Botryosphaeran, [(1 → 3)(1 → 6)-Β-d-glucan], Induces Apoptosis-Like Death in Promastigotes of Leishmania Amazonensis, and Exerts a Leishmanicidal Effect on Infected Macrophages by Activating NF-kB and Producing Pro-Inflammatory Molecules.

Leishmaniasis is an infectious-parasitic disease caused by the protozoan Leishmania spp. The available treatments are based upon expensive drugs bearing adverse side-effects. The search for new therapeutic alternatives that present a more effective action without causing adverse effects to the patient is therefore important. The objective of this study was to evaluate the in vitro effect of botryosphaeran, a (1 -> 3)(1 -> 6)-beta-D-glucan, on the promastigote and intracellular amastigote forms of Leishmania amazonensis. The direct activity of botryosphaeran on promastigote forms was evaluated in vitro and inhibited proliferation, the IC50 7 mu g/mL in 48 h was calculated. After 48 h treatment, botryosphaeran induced nitric oxide production (NO), caused mitochondrial membrane hyperpolarization, increased reactive oxygen species (ROS), and accumulation of lipid vesicles in promastigotes, resulting in apoptosis, necrosis and autophagy, and was accompanied by morphological and ultrastructural changes. The range of concentrations used did not alter the viability of peritoneal macrophages from BALB/c mice and erythrocytes of sheep. Botryosphaeran was able to reduce the number of infected macrophages and the number of amastigotes per macrophage at 12.5 mu g/mL (50.75% +/- 6.48), 25 mu g/mL (55.66% +/- 3.93) and 50 mu g/mL (72.9% +/- 6.98), and IC50 9.3 mu g/mL (+/- 0.66) for intracellular amastigotes forms. The leishmanicidal effect was due to activation of NF-kappa B and promoted an increase in pro-inflammatory cytokines (TNF-alpha and IL-6), iNOS and microbial-derived ROS and NO, in addition to decreasing the levels of SOD. Based upon the data obtained, we infer that botryosphaeran exerted an active leishmanicidal and immunomodulatory effect, acting on promastigotes through autophagic, apoptotic and necrosis processes, and in the intracellular amastigote form, through the action of ROS and NO.