Panicolytic-like effects of environment enrichment on male mice threatened by Bothrops jararaca lancehead pit vipers.

Environment enrichment (EE) is a well-known eustress model showing beneficial effects in different psychiatric diseases, but its positive properties in panic disorders are not yet established. The confrontation between prey and predator in complex arenas has been validated as a putative panic attack model. The principal aim of this work was to investigate the role of the EE on panic-like defensive responses elicited by mice threatened by venomous snakes. After 6 weeks of exposure either to an enriched or standard environments, 36 male mice were habituated in a complex polygonal arena for snakes containing an artificial burrow and elevated platforms for escape. The animals were confronted by Bothrops jararaca for 5 min, and the following antipredatory responses were recorded: defensive attention, stretched attend posture, flat back approach, prey versus predator interaction, oriented escape behavior, time spent in a safe place, and number of crossings. Mice threatened by snakes displayed several antipredatory reactions as compared to the exploratory behavior of those animals submitted to a nonthreatening situation (toy snake) in the same environment. Notably, EE causes anxiolytic- and panicolytic-like effects significantly decreasing the defensive attention and time spent in safe places and significantly increasing both prey versus predator interaction and exploratory behavior. In conclusion, our data demonstrate that EE can alter the processing of fear modulation regarding both anxiety- and panic-like responses in a dangerous condition, significantly modifying the decision-making defensive strategy.

BjSP, a novel serine protease from Bothrops jararaca snake venom that degrades fibrinogen without forming fibrin clots.

Snake venom serine proteases (SVSPs) are commonly described as capable of affecting hemostasis by interacting with several coagulation system components. In this study, we describe the isolation and characterization of BjSP from Bothrops jararaca snake venom, a serine protease with distinctive properties. This enzyme was isolated by three consecutive chromatographic steps and showed acidic character (pI 4.4), molecular mass of 28 kDa and N-carbohydrate content around 10%. Its partial amino acid sequence presented 100% identity to a serine protease cDNA clone previously identified from B. jararaca venom gland, but not yet isolated or characterized. BjSP was significantly inhibited by specific serine protease inhibitors and showed high stability at different pH values and temperatures. The enzyme displayed no effects on washed platelets, but was able to degrade fibrin clots in vitro and also the Aα and Bß chains of fibrinogen differently from thrombin, forming additional fibrinopeptides derived from the Bß chain, which should be related to its inability to coagulate fibrinogen solutions or platelet-poor plasma. In the mapping of catalytic subsites, the protease showed high hydrolytic specificity for tyrosine, especially in subsite S1. Additionally, its amidolytic activity on different chromogenic substrates suggests possible effects on other factors of the coagulation cascade. In conclusion, BjSP is a serine protease that acts nonspecifically on fibrinogen, generating different Bß fibrinopeptides and thus not forming fibrin clots. Its distinguished properties in comparison to most SVSPs stimulate further studies in an attempt to validate its potential as a defibrinogenating agent.

Potential Utilization of a Polysaccharide from the Marine Algae Gayralia oxysperma, as an Antivenom for Viperidae Snakebites.

Worldwide, snakebites have serious implications for human health. The administration of antivenom is the official treatment used to reverse the toxic activities of envenomation. However, this therapy is not efficient to treat the local effects, leading to the amputation or deformity of affected limbs. As such, alternative treatments are needed. Here, we analyze the ability of a polysaccharide from the green marine alga Gayralia oxysperma (Go3) to inhibit the effects of venom from Bothrops jararaca and Lachesis muta. B. jararaca or L. muta venoms were incubated together with sulfated heterorhamnans from Go3, and the in vitro (coagulation, proteolytic, and hemolytic) and in vivo (hemorrhagic, myotoxic, edematogenic, and lethal) activities of venoms were assessed. Additionally, Go3 was injected before and after the injection of venoms, and the toxic activities were further tested. When incubated with the venoms, Go3 inhibited all activities, though results varied with different potencies. Moreover, Go3 neutralized hemorrhagic, myotoxic, and edematogenic activities when injected before or after injection with B. jararaca and L. muta venom. Go3 also blocked the coagulation of plasma in mice caused by the venoms in an ex vivo test. Therefore, Go3 has the potential to be used as antivenom for B. jararaca and L. muta bites, notably exhibiting higher efficacy on L. muta venom.

Dynamic Rearrangement in Snake Venom Gland Proteome: Insights into Bothrops jararaca Intraspecific Venom Variation.

We carried out an analysis of the venom gland proteome of Bothrops jararaca taking into account two distinct phases of its ontogenetic development (i.e., newborn and adult) and the marked sexual dimorphism recently reported on its venom proteome. Proteomic data analysis showed a dynamic rearrangement in the proteome landscape of B. jararaca venom gland upon development and gender-related changes. Differentially expressed proteins covered a number of biological pathways related to protein synthesis, including proteins associated with transcription and translation, which were found to be significantly higher expressed in the newborn venom gland. Our results suggest that the variation in the expression levels of cellular proteins might give rise to an even higher variation in the levels of the expressed toxins. Upon aging, the venom gland proteome repertoire related to the protein synthesis together with ecological traits would have an impact on the toxin repertoire, which, in the case of B. jararaca species, would enable the species to deal with different prey types during its lifespan. Proteomic data are available via ProteomeXchange with identifier PXD004186.

Venom-related transcripts from Bothrops jararaca tissues provide novel molecular insights into the production and evolution of snake venom.

Attempts to reconstruct the evolutionary history of snake toxins in the context of their co-option to the venom gland rarely account for nonvenom snake genes that are paralogous to toxins, and which therefore represent important connectors to ancestral genes. In order to reevaluate this process, we conducted a comparative transcriptomic survey on body tissues from a venomous snake. A nonredundant set of 33,000 unigenes (assembled transcripts of reference genes) was independently assembled from six organs of the medically important viperid snake Bothrops jararaca, providing a reference list of 82 full-length toxins from the venom gland and specific products from other tissues, such as pancreatic digestive enzymes. Unigenes were then screened for nontoxin transcripts paralogous to toxins revealing 1) low level coexpression of approximately 20% of toxin genes (e.g., bradykinin-potentiating peptide, C-type lectin, snake venom metalloproteinase, snake venom nerve growth factor) in body tissues, 2) the identity of the closest paralogs to toxin genes in eight classes of toxins, 3) the location and level of paralog expression, indicating that, in general, co-expression occurs in a higher number of tissues and at lower levels than observed for toxin genes, and 4) strong evidence of a toxin gene reverting back to selective expression in a body tissue. In addition, our differential gene expression analyses identify specific cellular processes that make the venom gland a highly specialized secretory tissue. Our results demonstrate that the evolution and production of venom in snakes is a complex process that can only be understood in the context of comparative data from other snake tissues, including the identification of genes paralogous to venom toxins.

Effect of photobiomodulation on endothelial cell exposed to Bothrops jararaca venom.

Bleeding is a common feature in envenoming caused by Bothrops snake venom due to extensive damage to capillaries and venules, producing alterations in capillary endothelial cell morphology. It has been demonstrated, in vivo, that photobiomodulation (PBM) decreases hemorrhage after venom inoculation; however, the mechanism is unknown. Thus, the objective was to investigate the effects of PBM on a murine endothelial cell line (tEnd) exposed to Bothrops jararaca venom (BjV). Cells were exposed to BjV and irradiated once with either 660- or 780-nm wavelength laser light at energy densities of 4 and 5 J/cm(2), respectively, and irradiation time of 10 s. Cell integrity was analyzed by crystal violet and cell viability/mitochondrial metabolism by MTT assay. The release of lactic dehydrogenase (LDH) was quantified as a measure of cell damage. In addition, cytokine IL1-ß levels were measured in the supernatant. PBM at 660 and 780 nm wavelength was able to increase cellular viability and decrease the release of LDH and the loss of cellular integrity. In addition, the concentration of pro-inflammatory cytokine IL1-ß was reduced after PBM by both wavelengths. The data reported herein indicates that irradiation with red or near-infrared laser resulted in protection on endothelial cells after exposure to Bothrops venom and could be, at least in part, a reasonable explanation by the beneficial effects of PBM inhibiting the local effects induced by Bothrops venoms, in vivo.

The Anti-Inflammatory Effects of the Methanolic Extract and Fractions from Davilla elliptica St. Hil. (Dilleniaceae) on Bothrops jararaca Envenomation.

Inflammation and haemorrhage are the main characteristics of tissue injury in botropic envenomation. Although some studies have shown that anti-venom prevents systemic reactions, it is not efficient in preventing tissue injury at the site of the bite. Therefore, this work was undertaken to investigate the anti-inflammatory effects of the methanolic extract and fractions from D. elliptica and to evaluate the role of matrix metalloproteinases (MMPs) in this process. Effects of the extract and fractions from D. elliptica were evaluated using a carrageenan-induced paw oedema model in rats, and leukocyte rolling was visualized by intravital. The quantification of MMPs activities (MMP-2 and MMP-9) extracted from the dermis of mice treated with extract and fractions alone or incubated with venom was determined by zymographic analyses. Our results show that intraperitoneal (i.p.) injection of fractions significantly reduced paw oedema after the carrageenan challenge. Treatment with the tannins fraction also resulted in considerable inhibition of the rolling of leukocytes and this fraction was able to decrease the activation of MMP-9. These results confirmed the anti-inflammatory activity of the methanolic extract and tannins fraction of D. elliptica and showed that the dermonecrosis properties of B. jararaca venom might be mediated through the inhibition of MMP-9 activity.

Molecular Aspects Involved in the Mechanisms of Bothrops jararaca Venom-Induced Hyperalgesia: Participation of NK1 Receptor and Glial Cells.

Accidents caused by Bothrops jararaca (Bj) snakes result in several local and systemic manifestations, with pain being a fundamental characteristic. The inflammatory process responsible for hyperalgesia induced by Bj venom (Bjv) has been studied; however, the specific roles played by the peripheral and central nervous systems in this phenomenon remain unclear. To clarify this, we induced hyperalgesia in rats using Bjv and collected tissues from dorsal root ganglia (DRGs) and spinal cord (SC) at 2 and 4 h post-induction. Samples were labeled for Iba-1 (macrophage and microglia), GFAP (satellite cells and astrocytes), EGR1 (neurons), and NK1 receptors. Additionally, we investigated the impact of minocycline, an inhibitor of microglia, and GR82334 antagonist on Bjv-induced hyperalgesia. Our findings reveal an increase in Iba1 in DRG at 2 h and EGR1 at 4 h. In the SC, markers for microglia, astrocytes, neurons, and NK1 receptors exhibited increased expression after 2 h, with EGR1 continuing to rise at 4 h. Minocycline and GR82334 inhibited venom-induced hyperalgesia, highlighting the crucial roles of microglia and NK1 receptors in this phenomenon. Our results suggest that the hyperalgesic effects of Bjv involve the participation of microglial and astrocytic cells, in addition to the activation of NK1 receptors.

Ultrasound and Gold Nanoparticles Improve Tissue Repair for Muscle Injury Caused by Snake Venom.

OBJECTIVE: To develop a treatment that enhances recovery from envenomation-induced lesions caused by Bothrops jararaca venom by using ultrasound in combination with gold nanoparticles (GNPs). METHODS: A total of 108 Swiss mice were arranged into nine groups. The animals underwent necrotic induction with 250 µg B. jararaca venom (BjV) and were treated with ultrasound (U) at 1 MHz frequency at an intensity of 0.8 W/cm² for 5 min, 30 mg/L GNPs, and anti-bothropic serum (AS) in the following combinations: saline solution (SS); BjV; BjV + AS; BjV + AS + U; BjV + GNPs + AS; BjV + GNPs + AS + U; BjV + GNPs; BjV + GNPs + U; and BjV + U. The necrotic area, histology, oxidative stress, oxidative damage, and anti-oxidant system were assessed to evaluate the effects of the treatments. RESULTS: Treatments that included GNPs, U, and/or AS demonstrated reductions in necrotic area, increases in angiogenesis and fibroblast means, decreases in inflammatory infiltrates, and improvements in collagen synthesis. Additionally, there was an increase in oxidants and oxidant damage within the gastrocnemius muscle, along with an increase in anti-oxidants. Furthermore, systemic effects appear to have been achieved, improving the anti-oxidant system at the cardiovascular and renal levels. CONCLUSION: The use of GNPs and U may be effective at treating lesions caused by B. jararaca snake venom.

Alkaloids from Siparuna (Siparunaceae) are predicted as the inhibitors of proteolysis and plasma coagulation caused by snake venom and potentially counteract phospholipase A2 activity of Bothrops jararaca.

ETHNOPHARMACOLOGICAL RELEVANCE: Snakebite envenomation (SBE) is the world's most lethal neglected tropical disease. Bothrops jararaca is the species that causes the greatest number of SBEs in the South and Southeastern of Brazil. The main symptoms are local (inflammation, edema, hemorrhage, and myonecrosis) and systemic (hemorrhage, hemostatic alterations with consumptive coagulopathy, and death) effects. Species of the genus Siparuna, Siparunaceae, are used in folk and traditional medicine to treat SBE. However, limited information is available concerning Brazilian Siparuna species against SBE. AIM OF THE STUDY: To investigate the correlation between the compounds present in the extracts of five Siparuna species as potential agents against proteolytic activity, plasma coagulation, and phospholipase A2 (PLA2) activity caused by B. jararaca venom, using data obtained by UHPLC-MS/MS, biological activity, and multivariate statistics. MATERIALS AND METHODS: The ethanol extracts from leaves of S. ficoides, S. decipiens, S. glycycarpa, S. reginae, and S. cymosa were fractionated by liquid-liquid extraction using different solvents of increasing polarity (hexane, dichloromethane, ethyl acetate, and n-butanol), affording their respective extracts, totaling 25 samples that were assayed through in vitro plasma coagulation and proteolytic activity assays. Moreover, the extracts were analyzed by UHPLC-MS/MS, using electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI) in negative and positive ionization modes. The data was processed in MZmine v. 2.53 and evaluated by multivariate statistical tests (PLS) using the software UnscramblerX v. 10.4. These data were also used to build molecular networks (GNPS), and some ions of interest could be annotated using the library of molecules on the GNPS platform. RESULTS: A total of 19 extracts inhibited B. jararaca-induced plasma coagulation, with emphasis on S. cymosa and S. reginae (800 s). The inhibition of the proteolytic activity was also promising, ranging from 16% (S. glycycarpa) to 99% (S. cymosa, S. decipiens, and S. reginae). In addition, most extracts from S. cymosa and S. reginae inhibited 70-90% of PLA2 activity. Based on data from positive mode APCI analyses, it was possible to obtain a statistic model with reliable predictive capacity which exhibited an average R2 of 0.95 and a Q2 of 0.88, indicating a robust fit. This process revealed five ions, identified as the alkaloids: coclaurine (1), stepholidine (2) O-methylisopiline (3), nornantenine (4) and laurolitsine (5). This is the first study to evidence the potential antivenom of alkaloids from Siparuna species. CONCLUSIONS: Altogether, our results give support to the popular use of Siparuna extracts in SBE accidents, suggesting their potential as an alternative or complementary strategy against envenoming by B. jararaca venom. The predicted ions in the chemometric analysis for the assayed activities can also be correlated with the blocking activity and encourage the continuation of this study for possible isolation and testing of individual compounds on the used models.

Anti-inflammatory, healing and antiophidic potential of Jatropha mollissima (Pohl) Baill. (Euphorbiaceae): From popular use to pharmaceutical formulation in gel.

Jatropha mollissima (Pohl) Baill. (Euphorbiaceae) is widely used in traditional medicine to treat inflammatory disorders. So, a topical gel containing the hydroethanolic extract of its leaves was developed and evaluated for its anti-inflammatory, wound healing, and antiophidic properties in mice. First, the chemical profile of different parts of the plant was characterized by liquid chromatography coupled to mass spectrometry (LC-MS) using molecular networking. In the leaf extract, 11 compounds were characterized, with a particular emphasis on the identification of flavonoids. The gel efficiently inhibited carrageenan-induced paw edema, as well as acute and chronic croton oil-induced ear edema models, thereby reducing inflammatory and oxidative parameters in inflamed tissues. Besides anti-inflammatory activity, the herbal gel showed significant wound healing activity. The edematogenic, hemorrhagic and dermonecrotic activities induced by Bothrops jararaca snake venom were effectively inhibited by the treatment with J. mollissima gel. The association with the herbal gel improved in up to 90% the efficacy of commercial snake antivenom in reduce venom-induced edema. Additionally, while antivenom was not able to inhibit venom-induced dermonecrosis, treatment with herbal gel reduced in 55% the dermonocrotic halo produced. These results demonstrate the pharmacological potential of the herbal gel containing J. mollissima extract, which could be a strong candidate for the development of herbal products that can be used to complement the current antivenom therapy against snake venom local toxicity.

Activation of M1 muscarinic acetylcholine receptors by proline-rich oligopeptide 7a (Bothrops jararaca snake venom rescues oxidative stress-induced neurotoxicity in PC12 cells.

Background: The bioactive peptides derived from snake venoms of the Viperidae family species have been promising as therapeutic candidates for neuroprotection due to their ability to prevent neuronal cell loss, injury, and death. Therefore, this study aimed to evaluate the cytoprotective effects of a synthetic proline-rich oligopeptide 7a (PRO-7a;

Understanding Local Reactions Induced by Bothrops jararaca Venom: The Role of Inflammatory Mediators in Leukocyte-Endothelium Interactions.

In recent years, extensive research has delved into the pathophysiology of local reactions triggered by Bothrops snake venoms. Even though antivenom works well at reducing death and systemic effects, it is still not very effective in treating local reactions because it cannot counteract damage that has already been triggered. This limitation might be attributed to certain molecules that amplify the venom-induced innate response. While evidence suggests endogenous mediators at the venom site play a role in this envenomation, in Brazil, the concurrent use of anti-inflammatory agents or other drugs alongside antivenom remains uncommon. This study evaluated the pharmacological mediation of alterations in leukocyte-endothelium interactions following the experimental envenomation of mice with Bothrops jararaca venom, the main culprit of snake-related accidents in Southeast Brazil. We treated envenomed mice with inhibitors of different pharmacological pathways and observed the cremaster muscle microcirculation with intravital microscopy. We found that eicosanoids related to cyclooxygenase pathways and nitric oxide significantly contributed to B. jararaca venom-induced alterations in leukocyte-endothelium interactions. Conversely, lipoxygenase-mediated eicosanoids, histamine, and serotonin had minimal participation. Notably, dexamethasone and antivenom treatment diminished B. jararaca venom-induced alterations in leukocyte-endothelium interactions. The limited efficacy of the antivenom in managing Bothrops venom-induced local reactions emphasizes the critical need for supplementary treatments to enhance therapeutic outcomes.

Peptide fraction from B. jararaca snake venom protects against oxidative stress-induced changes in neuronal PC12 cell but not in astrocyte-like C6 cell.

Venom-derived proteins and peptides have prevented neuronal cell loss, damage, and death in the study of neurodegenerative disorders. The cytoprotective effects of the peptide fraction (PF) from Bothrops jararaca snake venom were evaluated against oxidative stress changes in neuronal PC12 cells and astrocyte-like C6 cells. PC12 and C6 cells were pre-treated for 4 h with different concentrations of PF, and then H2O2 was added (0.5 mM in PC12 cells; 0.4 mM in C6 cells) and incubated for 20 h more. In PC12 cells, PF at 0.78 µg mL-1 increased viability (113.6 ± 6.3%) and metabolism (96.3 ± 10.3%) cell against H2O2-induced neurotoxicity (75.6 ± 5.8%; 66.5 ± 3.3%, respectively), reducing oxidative stress markers such as ROS generation, NO production, and arginase indirect activity through urea synthesis. Despite that, PF showed no cytoprotective effects in C6 cells, but potentiated the H2O2-induced damage at a concentration lower than 0.07 µg mL-1. Furthermore, the role of metabolites derived from L-arginine metabolism was verified in PF-mediated neuroprotection in PC12 cells, using specific inhibitors of two of the key enzymes in the L-arginine metabolic pathway: the α-Methyl-DL-aspartic acid (MDLA) to argininosuccinate synthetase (AsS), responsible for the recycling of L-citrulline to L-arginine; and, L-NΩ-Nitroarginine methyl ester (L-Name) to nitric oxide synthase (NOS), which catalyzes the synthesis of NO from L-arginine. The inhibition of AsS and NOS suppressed PF-mediated cytoprotection against oxidative stress, indicating that its mechanism is dependent on the production pathway of L-arginine metabolites such as NO and, more importantly, polyamines from ornithine metabolism, which are involved in the neuroprotection mechanism described in the literature. Overall, this work provides novel opportunities for evaluating whether the neuroprotective properties of PF shown in particular neuronal cells are sustained and for exploring potential drug development pathways for the treatment of neurodegenerative diseases.

A snapshot of Bothrops jararaca snake venom gland subcellular proteome.

Snake venom protein synthesis undergoes finely regulated processes in the specialized secretory epithelium within the venom gland. Such processes occur within a defined period in the cell and at specific cellular locations. Thus, the determination of subcellular proteomes allows the characterization of protein groups for which the site may be relevant to their biological roles, thereby allowing the deconvolution of complex biological circuits into functional information. In this regard, we performed subcellular fractionation of proteins from B. jararaca venom gland, focusing on nuclear proteins since this cellular compartment comprises key effectors that shape gene expression. Our results provided a snapshot of B. jararaca's subcellular venom gland proteome and pointed to a 'conserved' proteome core among different life stages (newborn and adult) and between sexes (adult male and female). Overall, the top 15 highly abundant proteins identified in B. jararaca venom glands mirrored the panel of highly expressed genes in human salivary glands. Therefore, the expression profile observed for such a protein set could be considered a conserved core signature of salivary gland secretory epithelium. Moreover, the newborn venom gland displayed a unique expression signature of transcription factors involved in regulating transcription and biosynthetic processes and may mirror biological constraints of the ontogenetic development of B. jararaca, contributing to venom proteome diversity.

Cationic PLGA Nanoparticle Formulations as Biocompatible Immunoadjuvant for Serum Production and Immune Response against Bothrops jararaca Venom.

Snakebite envenoming represents a worldwide public health issue. Suitable technologies have been investigated for encapsulated recombinant or native proteins capable of inducing an effective and long-lasting adaptive immune response. Nanoparticles are colloidal dispersions that have been used as drug delivery systems for bioactive biological compounds. Venom-loaded nanoparticles modulate the protein release and activate the immune response to produce specific antibodies. In this study, biocompatible cationic nanoparticles with Bothrops jararaca venom were prepared to be used as a novel immunoadjuvant that shows a similar or improved immune response in antibody production when compared to a conventional immunoadjuvant (aluminum hydroxide). We prepared stable, small-sized and spherical particles with high Bothrops jararaca venom protein association efficiency. The high protein loading efficiency, electrophoresis, and zeta potential results demonstrated that Bothrops jararaca venom is adsorbed on the particle surface, which remained as a stable colloidal dispersion over 6 weeks. The slow protein release occurred and followed parabolic diffusion release kinetics. The in vivo studies demonstrated that venom-loaded nanoparticles were able to produce an immune response similar to that of aluminum hydroxide. The cationic nanoparticles (CNp) as carriers of bioactive molecules, were successfully developed and demonstrated to be a promising immunoadjuvant.

Local envenomation caused by a bioactive peptide fraction of Bothrops jararaca snake venom induces leukocyte influx in the lung and changes in pulmonary mechanics.

The crude venom of the Bothrops jararaca snake (Bj-CV) is a complex mixture of biologically active proteins that includes a variety of peptides in the low molecular weight fraction (Bj-PF). We investigated how an intramuscular injection of Bj-CV (1.2 mg kg-1) and Bj-PF (0.24 mg kg-1) influenced lung mechanics and lung and muscle inflammation in male Swiss mice 15 min, 1, 6, and 24 h after inoculation. Pressure dissipation against lung resistive components (ΔP1) rose significantly from 1 to 24 h after Bj-CV and 6-24 h after Bj-PF inoculation. Both Bj-CV and Bj-PF increased the total pressure variation of the lung (ΔPtot) 24 h after injection. Lung static elastance increased significantly after injection in all time periods investigated by Bj-CV and from 6 to 24 h by Bj-PF. Lung static elastance increased significantly after injection in all time periods investigated by Bj-CV and from 6 to 24 h by Bj-PF. Furthermore, intramuscular inoculation of Bj-CV and Bj-PF resulted in an increase in muscle and pulmonary inflammation, as evidenced by an increase in leukocyte influx when compared to the control group. Finally, both Bj-CV and Bj-PF cause acute lung injury, as shown by pulmonary inflammation and decreased lung mechanics. Furthermore, the fact that Bj-PF produces mechanical alterations in the lungs and muscular inflammation implies that non-enzymatic compounds can cause inflammation.

Bothrops jararaca Snake Venom Inflammation Induced in Human Whole Blood: Role of the Complement System.

The clinical manifestations of envenomation by Bothrops species are complex and characterized by prominent local effects that can progress to tissue loss, physical disability, or amputation. Systemic signs can also occur, such as hemorrhage, coagulopathy, shock, and acute kidney failure. The rapid development of local clinical manifestations is accompanied by the presence of mediators of the inflammatory process originating from tissues damaged by the bothropic venom. Considering the important role that the complement system plays in the inflammatory response, in this study, we analyzed the action of Bothrops jararaca snake venom on the complement system and cell surface receptors involved in innate immunity using an ex vivo human whole blood model. B. jararaca venom was able to induce activation of the complement system in the human whole blood model and promoted a significant increase in the production of anaphylatoxins C3a/C3a-desArg, C4a/C4a-desArg, C5a/C5a-desArg and sTCC. In leukocytes, the venom of B. jararaca reduced the expression of CD11b, CD14 and C5aR1. Inhibition of the C3 component by Cp40, an inhibitor of C3, resulted in a reduction of C3a/C3a-desArg, C5a/C5a-desArg and sTCC to basal levels in samples stimulated with the venom. Exposure to B. jararaca venom induced the production of inflammatory cytokines and chemokines such as TNF-α, IL-8/CXCL8, MCP-1/CCL2 and MIG/CXCL9 in the human whole blood model. Treatment with Cp40 promoted a significant reduction in the production of TNF-α, IL-8/CXCL8 and MCP-1/CCL2. C5aR1 inhibition with PMX205 also promoted a reduction of TNF-α and IL-8/CXCL8 to basal levels in the samples stimulated with venom. In conclusion, the data presented here suggest that the activation of the complement system promoted by the venom of the snake B. jararaca in the human whole blood model significantly contributes to the inflammatory process. The control of several inflammatory parameters using Cp40, an inhibitor of the C3 component, and PMX205, a C5aR1 antagonist, indicates that complement inhibition may represent a potential therapeutic tool in B. jararaca envenoming.

Study on the synthesis and structure-activity relationship of 1,2,3-triazoles against toxic activities of Bothrops jararaca venom.

Snakebite envenoming is a health concern and has been a neglected tropical disease since 2017, according to the World Health Organization. In this study, we evaluated the ability of ten 1,2,3-triazole derivatives AM001 to AM010 to inhibit pertinent in vitro (coagulant, hemolytic, and proteolytic) and in vivo (hemorrhagic, edematogenic, and lethal) activities of Bothrops jararaca venom. The derivatives were synthesized, and had their molecular structures fully characterized by CHN element analysis, Fourier-transform infrared spectroscopy and Nuclear magnetic resonance. The derivatives were incubated with the B. jararaca venom (incubation protocol) or administered before (prevention protocol) or after (treatment protocol) the injection of B. jararaca venom into the animals. Briefly, the derivatives were able to inhibit the main toxic effects triggered by B. jararaca venom, though with varying efficacies, and they were devoid of toxicity through in vivo, in silico or in vitro analyses. However, it seemed that the derivatives AM006 or AM010 inhibited more efficiently hemorrhage or lethality, respectively. The derivatives were nontoxic. Therefore, the 1,2,3-triazole derivatives may be useful as an adjuvant to more efficiently treat the local toxic effects caused by B. jararaca envenoming.

CB1 receptor signalling mediates cannabidiol-induced panicolytic-like effects and defensive antinociception impairment in mice threatened by Bothrops jararaca lancehead pit vipers.

BACKGROUND: Cannabis sativa-derived substances such as cannabidiol (CBD) have attracted increasing clinical interest and consist in a new perspective for treating some neurological and psychiatric diseases. AIMS: The aim of this work was to investigate the effect of acute treatment with CBD on panic-like defensive responses displayed by mice threatened by the venomous snake Bothrops jararaca. METHODS: Mice were habituated in the enriched polygonal arena for snake panic test. After recording the baseline responses of the tail-flick test, the prey were pretreated with intraperitoneal (i.p.) administrations of the endocannabinoid type 1 receptor (CB1) antagonist AM251 (selective cannabinoid 1 receptor antagonist with an IC50 of 8 nM) at different doses, which were followed after 10 min by i.p. treatment with CBD (3 mg/kg). Thirty minutes after treatment with CBD, mice were subjected to confrontations by B. jararaca for 5 min, and the following defensive responses were recorded: risk assessment, oriented escape behaviour, inhibitory avoidance and prey-versus-snake interactions. Immediately after the escape behaviour was exhibited, the tail-flick latencies were recorded every 5 min for 30 min. OUTCOMES: Mice threatened by snakes displayed several anti-predatory defensive and innate fear-induced antinociception responses in comparison to the control. CBD significantly decreased the risk assessment and escape responses, with a consequent decrease in defensive antinociception. The CBD panicolytic effect was reversed by i.p. treatment with AM251. CONCLUSIONS: These findings suggest that the anti-aversive effect of CBD depends at least in part on the recruitment of CB1 receptors.