Reação inflamatória induzida por duas SVMPs isoladas do veneno de Bothrops atrox e por fragmentos peptídicos gerados da hidrólise de componentes da membrana basal por estas enzimas

Abstract

Snake venom metalloproteases (SVMPs) are key toxins in local and systemic effects observed in victims of snakebites. The mechanisms involved in SVMPs action are related to the hydrolysis of blood vessels basement membrane components (BM), well as their direct action on platelets, endothelial and inflammatory cell receptors, and coagulation factors. Some evidence suggests that endogenous factors derived from BM hydrolysis increase the local effects induced by SVMPs and, consequently, the severity of snakebites. In this study we evaluated the role of two SVMPs of class P-I and P-III named Atroxlysin-Ia (ATXL) and Batroxrhagin (BATXH), respectively, isolated from the Bothrops atrox venom, and its hydrolysis products on Matrigel in experimental models of inflammation. Both toxins induced inflammatory reactions in mice models. The edema formed in the mice paws after the injection of different doses of SVMPs did not indicate a statistical difference between the tested doses or between the toxins. The lesions were similar despite the ATXL having a lower molecular mass than BATXH. In the kinetics tests of edema and accumulation of peritoneal leukocytes, a dose of 2 µg / animal was used. Both toxins induced the formation of an edema peak between 30 min and 1 h, with a 70% increase in the size of the paws. ATXL and BATXH induced an increase in leukocyte influx 4 h after intraperitoneal injection. For the evaluation of possible inflammatory mediators involved in these reactions, we stimulated Murine Peritoneal Adherent Cells (MPACs) with 40 µg / mL of the SVMPs or B. atrox venom in different time´s period. The cytokine TNF-α was identified in the supernatant of cells stimulated with BATXH or with B. atrox. The venom peaked between 2 h and 6 h with levels of 800 pg/mL, and BATXH induces secretion of 200 pg/mL in 4 h. The same was not observed with ATXL. To test the hypothesis that peptides resulting from MB hydrolysis by SVMPs could participate in the inflammatory process, Matrigel was incubated with the toxins for 1 h at 37 ° C, and the hydrolysis products were identified by LC-MS/MS. In parallel, peptides resulting from this incubation were isolated on centrifugal filters with a 10 kDa cut off. The reaction generated different fragments, mainly Laminin peptides. Isolated peptides in the filters induced the formation of edema with a 30% increase in the size of the paws and promoted leukocyte accumulation in the peritoneal cavity (4-5 x 106 cells/mL). Thus, the results suggest that ATXL and BATXH, together with peptides resulting from Matrigel´s hydrolysis, play an important role in inflammatory reaction observed in envenoming by B. atrox. Generally, envenoming causes irreversible local injuries, even after the administration of antivenom. In this context, we present tests with two inhibitors derived from the Jararhagin pro-domain that were able to neutralize the catalytic, fibrinolytic and hemorrhagic activity induced by SVMP, indicating a possible complementary treatment with antibotropic serum.