Crotoxin induces cytotoxic effects in human malignant melanoma cells in both native and detoxified forms.

Introduction: Melanoma, a highly aggressive skin cancer originating in melanocytes, poses a significant threat due to its metastatic potential. While progress has been made in treating melanoma with targeted therapies and immunotherapies, challenges persist. Crotoxin (CTX), the principal toxin in Crotalus durissus terrificus snake venom, exhibits various biological activities, including anti-tumoral effects across multiple cancers. However, its clinical use is limited by toxicity. Thus, exploring alternatives to mitigate adverse effects is crucial. Methods and Results: This study investigates the antitumoral potential of CTX in its native and in a detoxified form, in melanoma cells. Firstly, we demonstrated that detoxified CTX presented reduced phospholipase activity. Both forms proved to be more cytotoxic to SK-MEL-28 and MeWo melanoma cells than non-tumoral cells. In SK-MEL-28 cells, where cytotoxic effects were more pronounced, native and detoxified CTX induced increased necrosis and apoptosis rates. We also confirmed the apoptosis death demonstrated by the activation of caspase-3 and 7, and the formation of apoptotic bodies. Furthermore, both CTX caused cell cycle arrest at the G2/M phase, interfering with melanoma cell proliferation. Cell migration and invasion were also suppressed by both CTX. These results confirm the antitumoral potential of CTX. Discussion: The maintenance of the antiproliferative effects in the detoxified version, with reduced enzymatic activity often liked to harm effects, supports further studies to identify active parts of the molecule responsible for the interesting effects without causing substantial toxic events, contributing to the future use of CTX-derived drugs with safety and efficacy.

Correction: Bufalo et al. Human Sensory Neuron-like Cells and Glycated Collagen Matrix as a Model for the Screening of Analgesic Compounds. Cells 2022, 11, 247.

In the original publication [...].

Environmental factors and their impact on chronic pain development and maintenance.

It is more than recognized and accepted that the environment affects the physiological responses of all living things, from bacteria to superior vertebrates, constituting an important factor in the evolution of all species. Environmental influences range from natural processes such as sunlight, seasons of the year, and rest to complex processes like stress and other mood disorders, infections, and air pollution, being all of them influenced by how each creature deals with them. In this chapter, it will be discussed how some of the environmental elements affect directly or indirectly neuropathic pain, a type of chronic pain caused by a lesion or disease of the somatosensory nervous system. For that, it was considered the edge of knowledge in translational research, thus including data from human and experimental animals as well as the applicability of such findings.

Crotoxin induces cytotoxic effects in human malignant melanoma cells in both native and detoxified forms

Introduction: Melanoma, a highly aggressive skin cancer originating in melanocytes, poses a significant threat due to its metastatic potential. While progress has been made in treating melanoma with targeted therapies and immunotherapies, challenges persist. Crotoxin (CTX), the principal toxin in Crotalus durissus terrificus snake venom, exhibits various biological activities, including anti-tumoral effects across multiple cancers. However, its clinical use is limited by toxicity. Thus, exploring alternatives to mitigate adverse effects is crucial. Methods and Results: This study investigates the antitumoral potential of CTX in its native and in a detoxified form, in melanoma cells. Firstly, we demonstrated that detoxified CTX presented reduced phospholipase activity. Both forms proved to be more cytotoxic to SK-MEL-28 and MeWo melanoma cells than non-tumoral cells. In SK-MEL-28 cells, where cytotoxic effects were more pronounced, native and detoxified CTX induced increased necrosis and apoptosis rates. We also confirmed the apoptosis death demonstrated by the activation of caspase-3 and 7, and the formation of apoptotic bodies. Furthermore, both CTX caused cell cycle arrest at the G2/M phase, interfering with melanoma cell proliferation. Cell migration and invasion were also suppressed by both CTX. These results confirm the antitumoral potential of CTX. Discussion: The maintenance of the antiproliferative effects in the detoxified version, with reduced enzymatic activity often liked to harm effects, supports further studies to identify active parts of the molecule responsible for the interesting effects without causing substantial toxic events, contributing to the future use of CTX-derived drugs with safety and efficacy.

Crotoxin induces cytotoxic effects in human malignant melanoma cells in both native and detoxified forms

Introduction: Melanoma, a highly aggressive skin cancer originating in melanocytes, poses a significant threat due to its metastatic potential. While progress has been made in treating melanoma with targeted therapies and immunotherapies, challenges persist. Crotoxin (CTX), the principal toxin in Crotalus durissus terrificus snake venom, exhibits various biological activities, including anti-tumoral effects across multiple cancers. However, its clinical use is limited by toxicity. Thus, exploring alternatives to mitigate adverse effects is crucial. Methods and Results: This study investigates the antitumoral potential of CTX in its native and in a detoxified form, in melanoma cells. Firstly, we demonstrated that detoxified CTX presented reduced phospholipase activity. Both forms proved to be more cytotoxic to SK-MEL-28 and MeWo melanoma cells than non tumoral cells. In SK-MEL-28 cells, where cytotoxic effects were more pronounced, native and detoxified CTX induced increased necrosis and apoptosis rates. We also confirmed the apoptosis death demonstrated by the activation of caspase-3 and 7, and the formation of apoptotic bodies. Furthermore, both CTX caused cell cycle arrest at the G2/M phase, interfering with melanoma cell proliferation. Cell migration and invasion were also suppressed by both CTX. These results confirm the antitumoral potential of CTX. Discussion: The maintenance of the antiproliferative effects in the detoxified version, with reduced enzymatic activity often liked to harm effects, supports further studies to identify active parts of the molecule responsible for the interesting effects without causing substantial toxic events, contributing to the future use of CTX-derived drugs with safety and efficacy.

Environmental factors and their impact on chronic pain development and maintenance

It is more than recognized and accepted that the environment affects the physiological responses of all living things, from bacteria to superior vertebrates, constituting an important factor in the evolution of all species. Environmental influences range from natural processes such as sunlight, seasons of the year, and rest to complex processes like stress and other mood disorders, infections, and air pollution, being all of them influenced by how each creature deals with them. In this chapter, it will be discussed how some of the environmental elements affect directly or indirectly neuropathic pain, a type of chronic pain caused by a lesion or disease of the somatosensory nervous system. For that, it was considered the edge of knowledge in translational research, thus including data from human and experimental animals as well as the applicability of such findings.

Snake and arthropod venoms: search for inflammatory activity in human cells involved in joint diseases

Most anti-inflammatory drugs currently adopted to treat chronic inflammatory joint diseases can alleviate symptoms but they do not lead to remission. Therefore, new and more efficient drugs are needed to block the course of joint inflammatory diseases. Animal venoms, rich in bioactive compounds, can contribute as valuable tools in this field of research. In this study, we first demonstrate the direct action of venoms on cells that constitute the articular joints. We established a platform consisting of cell-based assays to evaluate the release of cytokines (IL-6, IL-8, TNFα, IL-1β, and IL-10) by human chondrocytes, synoviocytes and THP1 macrophages, as well as the release of neuropeptides (substance-P and β-endorphin) by differentiated sensory neuron-like cells, 24 h after stimulation of cells with 21 animal venoms from snake and arthropod species, sourced from different taxonomic families and geographic origins. Results demonstrated that at non-cytotoxic concentrations, the venoms activate at varying degrees the secretion of inflammatory mediators involved in the pathology of articular diseases, such as IL-6, IL-8, and TNF-α by chondrocytes, synoviocytes, and macrophages and of substance P by neuron-like cells. Venoms of the Viperidae snake family were more inflammatory than those of the Elapidae family, while venoms of Arthropods were less inflammatory than snake venoms. Notably, some venoms also induced the release of the anti-inflammatory IL-10 by macrophages. However, the scorpion Buthus occitanus venom induced the release of IL-10 without increasing the release of inflammatory cytokines by macrophages. Since the cell types used in the experiments are crucial elements in joint inflammatory processes, the results of this work may guide future research on the activation of receptors and inflammatory signaling pathways by selected venoms in these particular cells, aiming at discovering new targets for therapeutic intervention.

Snake and arthropod venoms: Search for inflammatory activity in human cells involved in joint diseases.

Most anti-inflammatory drugs currently adopted to treat chronic inflammatory joint diseases can alleviate symptoms but they do not lead to remission. Therefore, new and more efficient drugs are needed to block the course of joint inflammatory diseases. Animal venoms, rich in bioactive compounds, can contribute as valuable tools in this field of research. In this study, we first demonstrate the direct action of venoms on cells that constitute the articular joints. We established a platform consisting of cell-based assays to evaluate the release of cytokines (IL-6, IL-8, TNFα, IL-1ß, and IL-10) by human chondrocytes, synoviocytes and THP1 macrophages, as well as the release of neuropeptides (substance-P and ß-endorphin) by differentiated sensory neuron-like cells, 24 h after stimulation of cells with 21 animal venoms from snake and arthropod species, sourced from different taxonomic families and geographic origins. Results demonstrated that at non-cytotoxic concentrations, the venoms activate at varying degrees the secretion of inflammatory mediators involved in the pathology of articular diseases, such as IL-6, IL-8, and TNF-α by chondrocytes, synoviocytes, and macrophages and of substance P by neuron-like cells. Venoms of the Viperidae snake family were more inflammatory than those of the Elapidae family, while venoms of Arthropods were less inflammatory than snake venoms. Notably, some venoms also induced the release of the anti-inflammatory IL-10 by macrophages. However, the scorpion Buthus occitanus venom induced the release of IL-10 without increasing the release of inflammatory cytokines by macrophages. Since the cell types used in the experiments are crucial elements in joint inflammatory processes, the results of this work may guide future research on the activation of receptors and inflammatory signaling pathways by selected venoms in these particular cells, aiming at discovering new targets for therapeutic intervention.

Cardiorespiratory/sedative effects of a peptide identified in crotalic venom compared to acepromazine and xylazine in horses

The purpose of this study was to investigate whether intravenous crotalphine produces significant sedation, as well as physiological changes, in healthy standing horses. Six mares, aged 8 years and weighing 415kg underwent three different treatments in a crossover design: TA (acepromazine: 50μg.kg-1), TC (crotalphine: 0.01μg.kg-1) and TX (xylazine: 1000μg.kg-1), intravenously. At various time points over 60 minutes, physiologic variables were recorded: heart rate, respiratory rate, and rectal temperature. The head height from the ground (HHG) was evaluated in centimeters. Data were analyzed using ANOVA followed by Dunnett’s test or Friedman followed by Dunn’s test, under 5% significance. Heart rate decreased significantly at M5 and M10 compared with Mb in TX (28±7, 26±6 and 40±8 beats/minute-1, respectively; p=0.0004). Respiratory rate and rectal temperature did not differ among groups or time points. The HHG significantly decreased in all groups compared with Mb at various time points (p<0.0001). In conclusion, crotalphine did not produce reliable and durable sedation in healthy standing mares and did not influence cardiorespiratory variables in a clinically relevant manner.

O objetivo deste estudo foi investigar se a administração de crotalfina intravenosa produz sedação significativa e alterações fisiológicas em equinos saudáveis. Seis éguas, idade média de oito anos e peso médio de 415kg, foram submetidas a três tratamentos distintos: TA (acepromazina: 50μg/kg), TC (crotalfina: 0,01μg/kg) e TX xilazina: 1000μg/kg), por via intravenosa. Em vários momentos, ao de longo de 60 minutos, as variáveis fisiológicas registradas foram frequência cardíaca, frequência respiratória e temperatura retal. A altura de cabeça ao solo (ACS) foi avaliada em centímetros. Os dados foram analisados pela ANOVA, seguida pelo teste de Dunnett ou de Friedman e, depois, pelo teste de Dunn, sob 5% de significância. A frequência cardíaca diminuiu significativamente em M5 e M10 em comparação com Mb em TX (28±7, 26±6 e 40±8 bpm, respectivamente; P=0,0004). A frequência respiratória e a temperatura retal não diferiram entre os grupos ou os pontos de tempo. O HHG diminuiu significativamente em todos os grupos em comparação com Mb em vários momentos (P <0,0001). Em conclusão, a crotalfina não produziu sedação confiável e durável em éguas saudáveis e não influenciou as variáveis cardiorrespiratórias de maneira clinicamente relevante.

Cytotoxic effect of crotoxin on cancer cells and its antitumoral effects correlated to tumor microenvironment: A review

Cancer is the second leading cause of death worldwide, and despite the effort of standard treatments, the search for new tools against this disease is necessary. Importantly, it is known that the tumor microenvironment plays a crucial role in tumor initiation, progression, and response to therapies. Therefore, studies of potential drugs that act on these components are as critical as studies regarding antiproliferative substances. Through the years, studies of several natural products, including animal toxins, have been conducted to guide the development of medical compounds. In this review, we present the remarkable antitumor activities of crotoxin, a toxin from the rattlesnake Crotalus durissus terrificus, highlighting its effects on cancer cells and in the modulation of relevant elements in the tumor microenvironment as well as the clinical trials conducted with this compound. In summary, crotoxin acts through several mechanisms of action, such as activation of apoptosis, induction of cell cycle arrest, inhibition of metastasis, and decrease of tumor growth, in different tumor types. Crotoxin also modulates tumor-associated fibroblasts, endothelial cells, and immune cells, which contribute to its antitumoral effects. In addition, preliminary clinical studies confirm the promising results of crotoxin and support its potential future use as an anticancer drug.

Crotalphine Modulates Microglia M1/M2 Phenotypes and Induces Spinal Analgesia Mediated by Opioid-Cannabinoid Systems.

Pain is a worldwide public health problem and its treatment is still a challenge since clinically available drugs do not completely reverse chronic painful states or induce undesirable effects. Crotalphine is a 14 amino acids synthetic peptide that induces a potent and long-lasting analgesic effect on acute and chronic pain models, peripherally mediated by the endogenous release of dynorphin A and the desensitization of the transient receptor potential ankyrin 1 (TRPA1) receptor. However, the effects of crotalphine on the central nervous system (CNS) and the signaling pathway have not been investigated. Thus, the central effect of crotalphine was evaluated on the partial sciatic nerve ligation (PSNL)-induced chronic neuropathic pain model. Crotalphine (100 µg/kg, p.o.)-induced analgesia on the 14th day after surgery lasting up to 24 h after administration. This effect was prevented by intrathecal administration of CB1 (AM251) or CB2 (AM630) cannabinoid receptor antagonists. Besides that, crotalphine-induced analgesia was reversed by CTOP, nor-BNI, and naltrindole, antagonists of mu, kappa, and delta-opioid receptors, respectively, and also by the specific antibodies for ß-endorphin, dynorphin-A, and met-enkephalin. Likewise, the analgesic effect of crotalphine was blocked by the intrathecal administration of minocycline, an inhibitor of microglial activation and proliferation. Additionally, crotalphine decreased the PSNL-induced IL-6 release in the spinal cord. Importantly, in vitro, crotalphine inhibited LPS-induced CD86 expression and upregulated CD206 expression in BV-2 cells, demonstrating a polarization of microglial cells towards the M2 phenotype. These results demonstrated that crotalphine, besides activating opioid and cannabinoid analgesic systems, impairs central neuroinflammation, confirming the neuromodulatory mechanism involved in the crotalphine analgesic effect.

Caspase-1 and Cathepsin B Inhibitors from Marine Invertebrates, Aiming at a Reduction in Neuroinflammation.

Neuroinflammation is a condition associated with several types of dementia, such as Alzheimer's disease (AD), mainly caused by an inflammatory response to amyloid peptides that induce microglial activation, with subsequent cytokine release. Neuronal caspase-1 from inflammasome and cathepsin B are key enzymes mediating neuroinflammation in AD, therefore, revealing new molecules to modulate these enzymes may be an interesting approach to treat neurodegenerative diseases. In this study, we searched for new caspase-1 and cathepsin B inhibitors from five species of Brazilian marine invertebrates (four cnidarians and one echinoderm). The results show that the extract of the box jellyfish Chiropsalmus quadrumanus inhibits caspase-1. This extract was fractionated, and the products monitored for their inhibitory activity, until the obtention of a pure molecule, which was identified as trigonelline by mass spectrometry. Moreover, four extracts inhibit cathepsin B, and Exaiptasia diaphana was selected for subsequent fractionation and characterization, resulting in the identification of betaine as being responsible for the inhibitory action. Both molecules are already found in marine organisms, however, this is the first study showing a potent inhibitory effect on caspase-1 and cathepsin B activities. Therefore, these new prototypes can be considered for the enzyme inhibition and subsequent control of the neuroinflammation.

Human Sensory Neuron-like Cells and Glycated Collagen Matrix as a Model for the Screening of Analgesic Compounds.

Increased collagen-derived advanced glycation end-products (AGEs) are consistently related to painful diseases, including osteoarthritis, diabetic neuropathy, and neurodegenerative disorders. We have recently developed a model combining a two-dimensional glycated extracellular matrix (ECM-GC) and primary dorsal root ganglion (DRG) that mimicked a pro-nociceptive microenvironment. However, culturing primary cells is still a challenge for large-scale screening studies. Here, we characterized a new model using ECM-GC as a stimulus for human sensory-like neurons differentiated from SH-SY5Y cell lines to screen for analgesic compounds. First, we confirmed that the differentiation process induces the expression of neuron markers (MAP2, RBFOX3 (NeuN), and TUBB3 (ß-III tubulin), as well as sensory neuron markers critical for pain sensation (TRPV1, SCN9A (Nav1.7), SCN10A (Nav1.8), and SCN11A (Nav1.9). Next, we showed that ECM-GC increased c-Fos expression in human sensory-like neurons, which is suggestive of neuronal activation. In addition, ECM-GC upregulated the expression of critical genes involved in pain, including SCN9A and TACR1. Of interest, ECM-GC induced substance P release, a neuropeptide widely involved in neuroinflammation and pain. Finally, morphine, the prototype opiate, decreased ECM-GC-induced substance P release. Together, our results suggest that we established a functional model that can be useful as a platform for screening candidates for the management of painful conditions.

How environmental enrichment balances out neuroinflammation in chronic pain and comorbid depression and anxiety disorders.

Depression and anxiety commonly occur in chronic pain states and the coexistence of these diseases worsens outcomes for both disorders and may reduce treatment adherence and response. Despite the advances in the knowledge of chronic pain mechanisms, pharmacological treatment is still unsatisfactory. Research based on exposure to environmental enrichment is currently under investigation and seems to offer a promising low-cost strategy with no side effects. In this review, we discuss the role of inflammation as a major biological substrate and aetiological factor of chronic pain and depression/anxiety and report a collection of preclinical evidence of the effects and mechanisms of environmental enrichment. As microglia participates in the development of both conditions, we also discuss microglia as a potential target underlying the beneficial actions of environmental enrichment in chronic pain and comorbid depression/anxiety. We also discuss how alternative interventions under clinical guidelines, such as environmental enrichment, may improve treatment compliance and patient outcomes. LINKED ARTICLES: This article is part of a themed issue on Building Bridges in Neuropharmacology. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.8/issuetoc.

Caspase-1 and cathepsin B inhibitors from marine invertebrates, aiming at a reduction in neuroinflammation

Neuroinflammation is a condition associated with several types of dementia, such as Alzheimer’s disease (AD), mainly caused by an inflammatory response to amyloid peptides that induce microglial activation, with subsequent cytokine release. Neuronal caspase-1 from inflammasome and cathepsin B are key enzymes mediating neuroinflammation in AD, therefore, revealing new molecules to modulate these enzymes may be an interesting approach to treat eurodegenerative diseases. In this study, we searched for new caspase-1 and cathepsin B inhibitors from five species of Brazilian marine invertebrates (four cnidarians and one echinoderm). The results show that the extract of the box jellyfish Chiropsalmus quadrumanus inhibits caspase-1. This extract was fractionated, and the products monitored for their inhibitory activity, until the obtention of a pure molecule, which was identified as trigonelline by mass spectrometry. Moreover, four extracts inhibit cathepsin B, and Exaiptasia diaphana was selected for subsequent fractionation and characterization, resulting in the identification of betaine as being responsible for the inhibitory action. Both molecules are already found in marine organisms, however, this is the first study showing a potent inhibitory effect on caspase-1 and cathepsin B activities. Therefore, these new prototypes can be considered for the enzyme inhibition and subsequent control of the neuroinflammation.

Crotalphine modulates microglia M1/M2 phenotypes and induces spinal analgesia mediated by opioid-cannabinoid systems

Pain is a worldwide public health problem and its treatment is still a challenge since clinically available drugs do not completely reverse chronic painful states or induce undesirable effects. Crotalphine is a 14 amino acids synthetic peptide that induces a potent and long-lasting analgesic effect on acute and chronic pain models, peripherally mediated by the endogenous release of dynorphin A and the desensitization of the transient receptor potential ankyrin 1 (TRPA1) receptor. However, the effects of crotalphine on the central nervous system (CNS) and the signaling pathway have not been investigated. Thus, the central effect of crotalphine was evaluated on the partial sciatic nerve ligation (PSNL)-induced chronic neuropathic pain model. Crotalphine (100 µg/kg, p.o.)-induced analgesia on the 14th day after surgery lasting up to 24 h after administration. This effect was prevented by intrathecal administration of CB1 (AM251) or CB2 (AM630) cannabinoid receptor antagonists. Besides that, crotalphine-induced analgesia was reversed by CTOP, nor-BNI, and naltrindole, antagonists of mu, kappa, and delta-opioid receptors, respectively, and also by the specific antibodies for β-endorphin, dynorphin-A, and met-enkephalin. Likewise, the analgesic effect of crotalphine was blocked by the intrathecal administration of minocycline, an inhibitor of microglial activation and proliferation. Additionally, crotalphine decreased the PSNL-induced IL-6 release in the spinal cord. Importantly, in vitro, crotalphine inhibited LPS-induced CD86 expression and upregulated CD206 expression in BV-2 cells, demonstrating a polarization of microglial cells towards the M2 phenotype. These results demonstrated that crotalphine, besides activating opioid and cannabinoid analgesic systems, impairs central neuroinflammation, confirming the neuromodulatory mechanism involved in the crotalphine analgesic effect.

Pain and cellular migration induced by Bothrops jararaca venom in mice selected for an acute inflammatory response: involvement of mast cells

Bothrops jararaca venom (BjV) can induce mast cell degranulation. In order to investigate the role of mast cells and the interference of the host genetic background in the inflammation induced by BjV, we have used mouse strains selected for maximal (AIRmax) or minimal (AIRmin) acute inflammatory response (AIR). Mice were pretreated with an inhibitor of mast cell degranulation, cromolyn (CROM), and injected in footpads or intraperitoneally (i.p.) with BjV. Pain was measured with von Frey hairs, cell migration in the peritoneum by flow cytometry, and reactive oxygen species (ROS) production by chemiluminescence assays. The nociceptive response to BjV was higher in AIRmax than AIRmin mice; however, this difference was abolished by pretreatment with CROM. BjV induced peritoneal neutrophil (CD11b+ GR-1+) infiltration and ROS secretion in AIRmax mice only, which were partially inhibited by CROM. Our findings evidence a role for mast cells in pain, neutrophil migration, and ROS production triggered by BjV in AIRmax mice that are more susceptible to the action of BjV.

Human sensory neuron-like cells and glycated collagen matrix as a model for the screening of analgesic compounds

Increased collagen-derived advanced glycation end-products (AGEs) are consistently related to painful diseases, including osteoarthritis, diabetic neuropathy, and neurodegenerative disorders. We have recently developed a model combining a two-dimensional glycated extracellular matrix (ECM-GC) and primary dorsal root ganglion (DRG) that mimicked a pro-nociceptive microenvironment. However, culturing primary cells is still a challenge for large-scale screening studies. Here, we characterized a new model using ECM-GC as a stimulus for human sensory-like neurons differentiated from SH-SY5Y cell lines to screen for analgesic compounds. First, we confirmed that the differentiation process induces the expression of neuron markers (MAP2, RBFOX3 (NeuN), and TUBB3 (β-III tubulin), as well as sensory neuron markers critical for pain sensation (TRPV1, SCN9A (Nav1.7), SCN10A (Nav1.8), and SCN11A (Nav1.9). Next, we showed that ECM-GC increased c-Fos expression in human sensory-like neurons, which is suggestive of neuronal activation. In addition, ECM-GC upregulated the expression of critical genes involved in pain, including SCN9A and TACR1. Of interest, ECM-GC induced substance P release, a neuropeptide widely involved in neuroinflammation and pain. Finally, morphine, the prototype opiate, decreased ECM-GC-induced substance P release. Together, our results suggest that we established a functional model that can be useful as a platform for screening candidates for the management of painful conditions.

PKCζ-Mitogen-Activated Protein Kinase Signaling Mediates Crotalphine-Induced Antinociception.

Crotalphine (CRP) is a structural analogue to a peptide that was first identified in the crude venom from the South American rattlesnake Crotalus durissus terrificus. This peptide induces a potent and long-lasting antinociceptive effect that is mediated by the activation of peripheral opioid receptors. The opioid receptor activation regulates a variety of intracellular signaling, including the mitogen-activated protein kinase (MAPK) pathway. Using primary cultures of sensory neurons, it was demonstrated that crotalphine increases the level of activated ERK1/2 and JNK-MAPKs and this increase is dependent on the activation of protein kinase Cζ (PKCζ). However, whether PKCζ-MAPK signaling is critical for crotalphine-induced antinociception is unknown. Here, we biochemically demonstrated that the systemic crotalphine activates ERK1/2 and JNK and decreases the phosphorylation of p38 in the lumbar spinal cord. The in vivo pharmacological inhibition of spinal ERK1/2 and JNK, but not of p38, blocks the antinociceptive effect of crotalphine. Of interest, the administration of a PKCζ pseudosubstrate (PKCζ inhibitor) prevents crotalphine-induced ERK activation in the spinal cord, followed by the abolishment of crotalphine-induced analgesia. Together, our results demonstrate that the PKCζ-ERK signaling pathway is involved in crotalphine-induced analgesia. Our study opens a perspective for the PKCζ-MAPK axis as a target for pain control.