Gallic acid anti-myotoxic activity and mechanism of action, a snake venom phospholipase A2 toxin inhibitor, isolated from the medicinal plant Anacardium humile.

Snakebite envenoming is the cause of an ongoing health crisis in several regions of the world, particularly in tropical and neotropical countries. This scenario creates an urgent necessity for new practical solutions to address the limitations of current therapies. The current study investigated the isolation, phytochemical characterization, and myotoxicity inhibition mechanism of gallic acid (GA), a myotoxin inhibitor obtained from Anacardium humile. The identification and isolation of GA was achieved by employing analytical chromatographic separation, which exhibited a compound with retention time and nuclear magnetic resonance spectra compatible with GA's commercial standard and data from the literature. GA alone was able to inhibit the myotoxic activity induced by the crude venom of Bothrops jararacussu and its two main myotoxins, BthTX-I and BthTX-II. Circular dichroism (CD), fluorescence spectroscopy (FS), dynamic light scattering (DLS), and interaction studies by molecular docking suggested that GA forms a complex with BthTX-I and II. Surface plasmon resonance (SPR) kinetics assays showed that GA has a high affinity for BthTX-I with a KD of 9.146 × 10-7 M. Taken together, the two-state reaction mode of GA binding to BthTX-I, and CD, FS and DLS assays, suggest that GA is able to induce oligomerization and secondary structure changes for BthTX-I and -II. GA and other tannins have been shown to be effective inhibitors of snake venoms' toxic effects, and herein we demonstrated GA's ability to bind to and inhibit a snake venom PLA2, thus proposing a new mechanism of PLA2 inhibition, and presenting more evidence of GA's potential as an antivenom compound.

Photobiomodulation induces murine macrophages polarization toward M2 phenotype.

Photobiomodulation using light-emitting diode (LED) treatment has analgesic and anti-inflammatory effects which can be an effective therapeutic associated with serum therapy for local treatment of snakebites. Here we explored the effects of LED treatment on isolated macrophage under Bothrops jararacussu venom. Results showed that LED induced IL-6 and TNF-α genes down-regulation and, TGF and ARG1 genes up-regulation which indicates a polarization of macrophages to an M2 phenotype contributing to both tissue repair and resolution of inflammation.

Effect of light emitting diode photobiomodulation on murine macrophage function after Bothrops envenomation.

Several reports have suggested that photobiomodulation, owing to its analgesic, anti-inflammatory, and healing effects, may be an effective therapeutic option for local effects of snakebites when the availability and accessibility of conventional serum therapy are inefficient and far from medical care centers. Although there have been studies that demonstrate the application of photobiomodulation in the treatment of local adverse events due to snakebites from snakes of the genus Bothrops, its role in the activation of leukocytes, particularly macrophages, has not been evaluated. Here, we assessed the effect of light-emitting diode (LED) treatment on macrophage activation induced by B. jararacussu venom (BjV). LED treatment caused an increase in the viability of macrophages incubated with BjV. This treatment reduced reactive oxygen species (ROS) and nitric oxide (NO) production by macrophages after incubation with BjV. However, LED treatment did not interfere with IL-1ß and IL-10 production by macrophages after incubation with BjV. In conclusion, this study showed that LED treatment has the potential to be used in combination with conventional serum therapy to prevent or minimize the progression of local to severe symptoms after Bothrops envenomation.

Isolation and structural characterization of bioactive compound from Aristolochia sprucei aqueous extract with anti-myotoxic activity.

A bioactive compound isolated from the stem extract of Aristolochia sprucei through High Performance Liquid Chromatography (HPLC) was identified via Nuclear Magnetic Resonance (NMR) as the aristolochic acid (AA). This compound showed an inhibitory effect over the myotoxic activity of Bothrops jararacussu and Bothrops asper venoms, being also effective against the indirect hemolytic activity of B. asper venom. Besides, AA also inhibited the myotoxic activity of BthTX-I and MTX-II with an efficiency greater than 60% against both myotoxins. Docking predictions revealed an interesting mechanism, through which the AA displays an interaction profile consistent with its inhibiting abilities, binding to both active and putative sites of svPLA2. Overall, the present findings indicate that AA may bind to critical regions of myotoxic Asp 49 and Lys49-PLA2s from snake venoms, highlighting the relevance of domains comprising the active and putative sites to inhibit these toxins.

Camelid Single-Domain Antibodies (VHHs) against Crotoxin: A Basis for Developing Modular Building Blocks for the Enhancement of Treatment or Diagnosis of Crotalic Envenoming.

Toxic effects triggered by crotalic envenoming are mainly related to crotoxin (CTX), composed of a phospholipase A2 (CB) and a subunit with no toxic activity (CA). Camelids produce immunoglobulins G devoid of light chains, in which the antigen recognition domain is called VHH. Given their unique characteristics, VHHs were selected using Phage Display against CTX from Crotalus durissus terrificus. After three rounds of biopanning, four sequence profiles for CB (KF498602, KF498603, KF498604, and KF498605) and one for CA (KF498606) were revealed. All clones presented the VHH hallmark in FR2 and a long CDR3, with the exception of KF498606. After expressing pET22b-VHHs in E. coli, approximately 2 to 6 mg of protein per liter of culture were obtained. When tested for cross-reactivity, VHHs presented specificity for the Crotalus genus and were capable of recognizing CB through Western blot. KF498602 and KF498604 showed thermostability, and displayed affinity constants for CTX in the micro or nanomolar range. They inhibited in vitro CTX PLA2 activity, and CB cytotoxicity. Furthermore, KF498604 inhibited the CTX-induced myotoxicity in mice by 78.8%. Molecular docking revealed that KF498604 interacts with the CA–CB interface of CTX, seeming to block substrate access. Selected VHHs may be alternatives for the crotalic envenoming treatment.

The effect of 3ß, 6ß, 16ß-trihydroxylup-20(29)-ene lupane compound isolated from Combretum leprosum Mart. on peripheral blood mononuclear cells.

BACKGROUND: The Combretum leprosum Mart. plant, popularly known as mofumbo, is used in folk medicine for inflammation, pain and treatment of wounds. From this species, it is possible to isolate three triterpenes: (3ß, 6ß, 16ß-trihydroxylup-20(29)-ene) called lupane, arjunolic acid and molic acid. In this study, through preclinical tests, the effect of lupane was evaluated on the cytotoxicity and on the ability to activate cellular function by the production of TNF-α, an inflammatory cytokine, and IL-10, an immuno regulatory cytokine was assessed. The effect of lupane on the enzymes topoisomerase I and II was also evaluated. METHODS: For this reason, peripheral blood mononuclear cells (PBMCs) were obtained and cytotoxicity was assessed by the MTT method at three different times (1, 15 and 24 h), and different concentrations of lupane (0.3, 0.7, 1.5, 6, 3 and 12 µg/mL). The cell function was assessed by the production of TNF-α and IL-10 by PBMCs quantified by specific enzyme immunoassay (ELISA). The activity of topoisomerases was assayed by in vitro biological assays and in silico molecular docking. RESULTS: The results obtained showed that lupane at concentrations below 1.5 µg/mL was not toxic to the cells. Moreover, lupane was not able to activate cellular functions and did not alter the production of IL-10 and TNF-α. Furthermore, the data showed that lupane has neither interfered in the action of topoisomerase I nor in the action of topoisomerase II. CONCLUSION: Based on preclinical results obtained in this study, we highlight that the compound studied (lupane) has moderate cytotoxicity, does not induce the production of TNF-α and IL-10, and does not act on human topoisomerases. Based on the results of this study and taking into consideration the reports about the anti-inflammatory and leishmanicidal activity of 3ß, 6ß, 16ß-trihydroxylup-20(29)-ene, we suggest that this compound may serve as a biotechnological tool for the treatment of leishmaniasis in the future.

Biodiversity as a source of bioactive compounds against snakebites.

Snakebites are a frequently neglected public health issue in tropical and subtropical countries. According to the World Health Organization, 5 million people are bitten annually including up to 2.5 million envenomations. Treatment with antivenom serum remains the only specific therapy for snakebite envenomation. However, it is heterologous and therefore liable to cause adverse reactions, such as early anaphylactic, pyrogenic and delayed reactions. In order to develop alternatives to the current therapy, researchers have been looking for natural products and plant extracts with antimyotoxic, anti-hemorrhagic and anti-inflammatory properties. Especially due to the role the physiopathological processes triggered by snake toxins, play in paralysis, bleeding disorders, kidney failure and tissue damage. Considering the fact that studies involving snake toxins and specific inhibitors, particularly on a molecular level, are the main key to understand neutralization mechanisms and to propose models or prototypes for an alternative therapy, this article presents efforts made by the scientific community in order to produce validated data regarding 87 compounds and plant extracts obtained from 79 species. These plants, which belong to 63 genera and 40 families, have been used by traditional medicine as alternatives or complements to the current serum therapy.

Activation of alpha(M)beta(2)-mediated phagocytosis by HF3, a P-III class metalloproteinase isolated from the venom of Bothrops jararaca.

The integrin alpha(M)beta(2) regulates important cell functions in inflammation being the primary phagocytic receptor on macrophages. HF3, a metalloproteinase isolated from Bothrops jararaca venom, is a potent hemorrhagic toxin. A cDNA encoding HF3 indicated that it is a multidomain molecule composed of a pro-domain, a catalytic domain with a zinc binding sequence, followed by disintegrin-like and cysteine-rich domains. It is known that metalloproteinases play a relevant role in the pathogenesis of venom-induced local tissue damage including inflammation. In this study we evaluated the effects of native HF3 and its recombinant disintegrin-like/cysteine-rich domains (DC-HF3) on alpha(M)beta(2)-mediated phagocytosis of opsonized-zymosan particles by macrophages. HF3 and DC-HF3 significantly increased phagocytosis and this activity was inhibited by anti-alpha(M) and anti-beta(2) antibodies. The data show the ability of P-III metalloproteinases to activate macrophages for phagocytosis through integrin alpha(M)beta(2) and suggest that the disintegrin-like/cysteine-rich domains are important for this effect. This is the first report on the activation of phagocytosis via alpha(M)beta(2) integrin by a metalloproteinase containing disintegrin-like/cysteine-rich domains.