Atividade imunomoduladora do esteroide cardiotônico BD-8 in vitro e in vivo

Abstract

Cardiotonic steroids (CTS) are compounds known to bind to the Na+/K+-ATPase and modulate various biological processes, including the immune response. γ-Benzylidene digoxin 8 (BD-8) is a synthetic CTS with immunomodulatory activity. Its action has been shown to reduce phagocytic activity, nitric oxide (NO) levels, and the pro-inflammatory cytokine IL-1β in murine peritoneal macrophages. Furthermore, BD-8 also reduced iNOS expression and the phosphorylation of NF-κB, ERK, and p38; however, further studies are needed to elucidate its action. Therefore, the objective of this study was to evaluate the immunomodulatory activity of BD-8 in murine peritoneal macrophages in vitro by analyzing Akt, mTOR, and Src proteins and in vivo in models of paw edema and acute lung injury (ALI). For in vitro tests, female Swiss albino mice were previously stimulated with an intraperitoneal (i.p.) injection of 4% thioglycolate. Macrophages from peritoneal lavage were cultured and stimulated with zymosan (0.2 mg/mL) and/or treated with BD-8 (10 μM) for 24 hours, for subsequent analysis of Akt, mTOR, and Src proteins by flow cytometry. For in vivo tests, in the paw edema model, the mice were treated with an i.p. injection of BD-8 (1.12 mg/kg; 0.56 mg/kg, and 0.28 mg/kg) and stimulated with an intraplantar injection of carrageenan (2.5%). Paw thickness was quantified using a digital caliper, and histological analysis of the paw was also performed. In the ALI model, the animals were stimulated with LPS via nasal instillation (5 mg/kg) and treated (i.p.) with BD-8 (0.56 mg/kg and 0.28 mg/kg) at 1, 24, and 48 hours after the LPS challenge. Bronchoalveolar lavage fluid (BALF) was collected and a blood smear was performed for analysis. As a result, it was seen that BD-8 negatively modulated Akt, mTOR, and Src proteins in vitro. Furthermore, this molecule also reduced paw edema at doses of 0.56 mg/kg and 0.28 mg/kg, showing a greater effect at the lowest dose tested; this result was also seen in the histological analysis. In the ALI model, BD-8, at the lowest dose tested, reduced cell migration, and at doses of 0.56 mg/kg and 0.28 mg/kg, it reduced neutrophils in bronchoalveolar lavage fluid (BALF). Additionally, the lowest dose tested also reduced total protein levels and the pro-inflammatory cytokine TNF-α in BALF. Histological analysis of lung tissue showed a reduction in ALI characteristics. Treatment with BD-8 did not modulate the number of blood leukocytes, indicating local action of the compound during the period tested. Therefore, this work contributes to the development of new substances with therapeutic potential, in addition to expanding the understanding of the BD-8 molecule and its ability to modulate biological and inflammatory processes.