Introduction: Snake venoms are rich sources of bioactive peptides with therapeutic potential, particularly against neurodegenerative diseases linked to oxidative stress. While the peptide fraction (<10 kDa) from Bothrops jararaca venom has shown in vitro neuroprotection, analogous fractions from related species remain unexplored in vivo. Methods: This study comparatively evaluated the neuroprotective effects of two peptide fractions (pf) from Daboia siamensis (pf-Ds) and B. jararaca (pf-Bj) against H₂O₂-induced oxidative stress using in vitro (PC12 cells) and in vivo (zebrafish, Danio rerio) models. Results: In vitro, pf-Ds (1 µg mL⁻¹) did not protect PC12 cells against H₂O₂-induced cytotoxicity, unlike previously reported effects of pf-Bj. In vivo, neither pf-Ds nor pf-Bj (1–20 µg mL⁻¹) induced significant developmental toxicity in zebrafish larvae up to 120 h post-fertilization (hpf). The neuroprotective effects of both pf were evaluated using two experimental models: (I) Larvae at 96 hpf were exposed to either pf-Ds or pf-Bj (10 µg mL⁻¹) for 4 h, followed by co-exposure to H₂O₂ (0.2 mmol L⁻¹) for an additional 10 h to induce oxidative stress (4–20 h model); (II) Embryos at 4 hpf were treated with pf-Ds or pf-Bj (10 µg mL⁻¹) continuously until 96 hpf, after which they were exposed to H₂O₂ (0.2 mmol L⁻¹) for another 24 h (96–120 h model). In a short-term treatment model, neither fraction reversed H₂O₂-induced deficits in metabolism or locomotor activity. However, in a prolonged treatment model, pf-Bj significantly reversed the H₂O₂-induced locomotor impairment, whereas pf-Ds did not confer protection. Conclusions: These findings demonstrate, for the first time, the in vivo neuroprotective potential of pf-Bj against oxidative stress-induced behavioral deficits in zebrafish, contingent on the treatment regimen. The differential effects between pf-Ds and pf-Bj highlight species-specific venom composition and underscore the value of zebrafish for evaluating venom-derived peptides.

Snake venoms are rich sources of bioactive peptides with therapeutic potential, particularly against neurodegenerative diseases linked to oxidative stress. While the peptide fraction (< 10 kDa) from Bothrops jararaca venom has shown in vitro neuroprotection, analogous fractions from related species remain unexplored in vivo. This study comparatively evaluated the neuroprotective effects of two peptide fractions (pf) from Daboia siamensis (pf-Ds) and B. jararaca (pf-Bj) against H₂O₂-induced oxidative stress using in vitro (PC12 cells) and in vivo (zebrafish, Danio rerio) models. In vitro, pf-Ds (1 µg⋅mL⁻¹) did not protect PC12 cells against H₂O₂-induced cytotoxicity, unlike previously reported effects of pf-Bj. In vivo, neither pf-Ds nor pf-Bj (1-20 µg⋅mL⁻¹) induced significant developmental toxicity in zebrafish larvae up to 120 hours post-fertilization (hpf). The neuroprotective effects of both pf were evaluated using two experimental models: (I) Larvae at 96 hpf were exposed to either pf-Ds or pf-Bj (10 µg⋅mL⁻¹) for 4 hours, followed by co-exposure to H₂O₂ (0.2 mmol⋅L⁻¹) for an additional 10 hours to induce oxidative stress (4–20 hours model); (II) Embryos at 4 hpf were treated with pf-Ds or pf-Bj (10 µg⋅mL⁻¹) continuously until 96 hpf, after which they were exposed to H₂O₂ (0.2 mmol⋅L⁻¹) for another 24 hours (96–120 hours model). In a short-term treatment model, neither fraction reversed H₂O₂-induced deficits in metabolism or locomotor activity. However, in a prolonged treatment model, pf-Bj significantly reversed the H₂O₂-induced locomotor impairment, whereas pf-Ds did not confer protection. These findings demonstrate, for the first time, the in vivo neuroprotective potential of pf-Bj against oxidative stress-induced behavioral deficits in zebrafish, contingent on the treatment regimen. The differential effects between pf-Ds and pf-Bj highlight species-specific venom composition and underscore the value of zebrafish for evaluating venom-derived peptides.

Oxidative and nitrosative stress are central mechanisms in the pathogenesis of neurodegenerative diseases, where excessive production of reactive oxygen and nitrogen species (ROS/RNS) leads to mitochondrial dysfunction, membrane damage, and neuronal death. In this study, we established and compared short-term (2 h) and long-term (20 h) exposure paradigms to sodium nitroprusside (SNP), used as a xenobiotic nitric oxide donor, in two neuronal cell lines (mHippoE-18 and PC12) and zebrafish larvae, aiming to provide a preclinical framework for neurodegenerative drug discovery. In vitro, SNP exposure caused concentration-dependent reductions in viability and alterations in oxidative balance, with mHippoE-18 cells exhibiting higher susceptibility than PC12 cells. In the short-term exposure paradigm, cytotoxicity was primarily associated with membrane disruption at higher concentrations, whereas oxidative stress contributed more strongly at intermediate doses. In the long-term exposure, mHippoE-18 cells showed strong integrated correlations between ROS, LDH release, and viability loss, highlighting their increased vulnerability to nitrosative stress. In zebrafish, SNP exposure impaired metabolic activity and swimming behavior in both paradigms. Long-term exposure led to consistent dose-dependent increases in ROS, accompanied by locomotor deficits tightly linked to energy metabolism. Overall, the higher sensitivity of mHippoE-18 cells compared with PC12 cells, together with the dose-dependent metabolic and behavioral impairments observed in zebrafish, indicates that cellular responses partially mirror in vivo outcomes. This integrative approach underscores the value of combining neuronal cell lines with zebrafish larvae to capture complementary aspects of SNP-induced neurotoxicity and to strengthen preclinical evaluation of candidate compounds with protective or therapeutic potential. These findings support the use of SNP as a xenobiotic model to probe nitrosative stress-driven neurotoxicity across cellular and organismal systems.

Oxidative and nitrosative stress are central mechanisms in the pathogenesis of neurodegenerative diseases, where excessive production of reactive oxygen and nitrogen species (ROS/RNS) leads to mitochondrial dysfunction, membrane damage, and neuronal death. In this study, we established and compared short-term (2 h) and long-term (20 h) exposure paradigms to sodium nitroprusside (SNP), used as a xenobiotic nitric oxide donor, in two neuronal cell lines (mHippoE-18 and PC12) and zebrafish larvae, aiming to provide a preclinical framework for neurodegenerative drug discovery. In vitro, SNP exposure caused concentration-dependent reductions in viability and alterations in oxidative balance, with mHippoE-18 cells exhibiting higher susceptibility than PC12 cells. In the short-term exposure, cytotoxicity was primarily associated with membrane disruption at higher concentrations, while oxidative stress contributed more strongly at intermediate doses. In the long-term exposure, mHippoE-18 cells showed strong integrated correlations between ROS, LDH release, and viability loss, highlighting their vulnerability to nitrosative stress. In zebrafish, SNP exposure impaired metabolic activity and swimming behavior in both paradigms. Long-term exposure led to consistent dose-dependent increases in ROS, accompanied by locomotor deficits tightly linked to energy metabolism. Overall, the higher sensitivity of mHippoE-18 cells compared with PC12, and the dose-dependent metabolic and behavioral impairments observed in zebrafish, indicate that cellular responses partially mirror the in vivo outcomes. This integrative approach underscores the value of combining neuronal cell lines with zebrafish larvae to capture complementary aspects of SNP-induced neurotoxicity and to strengthen preclinical evaluation of candidate compounds with protective or therapeutic potential. These findings support the use of SNP as a xenobiotic model to probe nitrosative stress–driven neurotoxicity across cellular and organismal systems.