Efeito do BjKgn (Bothrops jararaca Kininogen) sobre proteases processadoras de citocinas pró-inflamatórias e sobre atividades biológicas de macrófagos

Abstract

The plasma of B.jararaca snake is rich in protease inhibitors. A potent inhibitor of cysteine-protease was isolated from its plasma and named BjKgn. This protein has similar characteristics to the human high molecular weight kininogen (HK), since it has a molecular mass of 110 kDa, releases a pharmacologically active peptide and besides that, It is an inhibitor of cysteine-protease. Both BjKgn and HK exhibit inhibitory activity on Jararhagin (JAR), hemorrhagic metalloprotease isolated from the venom of the same species snakes. JAR is an enzyme capable of inducing the release of tumor necrosis factor-alpha (TNF-α), cytokine-related to dermonecrosis activity of Bothops snake venom. This cytokine is expressed on the cell surface and cleaved by a metalloprotease, called TACE (TNF-α converting enzyme) resulting in the release of soluble TNF-α. Another important cytokine that participates during the poisoning by Bothrops snakes is the Interleukin-1β (IL-1β). It is intracellularly processed by ICE (Interleukin converting enzyme), a cysteine-proteinase which belongs to the family of caspases. The objectives of this study were to evaluate the possible inhibitory effect of BjKgn on ICE, TACE, and the effect on some functions of peritoneal adherent cells, such as spreading, phagocytosis, production of hydrogen peroxide (H2O2), nitric oxide ( NO) and TNF-α and IL-1β. The tests with TACE and ICE were performed using fluorescent substrates corresponding to their respective enzymes and different concentrations of BjKgn. Spreading and phagocytosis tests were conducted using adherent peritoneal cells stimulated or not, and different concentrations of BjKgn (0.5 µg, 1μg, 1.5 µg, 2 µg and 3μg). Tests for production of H2O2, NO and cytokine release were performed only with stimulated cells and 3 µg of BjKgn. Cells were adhered and incubated in the presence or absence of BjKgn for 1 hour in all ex vivo experiments but the ones for the cytokine release, which the cells were remained on contact with the protein for 12 hours. The results showed that BjKgn, on the used doses, was able to competitively inhibit TACE and ICE enzymes with the inhibition constants of 2 mM and 370 mM, respectively. Regarding the activities of peritoneal exudate cells, the protein did not affect any of the activities at doses of BjKgn used, since the cells presented the ability of spreading, phagocytosis, production of H2O2 and NO similar to those displayed by cells of the control group. Despite ICE and TACE inhibition in vitro, BjKgn did not influence on the release of TNF-α and IL-1β. We have concluded that BjKgn protein is an inhibitor of cysteineproteases and metalloproteases since it inhibited TACE and ICE in vitro and that, at least at the doses used, the protein does not affect the metabolic or biological adherent peritoneal cells function and it was not able to inhibit release of TNF- α and IL-1β by these cells.