Estudo in silico, in vitro e in vivo dos possíveis mecanismos de ação anti-inflamatória de derivados n-acilhidrazônicos substituidos

Abstract

The inflammatory process is a reaction of the body to an infection or tissue injury. NOS and COX-2 enzymes are key enzymes in the inflammatory response when stimulated primarily by pro-inflammatory cytokines. Compounds that act to modulate the activity of these enzymes are promising in the fight against inflammation, for example the N-acylhydrazonic derivatives that prove to be effective in classic models of inflammation in vivo and in vitro. This work aimed to evaluate in vitro, in vitro and in vivo, the possible mechanisms of anti-inflammatory action of the derivatives 2-cyanoacetohydrazide of the JR series (04, 09 and 19). Initially, the in silico study of the three compounds and in vitro (from JR04, JR09 and JR19) against cyclooxygenase isoforms (COXs) was performed. Next, gastric toxicity in mice and cytotoxicity in human erythrocytes were evaluated with compounds JR09 and 19. To investigate the participation of the nitric oxide / guanylate cyclase pathway of compound JR19, the carrageenan-induced peritonitis assay was performed in pre-treatment animals with L-NAME (non-specific nitric oxide synthase blocker) or methylene blue (non-specific guanylate cyclase blocker). In the same model the cytokine dosage was performed. The in silico results showed that JR04, JR09 and JR9 interacted specifically with the catalytic site of the COXs, and the compound JR04 had better affinity for COX-1. The in vitro test for the molecules JR04 and JR09 and JR19 corroborated with the in silico assay, where JR04 was shown to be more selective for COX-1 with a selectivity index (IS) of 2.22 for COX-2, whereas the molecules JR09 and 19 showed an IS of 1.67 and IS of 1.51, respectively with a percentage of inhibition of 100% of COX-2 in concentration of 25mM. In the gastric toxicity test the two compounds (100 mg / kg oral and subcutaneous) did not induce ulcerative lesions, even though they were able to interact with COX-1. In the erythrocyte cytotoxicity assay the compounds JR09 and JR19 showed low haemolytic activity, with an IC 50 of 51.42 and 85.02 mg / mL, respectively. In carrageenan-induced peritonitis, the compound JR19 was able to significantly reduce leukocyte counts of the peritoneal exudate by 48.3% compared to the control group, but this effect was reversed in animals pretreated with L-NAME or methylene blue, showing that this effect is dependent on the NO / GCs pathway. The levels of proinflammatory cytokines TNF-α, IFN-γ, IL-6 and IL-17 were also significantly reduced with administration of compound JR19 and reversed in animals pretreated with L-NAME, corroborating the probable participation of anti-inflammatory effect of this compound. The concentrations of IL-2 and IL-4 cytokines were not altered. Thus, the compounds JR04, JR09 and JR19 probably act in interaction with the COXs, compounds JR09 and 19 acting selectively on COX-2, without gastric toxic effects. In addition, compound JR19 has its anti-inflammatory action likely linked to maintenance of NO levels and reducing proinflammatory cytokines.