Estudo da interação da metaloprotease BmooMPα-I, isolada da peçonha de Bothrops moojeni (HOGE, 1965; Viperidae),com a citocina pró-inflamatória TNF-α.

Abstract

Inflammation is an essential process for maintaining homeostasis, involving vascular events, migration and activation of leukocytes. However, its manifestations exacerbated, usually mediated by increased levels of TNF-α, can result in chronic diseases such as rheumatoid arthritis, pulmonary fibrosis and hypersensitivity reactions. The present study aimed to assess whether the enzyme BmooMPα-I, a metalloprotease isolated from the venom of Bothrops moojeni, could play any role in the inflammatory response in experimental models in vitro and in vivo. We carried out the isolation of the enzyme BmooMPα-I from crude venom of the Bothrops moojeni, using DEAE Sephacel column chromatography, Sephadex G-75 and Benzamidine-Sepharose. The 2D electrophoresis showed purity, molecular mass (Mr) and isoelectric point (pI) of a proteic trimer under reducing conditions (~23.6 kDa, ~ 21.2 kDa and ~18.7 kDa, pI in the range of 7.24-7.33) and nonreducing conditions only one spot protein can be demonstrated (~23.0 kDa; pI 6.82). Immunization was performed in New Zealand rabbits to obtain polyclonal IgG anti-BmooMPα-I, enzyme immune assay (iELISA) confirmed the presence of polyclonal IgG anti-BmooMPα-I in serum of immunized rabbits. Their specificity and cross reactivity with antigens (enzyme BmooMPα-I and crude venom of B. moojeni) was confirmed by immunoblot 1D. The polyclonal antibody anti-BmooMPα-I neutralized azoproteolytic activity induced by crude venom of B. moojeni and pure enzyme (BmooMPα-I) in the 27% and 100%, respectively, in 1:15 (w/w) ratios. BALB/c mice in the presence of the metalloproteinase BmooMPα-I exhibited a reduction in leukocyte migration into the peritoneal cavity, suggesting a profile of anti-inflammatory response. The levels of TNF-α determined by ELISA were significantly reduced in the presence of the enzyme BmooMPα-I, suggesting that it exerts a proteolytic role on TNF-α, which was confirmed by the disappearance of the its bands on SDS-PAGE profile. Further, experiments done with activated macrophages PRRs agonists demonstrated a reduction of TNF-α secreted by macrophages after treatment with the enzyme BmooMPα-I. In conclusion, the enzyme BmooMPα-I presents a profile of anti-inflammatory immune response, resulting from direct action on the pro-inflammatory cytokine TNF-α.