols and procedures involving live animals which were implemented in light of the international regulations for animal research.

Molecular Cloning and Characterization of cDNA
Encoding Brevinin-1FL. The skin mRNA and double-strand cDNA were prepared as previously reported by us [10]. The chemical parameters of brevinin-1FL were analyzed with the ExPASy Bioinformatics Resource Portal (http:// www.expasy.org/tools/). The assembled sequences were aligned with ClustalW (http://embnet.vital-it.ch/software/ ClustalW.html) on the basis of similarity with previously reported antimicrobial peptides (AMPs) from different amphibian species including brevinin-1FL.

Peptide Internalization Analysis.
To ensure whether brevinin-1FL can enter into the cells to exert antioxidant effects, PC12 cells (1 × 10 5 cells/well) were grown in a 24well plate overnight and then incubated with FITC-labeled brevinin-1FL at the final concentrations of 2, 4, and 8 μM at 37°C for 6 h before the cells were collected and detected by flow cytometry (BD FACSCanto II, MA, USA). To identify the effects of heparin on peptide internalization, 8 μM brevinin-1FL was preincubated with 10, 20, or 40 μg/mL heparin for 30 min, and then, the mixture was incubated with PC12 cells for 6 h before flow cytometry analysis. To identify the effects of the cellular energy state on the internalization, PC12 cells were preincubated with 10, 20, or 40 μM of sodium azide (NaN 3 ) or 12.5, 25, or 50 mM of ammonium chloride (NH 4 Cl) for 30 min and then incubated with 8 μM FITC-labeled brevinin-1FL for another 6 h before flow cytometry analysis. To examine the effects of time and temperature on the internalization, PC12 cells were incubated with 8 μM FITC-labeled brevinin-1FL for 1 h or 6 h at 4°C or 37°C. To identify the effects of H 2 O 2 on internalization, PC12 cells were incubated with 8 μM FITClabeled brevinin-1FL in the presence of 0.25 mM H 2 O 2 for 6 h before flow cytometry analysis. All experiments were performed in triplicate.

Antioxidant
Activity Measurement in Vitro. The ABTS radical scavenging activity was measured with a commercial kit according to the manufacturer's instruction (Beyotime, Shanghai, China). Briefly, 10 μL brevinin-1FL (0-20 μM) and 200 μL ABTS working solution were mixed in a 96-well plate and placed at room temperature for 21 min. 10 μL of distilled water was used as the negative control. The absorbance was measured at 734 nm with a microplate reader (Infinite M1000 Pro, Tecan Company, Switzerland). ABTS radical scavenging activity was computed as follows: ABTS scavenging activity ð%Þ = ðA blank − A sample Þ/A blank × 100, where A blank is the absorbance of ABTS solution with distilled water and A sample is the absorbance in the presence of brevinin-1FL. All samples were analyzed in triplicate and averaged.
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicalscavenging activity was measured with the method described previously by us [11]. A 10 μL aliquot of brevinin-1FL (0-20 μM) was mixed with a 100 μL methanolic solution of DPPH radical at the final concentration of 0.2 mM before 2 Oxidative Medicine and Cellular Longevity being shaken vigorously and left to stand at room temperature for 21 min in the dark. The absorbance was determined at 517 nm with a microplate reader. All samples were analyzed in triplicate and averaged. The NO scavenging activity of brevinin-1FL was measured with the Griess reagent. In short, 2.5 mM sodium nitroprusside was incubated with brevinin-1FL (0-40 μM) in a 96-well plate for 1 h at room temperature. NO generation was quantified with the Griess reagent, and the scavenging rate was calculated as the decrease in NO production compared to the group without brevinin-1FL treatment.
The reducing power of brevinin-1FL was evaluated with the ferric reducing antioxidant power (FRAP) method as previously reported by us with minor modification [11]. The activated FRAP working reagent (180 μL) was loaded into a 96-well plate and incubated with 5 μL of brevinin-1FL (0-40 μM) or distilled water for 5 min at 37°C. The absorbance at 593 nm was measured with a microplate reader. Total antioxidant capacity was counted with the standard curve and expressed as the corresponding concentration of FeSO 4 solution.
2.6. Cell Viability Measurement. PC12 cells were cultured in RPMI-1640 medium (Gibco, Chicago, USA) containing 10% fetal bovine serum (Gibco, Chicago, USA) and 100 U/mL penicillin-streptomycin at 37°C in a 5% CO 2 incubator. Cell viability was determined using the MTT assay kit as previously reported by us with small modification [11]. In short, 1 × 10 4 cells/well PC12 cells were plated in 96-well plates and grown overnight. Next, PC12 cells were incubated with H 2 O 2 (0, 0.125, 0.5, and 1 mM) for 6 h or with brevinin-1FL (0-40 μM) for 24 h or with brevinin-1FL (0, 2, 4, and 8 μM) for 30 min before further coincubation with H 2 O 2 for 6 h at 37°C, respectively. 10 μL of MTT solution was loaded into each well and reincubated in the dark for 4 h. The culture supernatants were then carefully discarded, and 200 μL of DMSO was applied to dissolve the formazan crystals in each well. The absorbance at 570 nm was determined.
2.7. LDH Release Assay. The LDH release assay was carried out with the LDH kit (Beyotime, Shanghai, China) in light of the manufacturer's manual. In brief, PC12 cells were preincubated with brevinin-1FL (0, 2, 4, and 8 μM) for 30 min prior to coincubation with H 2 O 2 for another 6 h at 37°C. After centrifugation at 2,000 rpm for 5 min, 100 μL of the supernatant was mixed with 60 μL of substrate solution in the dark for 30 min, and the absorbance at 490 nm was determined with the microplate reader (Infinite M1000 Pro, Tecan Company, Switzerland). LDH release rate ð%Þ = where A blank is the absorbance of cell-free culture medium and A sample is the absorbance in supernatant of cells. All experiments were conducted in triplicate.
2.8. Cell Morphology Assessment. PC12 cells at a density of 5 × 10 5 cells/well were plated to 6-well plates and allowed to grow overnight before being pretreated with brevinin-1FL (0, 2, 4, and 8 μM) for 30 min prior to exposure to 0.25 mM H 2 O 2 for 6 h. Morphological observation was carried out with an inverted phase contrast microscope (CKX41, Olympus, Tokyo, Japan) at 100× magnification. About 4-5 single-plane photographs per well were acquired.
2.9. Antioxidant Capacity Measurement. The SOD and CAT activities as well as GSH and MDA contents in PC12 cells treated with brevinin-1FL were measured to further identify its antioxidant capacities. In brief, 5 × 10 5 cells/well PC12 were seeded into 6-well plates and allowed to grow overnight. Next, cells were incubated with brevinin-1FL (0, 2, 4, and 8 μM) for 30 min prior to incubation with 0.25 mM H 2 O 2 for another 6 h. After washing three times with cold PBS, the cells were harvested by centrifugation and lysed on ice. The supernatant was transferred to fresh tubes and stored on ice or frozen at -80°C before the measurement of MDA, SOD, CAT, and GSH using commercial assay kits (S0131, S0101, S0051, and S0052; Beyotime Institute of Biotechnology, Shanghai, China), respectively. The assay for SOD activity was based on the ability of SOD to inhibit water-soluble tetrazolium salt (WST-8) reduction by superoxide. Briefly, 20 μL of the supernatant was mixed with 160 μL of WST-8/enzyme working solution and 20 μL reacting solution and then incubated at 37°C for 30 min. Finally, the absorbance at 450 nm was determined with the microplate reader. One unit of SOD activity was defined as the amount of protein showing a 50% inhibitory effect on WST-8. The CAT activity was detected by a chromogenic substrate method. Briefly, 10 μL of the supernatant was treated with excess H 2 O 2 for 5 min. The remaining H 2 O 2 coupled with a chromogenic substrate was catalyzed with peroxidase to generate red N-4-antipyryl-3-chloro-5-sulfonate-p-benzoquinonemonoimine. The absorbance at 520 nm was detected by using a microplate reader. One unit of CAT activity is defined as the amount of enzyme catalyzing 1 μM of H 2 O 2 per mg per min at 25°C. The total GSH level was measured with the enzymatic recycling method based on the fact that GSH can be oxidized by 5,5-dithiobis-2-nitrobenzoic acid (DTNB) to generate yellow 2-nitro-5-thiobenzoic acid and reduced by NADPH in the presence of glutathione reductase. 10 μL of the supernatant was mixed with 150 μL of DNTB solution for 5 min at room temperature and then incubated with 50 μL of reaction solution containing NADPH and glutathione reductase for another 5 min. Finally, GSH concentration can be determined by measuring the absorption at 412 nm. The intracellular level of GSH was calculated based on cellular protein concentration. The concentration of MDA which is an indicator of lipid peroxidation in cells was measured using the thiobarbituric acid (TBA) method. In brief, 100 μL of the supernatant was mixed with 200 μL of the TBA reagent in a boiling water 3 Oxidative Medicine and Cellular Longevity bath for 15 min. After centrifugation at 2,000 rpm at room temperature for 10 min, the supernatant was measured at a wavelength of 530 nm under a microplate reader. MDA level unit was expressed as nmol/mg of protein. All experiments are repeated three times.
2.10. Cell Cycle and Apoptosis Measurement. The effects of brevinin-1FL on the cycle distribution and apoptosis of PC12 cells stimulated by H 2 O 2 were measured with flow cytometry (BD FACSCanto II, MA, USA). In brief, PC12 cells were seeded into 6-well plates at the density of 5 × 10 5 cells/well and allowed to grow overnight. The medium was discarded, and cells were subsequently incubated with brevinin-1FL (2, 4, and 8 μM) for 30 min before 0.25 mM H 2 O 2 was added. After cells fixed in cold 70% ethanol, the cells were collected by centrifugation and stained with propidium iodide and RNase (Beyotime, Shanghai, China) for 30 min at 37°C to measure the proportions of cell cycle distribution. For the apoptosis assay, after being grown in normal medium and treated with H 2 O 2 and brevinin-1FL (2, 4, and 8 μM) for 6 h, the cells were collected and stained with propidium iodide and Annexin V-FITC at room temperature for 30 min in the dark in light of the manufacturer's manual (Beyotime, Shanghai, China). All stained cells were analyzed by flow cytometry with a minimum of 10,000 cells. All experiments were performed in triplicate. The data were analyzed using FlowJo (ver. 7.6).

ROS Detection.
To evaluate the effect of brevinin-1FL on ROS generation in PC12 cells, DCFH-DA was used to examine the ROS level in differentially treated PC12 cells in light of the manufacturer's manual (Sigma-Aldrich; Darmstadt, Germany). In brief, 5 × 10 5 PC12 cells were incubated with brevinin-1FL (0, 2, 4, and 8 μM) for 30 min before 0.25 mM H 2 O 2 was added. After 6 h incubation, the cells were collected and gently washed with PBS. Then, cells were incubated with DCFH-DA for 30 min in the dark at 37°C, washed three times, and measured by flow cytometry (BD FACSCanto II, Mansfield, MA, USA). There were three samples in each group for the ROS detection assay.
2.12. Mitochondrial Membrane Potential (ΔΨm) Assay. ΔΨ m was determined with the JC-1 detection kit according to the manufacturer's manual (Beyotime, Shanghai, China). Briefly, the treated cells were collected and incubated with 500 μL JC-1 working solution at 37°C with 5% CO 2 for 20 min. Finally, the cells were washed before observation under the fluorescence microscopy at 400× magnification, and about 5-10 single-plane images per randomly selected area were acquired.
2.13. Western Blot Analysis. Western blots were carried out according to the method reported previously by us with minor modification [11]. In brief, cells were harvested and lysed with RIPA lysis buffer containing protease and phosphatase inhibitors (FDbio, Hangzhou, China) at 4°C for 15 min to obtain the sample for western blot analysis. Primary antibodies against phospho-AKT, AKT, phospho-ERK, ERK, phospho-JNK, JNK, phospho-p38, p38, p65, Bax, Bcl-2, PARP, cleaved PARP, caspase 3, cleaved caspase 3, and β-actin (4°C, 16 h, 1 : 2000; Cell Signaling Technology, Massachusetts, USA) and HRP-conjugated secondary antibodies (26°C, 1 h, 1 : 2000; Cell Signaling Technology, Massachusetts, USA) were used for western blot analysis. The band densities were quantified by using ImageJ software, and all experiments were performed in triplicate. Cells treated with medium alone were used as the negative control.
2.14. Carrageenan-induced Paw Edema Assay. The antiinflammatory and antioxidant activities were evaluated with the paw edema induced by carrageenan based on the method previously reported by us with minor modifications [12]. In short, the mice paw volume up to the ankle joint was measured with a plethysmometer (Taimeng PV-200 7500, Chengdu, China) as the baseline value before mice were given intraperitoneal injection with brevinin-1FL (10 mg/ kg), saline, or indomethacin (10 mg/kg). After 1 h, the plantar side of the right hind paw was injected with 50 μL of 1% carrageenan suspended in saline or saline to induce swelling and edema. The paw volume was then determined at 0, 1, 2, 3, 4, and 5 h with a plethysmometer. The animals were euthanized with an overdose of pentobarbital (200 mg/kg intraperitoneally) 5 hours after injection, and the right hind paws of all mice were surgically cut off to prepare sample for the measurement of inflammatory factors (IL-1β, IL-6, and TNF-α), oxidative stress-related indicators (SOD and CAT activity and GSH level as well as MDA content), and myeloperoxidase (MPO) activity and histological analysis.
2.15. Statistical Analysis. All experiments were repeated at least three times. All data were analyzed using the GraphPad Prism software version 5.03 (GraphPad Software, CA, USA) and expressed as the mean ± SD. One-way ANOVA with the Tukey multiple comparison posttest was used for multiplegroup analysis. A value of p < 0:05 was considered to represent a statistically significant difference.

Identification and Characterization of Brevinin-1FL.
The cDNA sequence encoding brevinin-1FL was cloned from the skin of F. limnocharis by the PCR-based method. As displayed in Figure 1, the cDNA of brevinin-1FL was 222 bp length and its deduced precursor contained 58 amino acid residues which possessed the classic sequence characteristic of amphibian defensive peptides, generally consisting of a predicted signal peptide sequence with 22 residues, an Nterminal acidic interval domain which was separated from the C-terminal mature peptide by a well-known KR protease cleavage site (Figure 1(a)). In light of the NCBI Basic Local Alignment Search Tool analysis, the deduced precursor shared high sequence identities to those of AMPs grouped into the brevinin-1 family. However, the amino acid sequence of its mature peptide named as brevinin-1FL, FWERCSRWLLN, lacked such similarity with any reported AMPs (Figure 1(b)). Brevinin-1FL had a theoretical PI of 9.24 with +3 net charge, and the aliphatic index was 104.33. Its relative mass was measured to 1509.82 Da. 4 Oxidative Medicine and Cellular Longevity 3.2. Antioxidant Activity of Brevinin-1FL in Vitro. ABTS and DPPH radical-scavenging assays were carried out to measure the antioxidant activity of brevinin-1FL. As illustrated in Figures 2(a) and 2(b), brevinin-1FL exhibited a dosedependent ABTS and DPPH radical-scavenging power across the measured concentrations (0, 2.5, 5, 10, and 20 μM). Brevinin-1FL eliminated approximately 64.20% of ABTS and 23.03% of DPPH at 21 min. NO, an important physiological mediator, has neurotoxic and proapoptotic effects when it is excessively generated [13]. As described in Figure 2(c), brevinin-1FL scavenged NO in a dosedependent manner. The total antioxidant activity of brevinin-1FL was also examined with the FRAP method. As shown in Figure 2(d), the ferric reducing ability of brevinin-1FL was increased with increasing concentrations. In addition, the antioxidant activity of brevinin-1FL was also tested with the DNA protection assay. Hydroxyl radical produced from the Fenton reaction can induce a single-strand break into supercoiled plasmid DNA and final formation of open circular DNA after incubation with plasmid DNA. As shown in Figure 2(

Internalization of Brevinin-1FL into PC12 Cells via
Endocytosis. Compared with the control group, brevinin-1FL could be rapidly internalized into PC12 cells as shown by the increasing fluorescence in the cells treated with FITC-labeled brevinin-1FL in dose-dependent manners (Figure 3(a)). Anionic heparin sulfate is an important component in the cell membrane and the extracellular matrix associated with the first step of the interaction between the membrane and a cell-penetrating peptide [14]. Thus, we further investigated the effects of heparin sulfate on the cellular internalization of brevinin-1FL. As displayed in Figure 3(b), the uptake of 8 μM FITC-labeled brevinin-1FL was reduced about 0.1%, 10.8%, and 15.6% after 1 h cotreatment with 10, 20, and 40 μg/mL heparin, respectively. Next, we examined whether the internalization of brevinin-1FL required energy. Both NH 4 Cl and NaN 3 are endocytic inhibitors because they can increase the pH of acidic endocytic vesicles and abolish ATP production within the cell membrane, respectively [15]. As shown in Figures 3(c) and 3(d), preincubation of PC12 cells with NaN 3 and NH 4 Cl for 1 h obviously inhibited the cellular uptake of brevinin-1FL in concentration-dependent manners. As a further confirmation assay, the effect of temperature on the cellular endocytosis of brevinin-1FL was also examined. As shown in Figure 3(e), the cellular uptake of brevinin-1FL at 37°C was significantly higher than that at 4°C after 1 h or 6 h incubation, suggesting an energy-dependent and thermo-sensitive endocytosis of brevinin-1FL into PC12 cells ( Figure S2).  Notably, there was no obvious difference between different incubation times under the same temperature, indicating that brevinin-1FL can fast be internalized into PC12 cells. The influence of 0.25 mM H 2 O 2 on the cellular penetration of brevinin-1FL was tested; coincubation with H 2 O 2 in PC12 cells for 6 h could not significantly influence the cellular uptake of brevinin-1FL. All these results indicate that brevinin-1FL can be internalized into PC12 cells via endocytosis (Figure 3(f)).

Effect of Brevinin-1FL on Oxidative Stress-induced Cell
Death. The cytotoxicity of a series of concentrations of H 2 O 2 towards PC12 cells was measured to ensure the protective effect of brevinin-1FL on oxidative stress-induced cell death. As shown by MTT results in Figure 4 (Figures 4(b) and 4(c)). Lactate dehydrogenase (LDH) release was investigated to evaluate the protective mechanism of brevinin-1FL against oxidative stress-induced cell death. As shown in Figure 4(d), brevinin-1FL reduced the LDH release of H 2 O 2 -treated cells in a dose-dependent manner. Notably, after PC12 cells were treated with 0.25 mM H 2 O 2 for 6 h, the discrepancy between the proliferation inhibition rate and the inhibition rate of LDH release indicating other mechanisms exists in cell death induced by H 2 O 2 except necrosis (Figures 4(c) and 4(d)). In line with the MTT results, morphological observation of PC12 cells showed that the unstimulated cells grew better than H 2 O 2 -treated cells, with a dendritic shape and even distribution while treatment with brevinin-1FL reversed these features (Figure 4(e)).

Effect of Brevinin-1FL on Intracellular SOD and CAT
Activity and MDA and GSH Content. MDA is a peroxidation product under oxidative stress while SOD, CAT, and GSH are antioxidants in cells. They are generally applied in the research of antioxidant drugs as pharmacodynamic indicators [1]. When compared with the control group, three antioxidant contents in H 2 O 2 -treated PC12 cells were restored

Effect of Brevinin-1FL on H 2 O 2 -induced ROS Production
and Mitochondrial Membrane Potential. ROS play the critical role in the regulation of cell proliferation as well as survival and mediate oxidative damage to lipids, DNA, and proteins, representing a key pathogenic role in neurodegenerative diseases [1,2]. Hence, the effect of brevinin-1FL on H 2 O 2 -induced total intracellular ROS accumulation in PC12 cells was examined. As shown in Figure 6(a), ROS contents were evidently increased after H 2 O 2 treatment. But this increase was significantly inhibited by pretreatment with brevinin-1FL in a concentration-dependent manner. The ROS production is closely associated with the mitochondrial membrane potential (Δψm) which can be used to evaluate mitochondrial function and test the protective effect of antioxidant peptides on cells suffering from oxidative stress. As shown by the fluorescence in Figure 6(b),   ROS can adjust AKT/MAPK/NF-κB signaling pathways which are involved in apoptosis and inflammation responses [1,2]. Therefore, western blot was carried out to examine the effects of brevinin-1FL on their activation in       In contrast, the contents of p65 as well as phosphorylated ERK and AKT in the cytoplasm were significantly downregulated, which suggest that AKT/MAPK/NF-κB pathways are responsible for H 2 O 2 -induced apoptosis in PC12 cells. Nevertheless, brevinin-1FL reversed the changes induced by H 2 O 2 , demonstrating that the protective effects of brevinin-1FL in PC12 cells treated with H 2 O 2 are associated with its regulation of AKT/MAPK/NF-κB signaling pathways.

Effect of Brevinin-1FL on Apoptosis and Cell Cycle Arrest.
Excessive ROS generation can cause cell cycle arrest and apoptosis due to its damage to mitochondrial functions in PC12 cells [11,16]. As displayed in Figure 7(a), the number of apoptotic cells was obviously increased in H 2 O 2 -treated cells when compared to the control. But it was reduced by brevinin-1FL in a concentration-dependent manner, and the apoptotic inhibition rates in the presence of brevinin-1FL at 2, 4, and 8 μM were around 24.30%, 35.23%, and 70.15%, respectively. Furtherly, H 2 O 2 markedly augmented the cell numbers accumulating in the G0/G1 phase, which was accompanied by the reduced cell numbers in the S phase in PC12 cells, whereas brevinin-1FL concentrationdependently ameliorated this effect induced by H 2 O 2 (Figure 7(b)). In agreement, exposure to H 2 O 2 for 6 h resulted in an obvious expression increase in Bax and decrease in Bcl-2. However, the addition of 8 μM brevinin-1FL to cells significantly counteracted these changes induced by H 2 O 2 . At the same time, brevinin-1FL significantly reduced PARP and caspase 3 cleavage upregulated by H 2 O 2 (Figures 7(c) and 7(d)). Overall, the present data prove that brevinin-1FL effectively inhibits the H 2 O 2 -induced cell cycle arrest and apoptosis, consequently increasing the viabilities of PC12 cells.

Antioxidant and Anti-Inflammatory Activity of
Brevinin-1FL in Vivo. Carrageenan-induced acute inflammation is known to be related to the accumulation of ROS, lipid peroxidation, and the impediment of antioxidant defense activities [17,18]. We therefore assessed the antiinflammatory and antioxidant ability of brevinin-1FL in the mouse paws injected by carrageenan as previously reported by us [12]. As illustrated in Figures 8(a) and 8(b), the volumes of paw edema were markedly increased by 1.81-fold after carrageenan injection in comparison with the control group. However, brevinin-1FL alleviated the paw swelling induced by carrageenan. In agreement, treatment with brevinin-1FL also suppressed the activity of MPO which is the indicator of neutrophil migration in carrageenan-injected paw tissues (Figure 8(c)). MDA is a mediator of inflammatory processes and a marker of cellular injury triggered by ROS and oxidative stress while endogenous antioxidant enzymes like SOD and CAT as well as GSH greatly contribute to eradicating the damaging effects of ROS and oxidative stress [19]. Carrageenan administration abated cellular SOD and CAT activity and GSH level and increased MDA concentration when compared to the control (Figures 8(d)-8(g)). The expression changes of IL-1β, TNF-α, and IL-6 were examined in serum and tissue of mice after carrageenan administration. As shown in Figure 8(h) and Figure S3, the above raised trends in the carrageenan-injected mice were reversed by brevinin-1FL and indomethacin. Consistently, histopathology analysis displayed that brevinin-1FL significantly weakened carrageenan-induced leukocyte infiltration (Figure 8(i)).
Notably, brevinin-1FL (10 mg/kg) compared to indomethacin possessed greater inhibition effect on the increase in MDA contents induced by carrageenan. Together, these data underscore that brevinin-1FL possesses the antioxidant and anti-inflammatory activities.

Discussion
Oxidative stress, the overproduction of ROS in cells and tissues, reflects an imbalance between antioxidants and free radicals and leads to the damage of cellular biomacromolecules such as DNA, proteins, and lipids, consequentially causing many human health disorders including neurological illness, inflammation, diabetes, cancer, and atherosclerosis [1,2]. Hence, natural and synthetic antioxidants protecting normal cells from damage derived from oxidants may have therapeutic potential and are increasingly recognized as a pivotal direction for the prevention and treatment of oxidation-associated diseases [1,3,5]. Some bioactive peptides identified from animals, especially from amphibian skin, have been found to display antioxidant and antiinflammatory activities [5,6]. However, there is no antioxidant peptide identified from F. limnocharis. Here, for the first time, we identify an antioxidant peptide from this tropical frog and explore its antioxidant effects plus underlying mechanism in H 2 O 2 -treated PC12 cells and carrageenanstimulated mice paws. Many antioxidant peptides containing different structures have been identified from amphibians, and the presence of critical residues of proline, leucine, phenylalanine, methionine, free cysteine, tyrosine, or tryptophan is responsible for their antioxidant activity [20][21][22][23][24][25][26]. In particular, cysteine containing the reducing thiol group provides more potent antioxidant capability than the above other amino acids to an antioxidant [27]. For brevinin-1FL, 1 free cysteine and 5 hydrophobic amino acids (2 leucine, 2 tyrosine, and 1 phenylalanine residues) are contained in its primary sequence (Figure 1(a)). Comparison with cathelicidin-OA1, antioxidin-I, and antioxidin-RL, brevinin-1FL contained higher proportion of hydrophobic amino acids and stronger antioxidant activity according to their radical-scavenging capability or protective effects on the cells with oxidative damage in vitro [21,28,29]. This result further demonstrates that the sequence of peptides has great effects on their antioxidant activity. It is a remarkable fact that the evolution and formation of antioxidant peptides in amphibians may be associated with long time exposure to sunshine and intense ultraviolet radiation [22,24]. The frog F. limnocharis captured from tropical Guangdong Province live in low altitude (23.12°N, 113.28°E) with long and strong sunlight radiation. Besides, these frogs generally live near pond ditches with few protections from sunlight, making them easy to receive sunlight radiation [30]. Thus, the present study supports the conclusion that the evolution of antioxidant peptides is associated with sunlight radiation as reported by other researchers [22,31,32]. Some brevinin-1-like peptides from the frog skin secretions like LFB, brevinin-1OS, and their N-terminal derivatives show potent antimicrobial activities [33,34]. Although it shares similar precursor structure with these peptides consisting of a signal peptide at the N-terminus followed by an acidic spacer region and a mature peptide at the C-terminus, brevinin-1FL like antioxidin-I and salamandrin-I does not show antibacterial activity against S. aureus ATCC 25923, E. coli ATCC 25922, and P. aeruginosa ATCC 27853 (data not shown) [24,35]. What is more, mature brevinin-1FL lacks structural similarity with any reported AMP which is consistent with our experimental antibacterial results and further proves the conclusion that a balance between hydrophobicity, positive charge, and degrees of α-helicity is crucial for keeping antimicrobial activity of peptide (Figure 1(b)) [34]. Thus, the presence of antioxidant peptide in this frog skin might reflect adaptation to the specific environment [36].
Antioxidants often keep homeostasis and prevent cells and tissues from oxidative stress-induced disorders by removing excessive free radicals [1,22]. MDA is a celldamaging peroxidation product of biomacromolecules on the surface of cell membranes caused by oxidative stress and can gradually lead to damage and dysfunction of all intracellular protein functions [37]. The hydroxyl group is thought as a DNA-damaging agent of physiological significance and can cause carcinogenesis or characteristic in the pathogenesis of neurodegenerative diseases like Alzheimer's and Parkinson's disease [3]. SOD, CAT, and GST are the key endogenous antioxidants which can reduce the contents of the oxidants and provide a first line of defense against their potentially damaging effects [19,38]. In this study, brevinin-1 FL has ability to scavenge free radicals like NO, hydroxyl radicals, ABTS + , and DPPH and reduce Fe 3+ in vitro (Figure 2), suggesting that it is an antioxidant peptide. The nervous system has high oxygen utilization, large amount of polyunsaturated fatty acids, and low contents of antioxidants, which make it very susceptible to oxidative assaults [3]. The PC12 cell line is particularly vulnerable to changes in O 2 concentration and usually used as a cellular model to research neuronal sensitivity to oxidative stress [39]. At present study, the rat differentiated PC12 cells are subjected to H 2 O 2 exposure for 6 h to mimic a neuronal in vitro model of oxidative injury. In agreement, H 2 O 2 significantly decreases the viability of PC12 cells via increasing ROS accumulation and MDA contents and decreasing the levels of endogenous antioxidants and Δψm (Figures 5 and 6), which suggest the neuronal sensitivity to oxidative damage. However, brevinin-1FL can be internalized into PC12 cells via endocytosis ( Figure 3) and successfully reverse the intracellular effects induced by H 2 O 2 , consequently decreasing the cycle arrest, apoptosis, and necrosis of PC12 cells caused