Interação entre uma metaloprotease purificada da peçonha de Bothrops moojeni (BmooMP-alfa-I) e o fator de necrose tumoral (TNF): modelos de estudo in vitro e in vivo.

Abstract

Inflammation is an essential process for maintaining homeostasis, involving vascular events, migration and activation of leukocytes. However, its exacerbated manifestations, usually mediated by increased levels of proinflammatory cytokines such as tumor necrosis factor (TNF), may result in chronic diseases such as rheumatoid arthritis, pulmonary fibrosis, and hypersensitivity reactions. The present study aimed to evaluate whether the enzyme BmooMPalpha-I, a metalloprotease isolated from Bothrops moojeni venom, has some role in the inflammatory response in experimental models in vitro and in vivo. Isolation of the BmooMPalpha-I enzyme from the crude venom of the B. moojeni snake was performed using DEAE Sephacel, Sephadex G-75 and Benzamidine-Sepharose chromatography columns. Twodimensional (2D) electrophoresis showed degree of purity, molecular mass (Mr) and isoelectric point (pI) in non-reducing conditions only one protein spot can be evidenced (~ 23.0 kDa; pI 6.82). The levels of TNF determined by the ELISA method were significantly reduced in the presence of the enzyme BmooMP-alpha-I, suggesting that it exerts a proteolytic action on TNF, which was confirmed by the disappearance of this cytokine among the profile of protein bands in SDS- PAGE and Western Blotting. The macrophages assayed with PRR agonists, demonstrated a reduction in levels of TNF secreted by macrophages after their treatment with the enzyme BmooMP-alpha-I. In vivo studies (septic shock test and DSSinduced colitis) have also shown that BmooMP-alpha-I is able to reduce levels of circulating TNF and ameliorate other aspects of the inflammatory bowel disease. In conclusion, BmooMPalpha-I metalloprotease presents an anti-inflammatory profile of immune response, resulting from its direct action on the proinflammatory cytokine TNF.