Novel ocellatin peptides mitigate LPS-induced ROS formation and NF-kB activation in microglia and hippocampal neurons

1 Scientific RepoRtS | (2020) 10:2696 | https://doi.org/10.1038/s41598-020-59665-1 www.nature.com/scientificreports novel ocellatin peptides Mitigate LpS-induced RoS formation and nf-kB Activation in Microglia and Hippocampal neurons nayara A. Sousa1, Guilherme A. L. oliveira1, Ana patrícia de oliveira 1, André Luís f. Lopes1, Bruno iles1, Kerolayne M. nogueira1, thiago S. L. Araújo1,3, Luan K. M. Souza1,3, Alyne R. Araújo2, Joilson Ramos-Jesus2,3, Alexandra plácido4,5, constança Amaral 6, Yuri D. M. campelo3, eder Alves Barbosa7, camila c. portugal4, Renato Socodato 4, Andrea Lobo4, Joao Relvas4, Marcelo Bemquerer8, peter eaton4,6, José Roberto S. A. Leite9 & Jand Venes R. Medeiros 1,2* cutaneous secretions of amphibians have bioactive compounds, such as peptides, with potential for biotechnological applications. therefore, this study aimed to determine the primary structure and investigate peptides obtained from the cutaneous secretions of the amphibian, Leptodactylus vastus, as a source of bioactive molecules. the peptides obtained possessed the amino acid sequences, GVVDiLKGAAKDLAGH and GVVDiLKGAAKDLAGHLASKV, with monoisotopic masses of [M + H]± = 1563.8 Da and [M + H]± = 2062.4 Da, respectively. The molecules were characterized as peptides of the class of ocellatins and were named as Ocellatin-K1(1–16) and Ocellatin-K1(1–21). Functional analysis revealed that Ocellatin-K1(1–16) and Ocellatin-K1(1–21) showed weak antibacterial activity. However, treatment of mice with these ocellatins reduced the nitrite and malondialdehyde content. Moreover, superoxide dismutase enzymatic activity and glutathione concentration were increased in the hippocampus of mice. In addition, Ocellatin-K1(1–16) and Ocellatin-K1(1–21) were effective in impairing lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) formation and nf-kB activation in living microglia. We incubated hippocampal neurons with microglial conditioned media treated with LPS and LPS in the presence of Ocellatin-K1(1–16) and Ocellatin-K1(1–21) and observed that both peptides reduced the oxidative stress in hippocampal neurons. furthermore, these ocellatins demonstrated low cytotoxicity towards erythrocytes. these functional properties suggest possible to neuromodulatory therapeutic applications. The skin of amphibians has been the subject of interest and study of several research groups as well as pharmaceutical industries, due to the abundance and diversity of bioactive molecules with potential biotechnological applications, especially for the production of new drugs1. The characteristic way of living of amphibians is divided between aquatic and the terrestrial environment2. They possess a highly sensitive skin that is essential to its respiration and is highly vulnerable to environmental aggressions, such as desiccation, attack of microorganisms, ultraviolet radiation, and injuries3. This vulnerability has culminated in the development of an innate defense system as a survival strategy based on the expression, production, accumulation, and secretion of bioactive 1Laboratório de Farmacologia da Inflamação e Doenças Gastrintestinais, Universidade Federal do Delta do Parnaíba, UFDPar, Piauí, Brazil. 2Núcleo de Pesquisa em Biodiversidade e Biotecnologia, Universidade Federal do Piauí, UFPI, Piauí, Brazil. 3Instituto de Educação Superior do Vale do Parnaíba, FAHESP/IESVAP/NRE, Parnaíba, Brazil. 4LAQV/ REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciencias da Universidade do Porto, Porto, Portugal. 5Instituto de Investigação e Inovação em Saúde and Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal. 6Instituto de Medicina Molecular, IMM, Universidade de Lisboa, Lisboa, Portugal. 7Laboratório de Síntese e Análise de Biomoléculas, LSAB, Instituto de Química, UnB, Brasília, Brazil. 8Embrapa Recursos Genéticos e Biotecnologia, Brasília, Brazil. 9Núcleo de Pesquisa em Morfologia e Imunonologia Aplicada, NuPMIA, Área Morfologia, Faculdade de Medicina, UnB, Brasília, Brazil. *email: [email protected] open 2 Scientific RepoRtS | (2020) 10:2696 | https://doi.org/10.1038/s41598-020-59665-1 www.nature.com/scientificreports www.nature.com/scientificreports/ molecules, such as peptides, by glands located in the dermis of these animals4. Nevertheless, this vulnerability may reappear in the presence of a global biotic threat such as the chytridiomycosis that promoted a huge reduction of Amphibian biodiversity affecting many species of the Leptodactylidae family5. Amphibian’s peptides are attractive candidates for investigating biological activities that may reveal detailed molecular defence mechanisms and high levels of functional diversity. They are known to function as antihypertensives and vasodilators, opioids, peptidase inhibitors, neuropeptides, peptides for wound healing, nitric oxide inhibitors, insulin releasers, myotropics, antitumoral, antimicrobial, and antioxidant peptides6; all these properties affect potential predators and pathogens3. Although a number of such defense peptides have been reported against biological injuries, very few peptides against abiotic injuries, such as those caused by exposure to ultraviolet radiation have been studied. In amphibians, exposure to ultraviolet radiation in the sensitive corneous area of the skin together with the difference in oxygen availability caused by the transition between the aquatic and terrestrial environment results in an accelerated endogenous production of reactive oxygen species (ROS)7. Under these conditions, when the environmental oxygen concentration is higher, the skin of the amphibians consumes more absorbed oxygen instead of satisfying the oxygen demands of other tissues. In addition, loss of body water is associated with increased oxidative damage2. Thus, one may suggest that the skin of amphibians can contribute to homeostasis against accelerated oxidative stress developed during changing environmental conditions for their survival. Among the strategies already known to protect amphibians from ultraviolet light are the proteins Melanopsin present in skin pigment cells8 and Pheomelanin found in the dorsal skin of Hymenochirus boettgeri9. Additionally, some studies have demonstrated antioxidant potential of peptides extracted from the cutaneous secretion of frogs, such as antioxidin-RL with a strong free radical scavenging ability2 and antioxidin-I that substantially attenuates the hypoxia-induced ROS production in living microglia, suggesting a potential neuroprotective role for this peptide10. Oxidative stress is a cellular or physiological condition with a high concentration of ROS that causes molecular damage to cellular structures11. Although cells contain a number of antioxidant defenses for minimizing ROS fluctuations, situations where ROS generation often exceed the antioxidant capacity of cells are correlated with the onset and progression of many diseases through mutations of DNA, protein oxidation, and lipid peroxidation with consequent functional alterations and loss of vital functions in several tissues or organs12,13. In this context, considering that the neuroanatomic region of the brain is highly vulnerable to oxidative stress, molecules such as amphibian antioxidant peptides, which rapidly exert their biological functions by eliminating free radicals within several seconds may serve as promising neuroprotective agents. Based on previously described data, the aim of this study is to identify and characterize Ocellatin-K1(1–16) and Ocellatin-K1(1–21) peptides isolated from cutaneous secretion of the tropical frog, Leptodactylus vastus, as a possible antioxidant agent in vivo and in vitro. Results isolation and structure characterization of new ocellatins. The identified and characterized ocellatins in this study were isolated from the amphibian, Leptodactylus vastus, which is found in an ecotonal region of the Brazilian northeast, undergoing great climatic variations, especially during periods of prolonged drought, often leading to mishaps during expeditions and dead individuals near temporary ponds (Fig.1A–C). The chromatogram obtained from the cutaneous secretion of L. vastus presented diverse components absorbing at 216 and 280 nm, suggesting that it contained several potential bioactive peptides (Fig.1D). The peptide identification strategy of this work was outlined for Ocellatin class molecules. The mass spectrometry analysis performed for chromatographic fractions eluted between 40–50 min revealed two ions with monoisotopic masses of [M + H]+ = 1563.9 and [M + H]+ = 2062.3 Da. De novo sequencing of these ions revealed the structures GVVDI/LI/LK/QGAAK/QDI/LAGH (Fig.2A) and GVVDI/LI/LK/QGAAK/QDI/LAGHI/LASK/QV (Fig.2B), respectively. Additionally, the C-terminal of both peptides were not post-translated modified being R-COOH. I/L and K/Q ambiguities were resolved by Edman degradation and the primary structures were confirmed as GVVDILKGAAKDLAGH and GVVDILKGAAKDLAGHLASKV. A search for similarities shows that both peptides reassembles structurally to ocellatins isolated from skin secretion of Leptodactylus frogs (Table1)14–22. The alignment shows that both peptides have structural similarity to Ocellatin-K1 (UniProtKB/Swiss-Prot: P86711.1), comprised of 25 amino acid residues, and isolated from the cutaneous secretion of Amazonian toad-frog Leptodactylus knudseni. L. knudseni, L. fallax, L. pentadactylus and L. vastus comprise the groups of species named Leptodactylus pentadactylus23. As both deduced peptides characterized here presented 16 (GVVDILKGAAKDLAGH) or 21 (GVVDILKGAAKDLAGHLASKV) amino acid residues identical to Ocellatin K1, and can be considered as truncated peptides of Ocellatin-K1, they were named Ocellatin-K1(1–16) and Ocellatin-K1(1–21), respectively. In fact, an m/z corresponding to the Ocellatin-K1 was identified but its characterization was not possible because its low intensity signal presented in the mass spectrum (data not showed). Furthermore, the peptides studied in this work are also very closely related to Ocellatin-F, Ocellatin-L1 and Ocellatin-L2, both isolated from cutaneous secretions of amphibians from Central America. The results of in silico analyses for molecular modeling and experimental data of circular dichroism show that these linear cationic peptides tend to possess an alpha-helix formation in the presence of TFE. In aqueous solution, they are randomized, but in TFE concentrations of 10 to 40%, the formation of secondary structures occurs (Fig.3). Antibacterial activity against strains of Escherichia coli and Staphylococcus aureus. To analyze the antibacterial activity of Ocellatin-K1(1–16) and Ocellatin-K1(1–21), we assayed the peptides against Gram-negative (Escherichia coli ATCC 25922) and Gram-positive (Staphylococcus aureus ATCC 25923) strains and MIC values were determined. The results obtained for both the ocellatins showed weak inhibitory activity 3 Scientific RepoRtS | (2020) 10:2696 | https://doi.org/10.1038/s41598-020-59665-1 www.nature.com/scientificreports www.nature.com/scientificreports/ against E. coli with MIC of 125 μg/mL (Fig.4) and inhibition percentage corresponding to 34.17 ± 11.66%. The optical density (630 nm) of E. coli decreased in a dose-dependent manner, showing significant reduction on viability for both the ocellatins at concentrations between 125 and 1000 μg/mL. The value 125 μg/mL of MIC is too high to be characterized as having significant antibacterial potential. Moreover, only Ocellatin-K1(1–16) showed any significant activity against S. aureus featuring MICs of 31.25 μg/mL and inhibition percentage corresponding to 30.79 ± 10.27%. This activity was not seen to be concentration dependent. Ocellatin-K1(1–21) did not exert antibacterial activity against S. aureus at any of the tested concentrations (31.25 to 1000 μg/mL). Effect of Ocellatin-K1(1–16) and Ocellatin-K1(1–21) on superoxide dismutase relative enzymatic activity. The results showed that SOD relative enzymatic activity in the hippocampal tissue homogenate of animals treated with saline (control) alone was 396.3 ± 54.29 U SOD/µg protein. Acute treatment with Ocellatin-K1(1–16) and Ocellatin-K1(1–21) at the tested doses (250 µg/kg) significantly (p < 0.05) stimulated SOD relative enzymatic activity (724.9 ± 88.90 U SOD/µg protein; and 616.0 ± 75.61 U SOD/µg protein, respectively) compared to those treated with saline. Ascorbic acid (250 mg/kg), an antioxidant standard, demonstrated Figure 1. The largest delta in open seas in the Americas and the third largest in the world, the Rio Parnaiba Delta covers 70 islands within its 2,700 km2 area, that includes dunes, mangrove forest, and streams (A) (ArcMap v10.3, http://desktop.arcgis.com/en/arcmap/10.3). A temporary pond located in the city of Ilha Grande, State of Piaui. This habitat is home to L. vastus in this region (B). Adult male specimen of L. vastus (Photo: Jose Roberto S. A. Leite) (C). Reverse-phase HPLC chromatogram of the crude extract from L. vastus skin secretion. Fractions containing Ocellatin-K1(1–16) and Ocellatin-K1(1–21) are shown (D). 4 Scientific RepoRtS | (2020) 10:2696 | https://doi.org/10.1038/s41598-020-59665-1 www.nature.com/scientificreports www.nature.com/scientificreports/ basal levels of the endogenous antioxidant SOD (324.3 ± 66.61 U SOD/µg protein) compared to those of control as shown in Fig.5A. Effect of Ocellatin-K1(1–16) and Ocellatin-K1(1–21) on nitrite reduction. Acute administration of Ocellatin-K1(1–16) and Ocellatin-K1(1–21) significantly reduced (p < 0.05) the basal content of nitrite (0.0991 ± 0.0004 µM; and 0.1001 ± 0.0009 µM, respectively) compared to those observed in the saline group (0.1021 ± 0.0007 µM). This result suggests that treatment with ocellatins protects the hippocampus. Furthermore, ascorbic acid significantly reduced (p < 0.05) the nitrite content (0.0980 ± 0.0004 µM) compared with that of the control group (Fig.5B). Thus, mice that were not treated with either ocellatins or ascorbic acid had higher contents of nitrite. Effect of Ocellatin-K1(1–16) and Ocellatin-K1(1–21) on glutathione concentration. Acute administration of both Ocellatin-K1(1–16) and Ocellatin-K1(1–21), cellular defence agents, significantly elevated (p < 0.05) the GSH concentration (157.4 ± 6.71 µg/g tissue and 146.6 ± 7.62 µg/g tissue, respectively) compared to those with saline group (105.6 ± 18.68 µg/g tissue). Therefore, the protective effect of ocellatins administration might be explained by a resultant increase in the hippocampal GSH concentration. Administration of ascorbic acid (164.1 ± 15.60 µg/g tissue) also elevated hippocampal GSH concentration in the experimental group when compared to those in the saline group, as shown Fig.5C. Effect of Ocellatin-K1(1–16) and Ocellatin-K1(1–21) on malondialdehyde concentration. Our results suggest that the administration of Ocellatin-K1(1–16) peptide maintained the basal concentration of MDA (163.0 ± 3.31 nmol/g tissue) and did not result in a significant difference in MDA concentration in the mice hippocampi when compared with those in saline group (169.5 ± 4.84 nmol/g tissue) as shown the Fig.5D. However, Ocellatin-K1(1–21) peptide significantly (p < 0.05) reduced the redox state of the hippocampi of animals in the experimental group to 138.6 ± 6.61 nmol/g tissue when compared with animals treated with saline. Furthermore, the ascorbic acid (143.1 ± 8.53 nmol/g tissue) treatment also showed a significant (p < 0.05) effect compared to that of control group. Effect of Ocellatin-K1(1–16) and Ocellatin-K1(1–21) on LPS-induced NF-kB activation in microglia. As demonstrated above, ocellatins decreased the basal ROS production and the oxidative state of the mice hippocampi. For a better understanding the role of the ocellatins redox regulation, we analysed the Ocellatin-K1(1–16) and Ocellatin-K1(1–21) effect on lipopolysaccharide (LPS)-induced NF-kB activation in living microglia using time-lapse video microscopy. To achieve this, we transfected the microglial cells with the Figure 2. MS/MS spectra for de novo sequencing of Ocellatin-K1(1–16), [M + H] ± = 1563.9 Da (A) and Ocellatin-K1(1–21), [M + H]± = 2062.3 Da (B) acquired using an MALDI-TOF/TOF UltraFlex Xtreme mass spectrometer. 5 Scientific RepoRtS | (2020) 10:2696 | https://doi.org/10.1038/s41598-020-59665-1 www.nature.com/scientificreports www.nature.com/scientificreports/ biosensor of NF-kB pathway inhibitor and observed that incubation with 100 μM of either Ocellatin-K1(1–16) or Ocellatin-K1(1–21) significantly (p < 0.01) prevented LPS-induced NF-kB activation in microglia when compared with LPS only-treated group (Fig.6) with no change in NF-kB basal activity. Effect of Ocellatin-K1(1–16) and Ocellatin-K1(1–21) on microglial-induced ROS formation in hippocampal neurons. The overactivated microglia released several molecules that could induce neuronal damage24. Since we observed that Ocellatin-K1(1–16) and Ocellatin-K1(1–21) blocked the LPS-induced activation of NF-kB by microglia, we hypothesized that ocellatins can protect hippocampal neurons from oxidative stress induced by microglial activation. Towards this objective, we treated hippocampal neurons with microglial conditioned medium, and observed a significant (p < 0.001) increase in ROS formation induced by the conditioned media obtained from LPS-treated microglia in comparison with conditioned media from control microglia (Fig.7). We observed that both Ocellatin-K1(1–16) and Ocellatin-K1(1–21) significantly (p < 0.001) reduced neuronal oxidative stress elicited by LPS-treated microglia. Name Sequence Mw¶(Da) pI µH Species Ocellatin-K1 (1–16) G V V D I L K G A A K D L A G H — — — — — — — — — — — — — — — — — 1563.82 6.75 0.483 L. vastus* Ocellatin-K1 (1–21) G V V D I L K G A A K D L A G H L A S K V — — — — — — — — — — — — 2062.44 8.51 0.399 L. vastus* Ocellatin-K1 G V V D I L K G A A K D L A G H L A S K V M N K I———————— a2549 9.53 0.256 L. knudseni-L. vastus Ocellatin-F (fallaxin) G V V D I L K G A A K D IA G H L A S K V M N K L———————— a2549 9.53 0.258 L. fallax/L. pentad-actylus Ocellatin-L1 (laticeptin) G V V D I L K G A A K D L A G H L A TK V M N K L— — — — — — — — 2563.1 9.53 0.244 L. laticeps Ocellatin-L2 G V V D I L K G A A K D L A G H L A TK V M D K L— — — — — — — — 2564 8.44 0.247 L. laticeps Ocellatin-S (syphaxin) G V LD I L K G A A K D L A G H VATK V I NK I ———————— a2545 9.53 0.223 L. syphax Ocellatin-V1 G V V D I L K G A GK D L L A HA L S K LS E K V———————— a2633.1 8.44 0.14 L. validus Ocellatin-V2 G V L D I L K G A GK D L L A HA L S K IS E K V ———————— a2576 8.44 0.274 L. validus Ocellatin-V3 G V LD I L TG A GK D L L A HA L S K LS E K V———————— a2549 6.75 0.295 L. validus Ocellatin-1 G V V D I L K G A GK D L L A H L V G K I S E K V———————— a2560 8.44 0.309 L. ocellatus Ocellatin-2 G V LD I F K D A A K Q I L A H A A E Q I ———————————— a2379.7 6.75 0.269 L. ocellatus Ocellatin-3 G V LD I L K N A A K N I L A H A A E Q I ———————————— a2202.5 6.75 0.438 L. ocellatus Ocellatin-4 G L LDF V T GVGK D IFA Q LIKQ I ———————————— a2275.7 5.96 0.473 L. ocellatus Ocellatin-5 G L LDF L K AAGK G L VTN L ———————————————— a1730.0 8.59 0.489 L. ocellatus Ocellatin-6 A V LDF I K AAGK G L VT N I M E K V G ——————————— a2274.7 8.54 0.344 L. ocellatus Ocellatin-P (penta-dactylin) G L LD T L K G A A K N V Vz G S L A S K V M E K L———————— a2543 9.53 0.324 L. pentadactylus Ocellatin-PT1 G V F D I IK D A GK Q L V A HA M G K I A E K V———————— a2639.1 8.44 0.267 L. pustullatus Ocellatin-PT2 G V F D I IK D A GK Q L V A HAT G K I A E K V———————— a260.9 8.44 0.272 L. pustullatus Ocellatin-PT3 G V ID I IK G A GK D L I A HA I G K L A E K V———————— a253.0 8.44 0.271 L. pustullatus Ocellatin-PT4 G V F D I IK G A GK Q L I A H A MG K I A E K V———————— a2595.1 9.53 0.252 L. pustullatus Ocellatin-PT5 G V F D I IK D A GR Q L V A H A MG G K I A EK V ——————— a2667.1 8.5 0.268 L. pustullatus Ocellatin-PT6 G V F D I IK G A GK Q L I A H A ME E K I A E K V G L N K D G N 3365.9 8.39 0.23 L. pustullatus Ocellatin-PT7 G V F D I IK G A GK Q L I A H A MG G K I A E K V G L N K D G N 3293.8 9.4 0.212 L. pustullatus Ocellatin-PT8 G V F D I K G A GK Q L I A R A MG K I A E K V G L N K D G N — 3312.9 9.82 0.214 L. pustullatus Table 1. Sequence, Mw, pI, and hydrophobic moment of ocellatins class peptides isolated from Leptodactylus genus. ∞Underline letters represent conservative substitutions. aC -terminus amidated peptide. *Peptides this work. 6 Scientific RepoRtS | (2020) 10:2696 | https://doi.org/10.1038/s41598-020-59665-1 www.nature.com/scientificreports www.nature.com/scientificreports/ Haemolytic Assays of Ocellatin-K1(1–16) and Ocellatin-K1(1–21). The haemolytic activities of ocellatins against human erythrocytes, as summarized in Fig.8, indicate that Ocellatin-K1(1–16) peptide did not give rise to lysis at the concentrations tested (7.8 to 500 µg/mL), and there was no significant difference between the effects of ocellatins and the negative control (saline). However, the highest concentration (500 μg/mL) of the Ocellatin-K1(1–21) analysed, demonstrated approximately 35% haemolysis. Thus, these results suggest that Ocellatin-K1(1–16) is less toxic than Ocellatin-K1(1–21) in lysing erythrocytes. Figure 3. Circular dichroism of peptides in aqueous solution and in 2,2,2-TFE. (A) Ocellatin-K1(1–16) and (B) Ocellatin-K1(1–21). (C) 3D structural model predictions of the ocellatins from Leptodactylus vastus (this study) compared to those of Ocellatin-K1 from Leptodactylus knudseni (Knudsen’s thin-toed frog). Figure 4. Assessment of anti-microbial activity of Ocellatin-K1(1–16) and Ocellatin-K1(1–21) by MIC assays against E. coli (A) and S. aureus (B) strains in a range of concentrations from 31.25 to 1000 μg/mL. The tests were performed in a single assay in triplicate. The results are expressed as mean ± SEM. *p < 0.05 vs. control group; **p < 0.01 vs. control group; ***p < 0.001 vs. control group; **** p < 0.0001 vs. control group. ANOVA and Sidak test. Abbreviations: K1(1–16): Ocellatin-K1(1–16); K1(1–21): Ocellatin-K1(1–21); OD: optical density. 7 Scientific RepoRtS | (2020) 10:2696 | https://doi.org/10.1038/s41598-020-59665-1 www.nature.com/scientificreports www.nature.com/scientificreports/ Effect of Ocellatin-K1(1–16) and Ocellatin-K1(1–21) on erythrocyte morphology and roughness. As demonstrated above, ocellatins showed low haemolytic activity in human erythrocytes. Thus, we analysed the human erythrocyte membrane morphology and roughness under AFM and the representative results are shown in Fig.9. Untreated erythrocytes appeared as typical biconcave shape (Fig.9A). After 30 minutes of incubation with Ocellatin-K1(1–16), no significant morphological changes in the cells were found at the concentrations tested (250, 500 and 1000 µg/mL; Fig.9B–F, respectively). However, erythrocytes treated with Ocellatin-K1(1–16) peptide at a concentration 500 µg/mL demonstrated a significant increase of roughness (7.64 ± 0.56 nm) compared to that observed in untreated samples (4.68 ± 0.22 nm), and this finding was statistically (p < 0.0001) significant (Fig.9H). In addition, it was observed that the biconcave topography of erythrocytes was altered by incubation with Ocellatin-K1(1–21) at a concentration 500 µg/mL for 30 minutes (Fig.9E). Thus, characteristic protrusions indicating damage to the erythrocyte membrane were observed, which were not seen in control cells. Furthermore, exposure to Ocellatin-K1(1–21) at a concentration of 1000 µg/mL resulted in a significant change in the cell shapes (Fig.9G) compared to that in control group. This damage could be confirmed by the significant increase in the membrane roughness (11.44 ± 0.74 nm) of erythrocytes treated with Ocellatin-K1(1–21) at concentration of 1000 µg/mL, which was significantly increased (p < 0.0001) as compared to that in control cells. Discussion Amphibian’s skins are a rich resource of peptides, which have diverse biological activities and are regarded as potential sources of new therapeutic agents25. Studies have focused on the purification of novel antimicrobial peptides due to their considerable association with innate defence mechanisms26. In the current study, we analysed the antioxidant activity of two peptides purified from skin secretions of L. vastus. According to the sequence alignment of the structures obtained, the peptides were designated as Ocellatin-K1(1–16) and Ocellatin-K1(1–21) because they were leptodactylid peptides with similarities in amino acid sequence to the ocellatins. Many studies involving characterization of leptodactylid antimicrobial peptides have also been described18. However, Ocellatin-K1(1–16) and Ocellatin-K1(1–21) showed weak antibacterial activity against the E. coli ATCC 25922 and S. aureus ATCC 25923 strains tested. Previous reports indicate that not all ocellatins have antibacterial activity. Studies with Leptodactylus pustulatus22 revealed that Ocellatin-PT2 did not inhibit Gram-negative bacterial strains; however, Ocellatin-PT7 and -PT8 demonstrated antibacterial activities against a Gram-positive strain with low antimicrobial potency. In this study, other homologous ocellatins, such as ocellatin-PT1, -PT3, -PT4, Figure 5. Oxidative parameters in hippocampus of mice acutely treated with Ocellatin-K1(1–16) and Ocellatin-K1(1–21). (A) SOD relative enzymatic activity, (B) nitrite content, (C) GSH, and (D) MDA concentration. Ascorbic acid was used as a standard antioxidant. The results are expressed as mean ± SEM of a minimum of six animals per group. *p < 0.05 vs. saline group employing ANOVA and Newman–Keuls test. Abbreviations: AA: ascorbic acid; GSH: reduced glutathione; K1(1–16): Ocellatin-K1(1–16); K1(1–21): Ocellatin-K1(1–21); MDA: malondialdehyde; SAL: saline; SOD: superoxide dismutase. 8 Scientific RepoRtS | (2020) 10:2696 | https://doi.org/10.1038/s41598-020-59665-1 www.nature.com/scientificreports www.nature.com/scientificreports/ -PT5 and -PT6 inhibited one or more Gram-negative and -positive bacterial strains. These peptides differ by only a few amino acid substitutions and present different bactericidal activities. In our study, employing sequence alignments, Ocellatin-K1(1–16) and Ocellatin-K1(1–21) peptides showed sequence similarities with such ocellatins obtained from L. pustulatus. Thus, it may be suggested that small differences in sequences can lead to important differences in the activity spectra of the peptides. In addition, the antimicrobial activity of peptides is determined by a set of factors, such as conformation, net charge, hydrophobicity, and amphipathicity27. Further, despite Ocellatin-K1(1–16) and Ocellatin-K1(1–21) tend to adopt α-helices conformation at a hydrophobic environment, a trait of amphibian’s antimicrobial peptide, they are truncated peptides of Ocellatin-K1 that possess the “additional” motif MNKL-NH2, when compared to Ocellatin-K1(1–21). The absence of the referred motif, as well as the lack of C-terminal amidation, may be promoting the reduced antimicrobial activities observed for Ocellatin-K1(1–16) and Ocellatin-K1(1–21). C-terminal amidation is directly associated to an improvement in the abilities of cationic peptides to interact with biological membranes, a prerequisite to action of membrane active antimicrobial peptides28–30. Finally, the study of truncated peptides was reported for Hypsiboas raniceps and was associated to the loss of antimicrobial activity when compared to the intact molecule, probably reflecting distinct protective role between stored and secreted peptides31. Considering that anura of L. vastus species live in the Parnaiba Delta Region, Northeast of Brazil, with exposure to strong and long periods of sunlight radiation, wherein their skins are exposed to elevated ultraviolet radiation, they are likely to possess a specific and highly effective antioxidant system. Thus, the main objective of this study was to test if the new peptides isolated from cutaneous secretion of L. vastus have antioxidant activity. In vivo studies were performed using isolated hippocampus due to its high sensitivity to oxidative stress. Neural tissue has a high rate of oxygen consumption and possesses a high metabolic activity, and therefore, is a tissue more vulnerable to lipid peroxidation as compared to other tissues32. Consequently, antioxidant peptides, which rapidly exert their biological functions, have a potential to prevent neuronal damage related to lipid peroxidation. Ocellatin-K1(1–16) and Ocellatin-K1(1–21) have small structures that help penetrate the blood–brain barrier (BBB) and exert antioxidant effects in the hippocampus. Indeed, some peptides cross the BBB through endocytic mechanisms involving receptor mediated transcytosis and/or adsorptive-mediated transcytosis. In recent years, BBB shuttle peptides have received growing attention because of their lower cost, reduced immunogenicity, biologic specificity and higher chemical versatility33. We demonstrated that Ocellatin-K1(1–16) and Ocellatin-K1(1–21) protected against oxidative stress at the concentrations tested. Our ocellatins were effective in increasing SOD activity and the basal concentration of GSH in hippocampal tissues. These markers protect cells against deleterious effects of free oxygen radicals, thus Figure 6. Ocellatin-K1(1–16) and Ocellatin-K1(1–21) prevented the LPS-induced NF-kB activation in living microglia. CHME3 human microglial cells expressing the biosensor of NF-kB pathway inhibitor were incubated with 100 μM Ocellatin-K1(1–16) or Ocellatin-K1(1–21) and challenged with 1 μg/mL LPS. Microglia were incubated only with Ocellatin-K1(1–16) and Ocellatin-K1(1–21) and compared with saline control group. Time-lapse fluorescence intensities for the NF-kB pathway inhibitor biosensor are shown (n = 17–19 cells pooled across two different experiments). The results are expressed as mean ± SEM. #p < 0.01 vs. LPS group; *p < 0.001 vs. CT not treated with LPS employing one-way ANOVA with Bonferroni post-test. Abbreviations: CT: control; LPS: lipopolysaccharide. 9 Scientific RepoRtS | (2020) 10:2696 | https://doi.org/10.1038/s41598-020-59665-1 www.nature.com/scientificreports www.nature.com/scientificreports/ providing a defence mechanism for the survival of aerobic organisms. SOD, a metalloenzyme, catalyses the dismutation of superoxide anions into oxygen and hydrogen peroxide. In addition, the tripeptide, GSH, protects cells against damage caused by reactive oxygen species, including free radicals and peroxides34. This study Figure 7. Ocellatin-K1(1–16) and Ocellatin-K1(1–21) protect hippocampal neurons from oxidative stress induced by LPS-treated-microglial conditioned media. Representative confocal images and quantification of ROS production in hippocampal neurons incubated with conditioned medium from microglia subjected to LPS-induced and 100 μM Ocellatin-K1(1–16) or Ocellatin-K1(1–21). Images show neurons expressing mVenus (green) and the Hyper Red ROS biosensor (red). The results are expressed as mean ± SEM calculated from 3 different cultures. *p < 0.001 vs. LPS group; #p < 0.001 vs. CT employing one-way ANOVA with the Bonferroni post-test. Abbreviations: CT: control; LPS: lipopolysaccharide; MCM: microglia conditioned medium. Figure 8. Haemolytic activity of Ocellatin-K1(1–16) and Ocellatin-K1(1–21) in human erythrocytes at concentrations ranging from 7.8 to 500 µg/mL. A positive control was determined using a 10% solution of Triton X-100. ANOVA and t-test were used for statistical analysis. 16 Scientific RepoRtS | (2020) 10:2696 | https://doi.org/10.1038/s41598-020-59665-1 www.nature.com/scientificreports www.nature.com/scientificreports/ 56. Socodato, R. C. et al. c-Src deactivation by the polyphenol 3-O-caffeoylquinic acid abrogates reactive oxygen species-mediated glutamate release from microglia and neuronal excitotoxicity. Free Radic. Biol. Med. 79, 45–55 (2015). 57. So cod ato, R . et al. Dopamine promotes NMDA receptor hypofunction in the retina through D1 receptor-mediated Csk activation, Src inhibition and decrease of GluN2B phosphorylation. Sci. Rep. 7, 40912 (2017). 58. Bignami, G. S. A rapid and sensitive hemolysis neutralization assay for palytoxin. Toxicon. 31, 817–820 (1993). Acknowledgements The authors thank Dr. Etielle Andrade for the illustrative scheme of the map of the region of the Parnaiba delta used in this work and Pedro Costa e Silva and Dr. Maria Feio for their help in scientific expeditions and knowledge of the region. Alexandra Plácido is a recipient of a post-doctoral grant from the project FCT (PTDC/ BII-BIO/31158/2017). Renato Socodato and Camila Cabral Portugal hold postdoctoral fellowships from FCT (Refs: SFRH/BPD/91833/2012 and FRH/BPD/91962/2012, respectively). This work was funded through project UID/QUI/50006/2013-POCI/01/0145/FEDER/007265 (LAQV/REQUIMTE) with financial support from FCT/ MEC through national funds and co-financed by FEDER, under the Partnership Agreement PT 2020. Author contributions J.V.R.M., J.R.S.A.L. and N.A.S. designed the project. N.A.S. participated substantially in all the stages of this research. J.R.S.A.L. and P.E. collected frogs for this study. A.P., Y.D.M.C., and E.A.B. performed the isolation, characterization and structural studies of the peptides. M.B. performed the peptide synthesis. A.R.A. performed antibacterial test. G.A.L.O., A.P.O., A.L.F.L., B.I., K.M.N., T.S.L.A. and L.K.M.S. participated in the antioxidant activity determination of peptides in mice hippocampus. J.R.J. participated haemolytic assays. P.E. and C.A. performed atomic force microscopy analyse on the human red blood cells. C.C.P., R.S., J.R. and A.L. participated in the microglia and neuron assays. The authors read and approved the final manuscript. competing interests The authors declare no competing interests. Additional information Correspondence and requests for materials should be addressed to J.V.R.M. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. 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