Avaliação da atividade pró-inflamatória de Bothropstoxina-I do veneno de Bothrops jararacussu em camundongos AIRmax e AIRmin.

Snakebites are characterized as a neglected disease worldwide. Envenomation by snakes of the Bothrops genus have been mostly studied, due to their prevalence and incidence of related accidents. Hemorrhage, edema, tissue damage, myonecrosis; in addition to systemic effects, such as renal failure and incoagulability are described in bothropic envenomation. Local inflammatory effects are evident and mainly correlated with the tissue damage triggered by the toxins from the whole venom. The in vitro experimental model of C2C12 myoblasts has been a tool to study the myotoxicity induced by distinct toxins as well as the repair and regeneration processes. Metalloproteinases are predominant among other components in most of Bothrops snakes, however, for the Bothrops jararacussu, phospholipases A2 (PLA2) are in greater quantity. This finding may be mainly related to the high myotoxicity verified in the envenomation involving this snake. B. jararacussu venom has two main PLA2: bothropstoxin-I and II (BthTX-I and BthTX-II, respectively). BthTX-I is a PLA2 Lys49 with a molecular mass of 13.7kDa that has a myotoxic and pro-inflammatory effect on site of the bite. In this work, we aimed to obtain the BthTX-I from the whole venom and to study the pro-inflammatory potential using in vivo and in vitro experimental models. For this, in in vivo model we evaluated the myotoxic action of BthTX-I in mice selected for high and low acute inflammatory response - AIRmax and AIRmin, respectively, and evaluated the ability of the toxin to induce the IL-1β and IL -6 production by peritoneal macrophages from these mice. Furthermore, we evaluated the myotoxic action of BthTX-I on differentiated C2C12 myotubes in vitro. The BthTX-I induced myotoxicity in AIRmax and AIRmin mice and the production of IL-1β and IL -6 by macrophages primed with LPS. In cultures of C2C12 myotubes, we observed morphological alterations of the fibers induced by the toxin.

Acidentes ofídicos estão caracterizados como uma doença negligenciada mundialmente. Os envenenamentos por serpentes do gênero Bothrops têm sido majoritariamente estudados, devido à sua prevalência e incidência de acidentes relatados. Dentro os sinais clínicos, podemos destacar presença de hemorragia, edema, comprometimento tecidual, mionecrose; além de efeitos sistêmicos, como insuficiência renal e incoagulabilidade. Os efeitos inflamatórios locais são evidentes e estão relacionados sobretudo ao dano tecidual causado por toxinas presentes no veneno total. A miotoxicidade pode ser estudada em modelo experimental in vitro com cultivo de linhagem de mioblasto C2C12, utilizada como modelo para estudos do processo de proliferação e diferenciação miogênica. O veneno da maioria das serpentes do gênero Bothrops apresenta prevalência de metaloproteases dentre outros componentes, porém, no caso de Bothrops jararacussu, as fosfolipases do tipo A2 (PLA2) estão em maior quantidade em relação aos demais. Esse achado pode estar relacionado sobretudo à alta miotoxicidade observada no envenenamento por essa serpente. O veneno de B. jararacussu apresenta duas fosfolipases A2 (PLA2 ): bothropstoxina I e II (BthTX-I e BthTX-II, respectivamente). A BthTX-I é uma PLA2 Lys49 com massa molecular de 13,7kDa que apresenta efeito miotóxico e pró-inflamatório local. Neste trabalho buscamos obter a BthTX-I para estudar o potencial pró-inflamatório da toxina utilizando modelos experimentais in vivo e in vitro. Para tanto, em modelo in vivo avaliamos a ação miotóxica da BthTX-I em camundongos selecionados para alta e baixa resposta inflamatória aguda - camundongos AIRmax e AIRmin, respectivamente e ainda, estudamos a capacidade da toxina de induzir a produção de IL-1β e IL-6 por macrófagos peritoneais obtidos dos camundongos. Além disso, avaliamos a ação miotóxica da BthTX-I sobre miotubos C2C12 diferenciados in vitro. A BthTX-I promoveu miotoxicidade em camundongos AIRmax e AIRmin e promoveu a produção de IL-1β e IL-6 em culturas de macrófagos primados com LPS. Nos miotubos C2C12, a BthTX-i foi capaz de induzir alterações morfológicas das culturas in vitro.

Obtenção da Bothropstoxina-I do veneno de Bothrops jararacussu para estudo do processo inflamatório em camundongos AIRMax e AIRMin

Around 70% of the ophidian accidents reported in Brazil (2021) are from Bothrops genus snakebites. Intense inflammatory reaction in the local tissue with, in severe cases of bothropic envenomation, dysfunction of affected organ. Bothropstoxin-I (BthTX-I), isolated from Bothrops jararacussu venom, is a phospholipase A2 (PLA2) involved in inflammatory process and tissue damage. Cooperation between tissue components and immune cells, such as the macrophage, is critical for the repair of the injured site. The inflammatory response is initiated by detecting signs of acute tissue damage due the disturbance in the homeostasis (DAMPs) and/or by recognition of pathogen-associated molecular patterns (PAMPs). Several receptors are involved in the detection of PAMPs and DAMPs, and among them are the inflammasome. Therefore, it has been reported that the inflammasome participates in the induction and resolution of inflammatory response induced by different microbial antigens and/or those. Mice genetically selected for maximum (AIRmax) and minimum (AIRmin) acute inflammatory response to the Biogel, have been useful for the studies of the mechanisms involved in the inflammatory response. Therefore, our focus is to evaluate the inflammatory process induced by BthTX-I in AIRmax and AIRmin mice. Therefore, the BthTX-I was obtained from the B.jararacussu venom in quantity and purity for the next experiments. Furthermore, the BthTX-I was able to induce myotoxicity in AIRMax and AIRMin mice, which will allow the development of next experiments and studies to contribute for the understanding the mechanisms involved in the inflammatory process.

Cerca de 70 % dos acidentes ofídicos relatados no Brasil, em 2021, são resultantes de picadas de serpentes do gênero Bothrops. Intensa reação inflamatória é observada na lesão tecidual com relatos de disfunção do órgão afetado em casos graves de envenenamento botrópico. Bothropstoxina-I (BthTX-I), isolada do veneno de Bothrops jararacussu, é uma fosfolipase A2 (FLA2) envolvida no processo inflamatório e lesão local. A cooperação entre componentes teciduais e células imunes, como o macrófago, é fundamental para o reparo do local lesionado. A resposta inflamatória é iniciada por meio da detecção de sinais resultantes do dano tecidual devido à quebra da homeostasia (DAMPs) e/ou por reconhecimento de padrões moleculares associados aos patógenos (PAMPs). Diversos receptores estão envolvidos na detecção de PAMPs e DAMPs e, dentre eles estão os inflamassomas. Portanto, têm sido demonstrada a participação do inflamassoma na indução e regulação da resposta inflamatória induzida por antígenos microbianos e/sinais de danos teciduais. Camundongos geneticamente selecionados para máxima (AIRmax) e mínima (AIRmin) resposta inflamatória aguda ao Biogel, têm sido utilizados como modelo experimental para o estudo dos mecanismos envolvidos na resposta inflamatória. Isto posto, temos como foco avaliar a ação da BthTX-I sobre o processo inflamatório em camundongos AIRmax e AIRmin. Portanto, a BthTX-I foi obtida a partir do veneno de B. jararacussu em quantidade e pureza para a realização dos experimentos. Além disso, a BthTX-I purificada foi capaz de induzir atividade miotóxica em camundongos AIRMax e AIRMin, o que permitirá o desenvolvimento de novos estudos e experimentos sobre os fatores/mecanismos que medeiam a resposta inflamatória.

Inflammatory effect of Bothropstoxin-I from the Bothrops jararacussu venom mediated by NLRP3 inflammasome involves ATP and P2X7 receptor

Muscle tissue damage is one of the local effects described in bothropic envenomations. Bothropstoxin-I (BthTX-I), from B. jararacussu venom, is a K49-phospholipase A2 that induces a massive muscle tissue injury, and, consequently, local inflammatory reaction. The NLRP3 inflammasome is a sensor that triggers inflammation by activating caspase 1 and releasing IL-1b and/or inducing pyroptotic cell death in response to tissue damage. We, therefore, aimed to address activation of NLRP3 inflammasome by BthTX-I-associated injury and the mechanism involved in this process. Intramuscular injection of BthTX-I results in infiltration of neutrophils and macrophages in gastrocnemius muscle, which is reduced in NLRP3- and Caspase-1-deficient mice. The in vitro IL-1β production induced by BthTX-I- inperitoneal macrophages requires caspase 1/11, ASC and NLRP3 and is dependent of ATP-induced K+ efflux and P2X7R. BthTX-I induces a dramatic release of ATP from C2C12 myotubes, therefore representing the major mechanism for P2X7R-dependent inflammasome activation in macrophages. A similar result was obtained when human monocyte-derived macrophages were treated with BthTX-I. These findings demonstrated the inflammatory effect of BthTX-I on muscle tissue, pointing out a role for the ATP released by damaged cells for the NLRP3 activation on macrophages, contributing to the understanding of the microenvironment of the tissue damage of the Bothrops envenomation.

Differential Macrophage Subsets in Muscle Damage Induced by a K49-PLA2 from Bothrops jararacussu Venom Modulate the Time Course of the Regeneration Process.

Bothrops snakes cause around 80% of snakebites in Brazil, with muscle tissue damage as an important consequence, which may cause dysfunction on the affected limb. Bothropstoxin-I (BthTX-I) from Bothrops jararacussu is a K49-phospholipase A2, involved in the injury and envenomation's inflammatory response. Immune system components act in the resolution of tissue damage and regeneration. Thus, macrophages exert a crucial role in the elimination of dead tissue and muscle repair. Here, we studied the cellular influx and presence of classical and alternative macrophages (M1 and M2) during muscle injury induced by BthTX-I and the regeneration process. BthTX-I elicited intense inflammatory response characterized by neutrophil migration, then increased influx of M1 macrophages followed by M2 population that declined, resulting in tissue regeneration. The high expressions of TNF-α and IL6 were changed by increased TGF-ß expression after BthTX-I injection, coinciding with the iNOs and arginase expression and the peaks of M1 and M2 macrophages in muscle tissue. A coordinated sequence of PAX7, MyoD, and myogenin expression involved in muscle regenerative process appeared after BthTX-I injection. Together, these results demonstrate a direct correlation between the macrophage subsets, cytokine microenvironment, and the myogenesis process. This information may be useful for new envenomation and muscular dysfunction therapies.

Differential macrophage subsets in muscle damage induced by a K49-PLA2 from Bothrops jararacussu venom modulate the time course of the regeneration process

Bothrops snakes cause around 80% of snakebites in Brazil, with muscle tissue damage as an important consequence, which may cause dysfunction on the affected limb. Bothropstoxin-I (BthTX-I) from Bothrops jararacussu is a K49-phospholipase A2, involved in the injury and envenomation’s inflammatory response. Immune system components act in the resolution of tissue damage and regeneration. Thus, macrophages exert a crucial role in the elimination of dead tissue and muscle repair. Here, we studied the cellular influx and presence of classical and alternative macrophages (M1 and M2) during muscle injury induced by BthTX-I and the regeneration process. BthTX-I elicited intense inflammatory response characterized by neutrophil migration, then increased influx of M1 macrophages followed by M2 population that declined, resulting in tissue regeneration. The high expressions of TNF-a and IL6 were changed by increased TGF-ß expression after BthTX-I injection, coinciding with the iNOs and arginase expression and the peaks of M1 and M2 macrophages in muscle tissue. A coordinated sequence of PAX7, MyoD, and myogenin expression involved in muscle regenerative process appeared after BthTX-I injection. Together, these results demonstrate a direct correlation between the macrophage subsets, cytokine microenvironment, and the myogenesis process. This information may be useful for new envenomation and muscular dysfunction therapies

Pharmacological Properties of Vochysia Haenkeana (Vochysiaceae) Extract to Neutralize the Neuromuscular Blockade Induced by Bothropstoxin-I (Lys49 Phospholipase A2) Myotoxin.

Purpose: Bothrops snakes are responsible for more than 70 % of snakebites every year in Brazil and their venoms cause severe local and systemic damages. The pharmacological properties of medicinal plants have been widely investigated in order to discover new alternative treatments for different classes of diseases including neglected tropical diseases as envenomation by snakebites. In this work, we have investigated the ability of Vochysia haenkeana stem barks extract (VhE) to neutralize the neuromuscular effects caused by Bothropstoxin-I (BthTX-I), the major phospholipase A2 (PLA2) myotoxin from B. jararacussu venom. Methods: The biological compounds of VhE were analysed under thin layer chromatography (TLC) and its neutralizing ability against BthTX-I was assessed through twitch-tension recordings and histological analysis in mouse phrenic nerve-diaphragm (PND) preparations. The antimicrobial activity of VhE was assessed against S. aureus, E. coli and P. aeruginosa strains. The aggregation activity of VhE was analysed under protein precipitation assay. Results: VhE showed the presence of phenolic compound visualized by blue trace under TLC. VhE abolished the neuromuscular blockade caused by BthTX-I applying the pre-toxin incubation treatment and partially neutralized the BthTX-I action under post-toxin incubation treatment; VhE contributed slightly to decrease the myotoxicity induced by BthTX-I. The neutralizing mechanism of VhE may be related to protein aggregation. VhE showed no antimicrobial activity. Conclusion: V. haenkeana extract which has no antimicrobial activity exhibited neutralizing ability against the neuromuscular blockade caused by BthTX-I and also contributed to decrease its myotoxicity. Protein aggregation involving phenolic compounds may be related in these protective effects.

Can anti-bothropstoxin-I antibodies discriminate between Bothrops jararaca and Bothrops jararacussu venoms?

BACKGROUND: Snakes of the genus Bothrops, popularly known as pit vipers, are responsible for most cases of snakebite in Brazil. Within this genus, Bothrops jararacussu and B. jararaca deserve special attention due to the severity of their bites and for inhabiting densely populated areas. Regarding the treatment of snakebites by Bothrops jararacussu, questions have been raised about the effectiveness of the specific bothropic antivenom in neutralizing myotoxic effects; however, there are no accurate data for humans. Thus, the development of a differential diagnostic kit for this species would be of great interest because it provides, for healthcare professionals, a tool that would allow us to determine whether the accident was caused by B. jararacussu or other species of the genus. It would also make it possible to evaluate the specificity of the treatment and to provide data for epidemiological studies. METHODS: First, we produced a species-specific polyclonal antibody - a potential biomarker of Bothrops jararacussu venom - against bothropstoxin-I (BthTx-I), which is also found in smaller quantities in the venoms of B. jararaca from southern Brazil. RESULTS: Polyclonal antibodies against bothropstoxin-I could be separated into several species-specific immunoglobulins. Then, aiming to develop a system of safe and standardized immunoassay, we produced monoclonal antibodies. Seven hybridomas were obtained. Five of them were specific to the venom of B. jararacussu and two recognized the venom of B. jararaca from the southeastern population. The use of monoclonal antibodies also made it possible to differentiate B. jararacussu from B. jararaca venom obtained from the southern population. Analyzing the reactivity of monoclonal antibodies against other bothropic venoms, we found mAb Bt-3 to be more specific than others for B. jararacussu venom. CONCLUSIONS: These results show the potential of BthTx-I for producing monoclonal antibodies that differentiate between B. jararacussu and other Bothrops species venoms.

Can anti-bothropstoxin-I antibodies discriminate between Bothrops jararaca and Bothrops jararacussu venoms?

Background Snakes of the genus Bothrops, popularly known as pit vipers, are responsible for most cases of snakebite in Brazil. Within this genus, Bothrops jararacussu and B. jararaca deserve special attention due to the severity of their bites and for inhabiting densely populated areas. Regarding the treatment of snakebites by Bothrops jararacussu, questions have been raised about the effectiveness of the specific bothropic antivenom in neutralizing myotoxic effects; however, there are no accurate data for humans. Thus, the development of a differential diagnostic kit for this species would be of great interest because it provides, for healthcare professionals, a tool that would allow us to determine whether the accident was caused by B. jararacussu or other species of the genus. It would also make it possible to evaluate the specificity of the treatment and to provide data for epidemiological studies. Methods First, we produced a species-specific polyclonal antibody - a potential biomarker of Bothrops jararacussu venom - against bothropstoxin-I (BthTx-I), which is also found in smaller quantities in the venoms of B. jararaca from southern Brazil. Results Polyclonal antibodies against bothropstoxin-I could be separated into several species-specific immunoglobulins. Then, aiming to develop a system of safe and standardized immunoassay, we produced monoclonal antibodies. Seven hybridomas were obtained. Five of them were specific to the venom of B. jararacussu and two recognized the venom of B. jararaca from the southeastern population. The use of monoclonal antibodies also made it possible to differentiate B. jararacussu from B. jararaca venom obtained from the southern population. Analyzing the reactivity of monoclonal antibodies against other bothropic venoms, we found mAb Bt-3 to be more specific than others for B. jararacussu venom. Conclusions These results show the potential of BthTx-I for producing monoclonal antibodies that differentiate between B. jararacussu and other Bothrops species venoms.(AU)

Can anti-bothropstoxin-I antibodies discriminate between Bothrops jararaca and Bothrops jararacussu venoms?

Abstract Background Snakes of the genus Bothrops, popularly known as pit vipers, are responsible for most cases of snakebite in Brazil. Within this genus, Bothrops jararacussu and B. jararaca deserve special attention due to the severity of their bites and for inhabiting densely populated areas. Regarding the treatment of snakebites by Bothrops jararacussu, questions have been raised about the effectiveness of the specific bothropic antivenom in neutralizing myotoxic effects; however, there are no accurate data for humans. Thus, the development of a differential diagnostic kit for this species would be of great interest because it provides, for healthcare professionals, a tool that would allow us to determine whether the accident was caused by B. jararacussu or other species of the genus. It would also make it possible to evaluate the specificity of the treatment and to provide data for epidemiological studies. Methods First, we produced a species-specific polyclonal antibody a potential biomarker of Bothrops jararacussu venom against bothropstoxin-I (BthTx-I), which is also found in smaller quantities in the venoms of B. jararaca from southern Brazil. Results Polyclonal antibodies against bothropstoxin-I could be separated into several species-specific immunoglobulins. Then, aiming to develop a system of safe and standardized immunoassay, we produced monoclonal antibodies. Seven hybridomas were obtained. Five of them were specific to the venom of B. jararacussu and two recognized the venom of B. jararaca from the southeastern population. The use of monoclonal antibodies also made it possible to differentiate B. jararacussu from B. jararaca venom obtained from the southern population. Analyzing the reactivity of monoclonal antibodies against other bothropic venoms, we found mAb Bt-3 to be more specific than others for B. jararacussu venom. Conclusions These results show the potential of BthTx-I for producing monoclonal antibodies that differentiate between B. jararacussu and other Bothrops species venoms.