Evaluation of Rhinella marina Extracts on the Effector Functions of Human Neutrophils

Neutrophils play a central role in the innate immune response. However, in inflammatory and autoimmune diseases, the exacerbated activation of these cells can result in excessive release of reactive oxygen species (ROS), proteases, and inflammatory mediators, contributing to tissue damage. In this context, natural compounds derived from amphibian venoms have attracted interest due to their pharmacological and immunomodulatory potential. This study aimed to evaluate in vitro the effects of the crude methanolic extract (CME) of Rhinella marina on the effector functions of human neutrophils. For this purpose, peripheral blood neutrophils from healthy individuals were treated with CME (0.5, 5, and 50 µg/mL) or untreated controls. Cytotoxicity, total ROS production, superoxide anion production, myeloperoxidase (MPO) activity, elastase degranulation, NET (Neutrophil Extracellular Trap) formation, and parameters related to cellular redox status, including antioxidant enzymes and markers of oxidative damage, were evaluated. CME showed concentration-dependent cytotoxicity. There were no changes in ROS production by luminol-dependent chemiluminescence after stimulation with myristoylphorbol acetate (PMA). However, it reduced superoxide anion production in neutrophils in both stimuli in the NBT assay. CME did not significantly modulate MPO activity or NET formation, but it reduced elastase release, suggesting modulation of neutrophil degranulation. Additionally, it increased glutathione-S-transferase (GST) activity and reduced glutathione (GSH) levels. It can be concluded that CME partially modulated neutrophil effector functions, such as superoxide anion production and degranulation, in addition to exhibiting partial protective effects on cellular redox state parameters. These findings suggest the extract's immunomodulatory potential and reinforce the need for further studies to clarify its mechanisms of action and possible applicability in neutrophil-mediated inflammatory conditions.