Marcelo A. Strauch

Ability of a synthetic coumestan to antagonize Bothrops snake venom activities.

We investigated a synthetic coumestan named LQB93 and similar compounds abilities to antagonize activities of Bothrops jararacussu and Bothrops jararaca crude venoms in different protocols. The antimyotoxic activity was evaluated in vitro by the rate of release of creatine kinase (CK) from isolated mouse extensor digitorum longus muscle (EDL) induced by B. jararacussu (25 g/ml). For in vivo studies, B. jararacussu venom (1.0 mg/kg) was preincubated with LQB93 (0.1-30 mg/kg), during 30 min, for later injection in mouse tight and evaluation of the antimyotoxic and anti-edematogenic effects. LQB93 antagonized in vitro, the increase of CK release from the EDL muscle (IC(50)=0.0291 M). It also showed in vivo, antimyotoxic and anti-edematogenic effects that were dose-dependent with ID50 of 0.17 mg/kg and 0.14 mg/kg, respectively. The hemorrhage induced by B. jararaca (1.0 mg/kg) venom in the mouse skin, was abolished by LQB93 (10.0 mg/kg) preincubated with venom. Like wedelolactone, LQB93 protected rat isolated heart on a Langendorff preparation, from the cardiotoxicity of B. jararacussu venom. LQB93 inhibit the effects of Bothrops venoms like wedelolactone, a natural compound isolated from the plant Eclipta prostrata.

Counteraction of Bothrops snake venoms by Combretum leprosum root extract and arjunolic acid.

ETHNOPHARMACOLOGICAL RELEVANCE Serotherapy against snakebite is often unavailable in some regions over Brazil, where people make use of plants from folk medicine to deal with ophidic accidents. About 10% of Combretum species have some ethnopharmacological use, including treatment of snakebites. MATERIALS AND METHODS We evaluated the ability of the extract of Combretum leprosum and its component arjunolic acid to reduce some in vivo and in vitro effects of Bothrops jararacussu and Bothrops jararaca venoms. The protocols investigated include phospholipase, proteolytic, collagenase, hyaluronidase, procoagulant, hemorrhagic, edematogenic, myotoxic and lethal activities induced by these venoms in Swiss mice. RESULTS Oral pre-treatment with arjunolic acid reduced the Bothrops jararacussu lethality in up to 75%, while preincubation prevented the death of all the animals. Hemoconcentration effect of Bothrops jararacussu venom was confirmed two hours after i.p. injection, while preincubation with arjunolic acid preserved the hematocrit levels. Both Combretum leprosum extract and arjunolic acid abolished the myotoxic action of Bothrops jararacussu venom. Preincubation of Bothrops jararacussu venom with the extract or arjunolic acid prevented the increase of plasma creatine kinase activity in mice. The hemorrhagic activity of Bothrops jararaca crude venom was reduced down to about 90% and completely inhibited by preincubation with 10 mg/kg or 100 mg/kg Combretum leprosum extract, respectively, while the preincubation and the pretreatment with 30 mg/kg of arjunolic acid reduced the venom hemorrhagic activity down to about 12% and 58%, respectively. The preincubation of the venom with both extract and 30 mg/kg arjunolic acid significantly reduced the bleeding amount induced by Bothrops jararacussu venom. The extract of Combretum leprosum decreased the edema formation induced by Bothrops jararacussu venom both in preincubation and pretreatment, but not in posttreatment. Similarly, arjunolic acid preincubated with the venom abolished edema formation, while pre- and posttreatment have been partially effective. Some enzymatic activities of Bothrops jararacussu and Bothrops jararaca venoms, i.e. phospholipase A2, collagenase, proteolytic and hyaluronidase activities, were to some extent inhibited by the extract and arjunolic acid in a concentration-dependent manner. CONCLUSIONS Altogether, our results show that Combretum leprosum extract can inhibit different activities of two important Brazilian snake venoms, giving support for its popular use in folk medicine in the management of venomous snakebites.

Occurrence of sulfated fucose branches in fucosylated chondroitin sulfate are essential for the polysaccharide effect preventing muscle damage induced by toxins and crude venom from Bothrops jararacussu snake

Snake envenoming is an important public health problem around the world, particularly in tropics. Beyond deaths, morbidity induced by snake venoms, such as myotoxicity, is of pivotal consequence to population. Bothrops jararacussu is the main venomous snake in southeast region of Brazil, and particularly presents strong myotoxic effect. The only available therapy, antibothropic antivenom, poorly affects venom-induced myotoxicity. The aim of this study is to assess the ability of fucosylated chondroitin sulfate (fucCS), a glycosaminoglycan with anticoagulant and antithrombotic properties, and its derivatives to inhibit toxic activities of B. jararacussu crude venom and its isolated toxins, named bothropstoxins (BthTX-I and BthTX-II). The in vitro myotoxic activities induced by crude venom, by BthTX-I alone and by toxins together were abolished by fucCS. Carboxyl reduction (fucCS-CR) kept this ability whereas defucosilation (defucCS) abrogates myoprotection. We observed the same pattern in the response of these polysaccharides in antagonizing the increase in plasma creatine kinase (CK) levels, the reduction of skeletal muscle CK content and the rise of myeloperoxidase (MPO) activity induced by crude venom and isolated toxins. FucCS inhibited edematogenic activity and partially prevented the reduction of total leukocytes in blood when pre-incubated with crude venom. Furthermore, the venom procoagulant effect was completely antagonized by increasing concentrations of fucCS, although this polyanion could stop neither the tail bleeding nor the skin hemorrhage induced by Bothrops jararaca venom. The B. jararacussu phospholipase, hyaluronidase, proteolytic and collagenase activities were inhibited in vitro. The results suggest that fucCS could be able to interact with both toxins, and it is able to inhibit BthTX-II phospholipase activity. Light microscopy of extensor digitorum longus muscle (EDL) muscle showed myoprotection by fucCS, once necrotic areas, edema and inflammatory cells were all decreased as compared to venom injection alone. Altogether, data show that fucCS was able to inhibit myotoxicity and inflammation induced by B. jararacussu venom and its phospholipase toxins, BthTX-I and BthTX-II. Thus, fucosylated chondroitin sulfate is a new polyanion with potential to be used as an adjuvant in the treatment of snakebites in the future.

Pulsed ultrasound therapy accelerates the recovery of skeletal muscle damage induced by Bothrops jararacussu venom

We studied the effect of pulsed ultrasound therapy (UST) and antibothropic polyvalent antivenom (PAV) on the regeneration of mouse extensor digitorum longus muscle following damage by Bothrops jararacussu venom. Animals (Swiss male and female mice weighing 25.0 ± 5.0 g; 5 animals per group) received a perimuscular injection of venom (1 mg/kg) and treatment with UST was started 1 h later (1 min/day, 3 MHz, 0.3 W/cm2, pulsed mode). Three and 28 days after injection, muscles were dissected and processed for light microscopy. The venom caused complete degeneration of muscle fibers. UST alone and combined with PAV (1.0 mL/kg) partially protected these fibers, whereas muscles receiving no treatment showed disorganized fascicules and fibers with reduced diameter. Treatment with UST and PAV decreased the effects of the venom on creatine kinase content and motor activity (approximately 75 and 48%, respectively). Sonication of the venom solution immediately before application decreased the in vivo and ex vivo myotoxic activities (approximately 60 and 50%, respectively). The present data show that UST counteracts some effects of B. jararacussu venom, causing structural and functional improvement of the regenerated muscle after venom injury.

Purinergic P2Y12 Receptor Activation in Eosinophils and the Schistosomal Host Response.

Identifying new target molecules through which eosinophils secrete their stored proteins may reveal new therapeutic approaches for the control of eosinophilic disorders such as host immune responses to parasites. We have recently reported the expression of the purinergic P2Y12 receptor (P2Y12R) in human eosinophils; however, its functional role in this cell type and its involvement in eosinophilic inflammation remain unknown. Here, we investigated functional roles of P2Y12R in isolated human eosinophils and in a murine model of eosinophilic inflammation induced by Schistosoma mansoni (S. mansoni) infection. We found that adenosine 5’-diphosphate (ADP) induced human eosinophils to secrete eosinophil peroxidase (EPO) in a P2Y12R dependent manner. However, ADP did not interfere with human eosinophil apoptosis or chemotaxis in vitro. In vivo, C57Bl/6 mice were infected with cercariae of the Belo Horizonte strain of S. mansoni. Analyses performed 55 days post infection revealed that P2Y12R blockade reduced the granulomatous hepatic area and the eosinophilic infiltrate, collagen deposition and IL-13/IL-4 production in the liver without affecting the parasite oviposition. As found for humans, murine eosinophils also express the P2Y12R. P2Y12R inhibition increased blood eosinophilia, whereas it decreased the bone marrow eosinophil count. Our results suggest that P2Y12R has an important role in eosinophil EPO secretion and in establishing the inflammatory response in the course of a S. mansoni infection.