Histological Analysis of the Skin of Pithecopus spp. (Anura) and Functional Characterization of the Tryptophillin PaT-2 as an Antioxidant Peptide ^†

Introduction: Amphibians of the family Phyllomedusidae are characterized by complex cutaneous secretions with ecological and pharmacological relevance. Here, we investigated the skin morphology of Pithecopus oreades and P. azureus, and further investigated the antioxidant function of the tryptophyllin-class peptide PaT-2 in microglial cells. Methodology: For this, the methodology involved the synthesis of the bioactive peptide PaT-2 and confirmation by LC-MS/MS. Cytotoxicity tests and antioxidant assays of PaT-2 were performed. Skin histology was also analyzed. Results: Histological and histochemical analyses revealed a conserved organization of epidermis and dermis, with abundant mucous and serous glands, with the latter being significantly larger in dorsal and cephalic regions. Staining confirmed the presence of polysaccharides and lipid components, suggesting a multifunctional role in hydration, chemical defense and ecological adaptation. In cellular assays, menadione induced oxidative stress in BV2 microglial cells, manifested as increased disulfide glutathione levels, and induced glutathione synthesis without affecting cell viability. Synthetic PaT-2 showed no cytotoxicity and attenuated menadione-induced glutathione oxidation and glutathione synthesis, indicating antioxidant activity in cells. Conclusions: This study provides the first histological description of P. oreades and demonstrates that PaT-2 can modulate redox metabolism, highlighting both the ecological significance of cutaneous glandular patterns and the potential of amphibian-derived peptides as novel therapeutic leads.