Abstract
Bothrops moojeni, a Brazilian lanced-head viper, presents a rich, but not well explored, venom composition. This venom is a powerful tool for the discovery of new molecular targets in many different biological processes. Osteoclasts (OC) are extremely important for bone maintenance, calcium physiology, and balance of tissue regeneration being involved in such diseases as osteoporosis and rheumatoid arthritis. The goal of our study was to evaluate the effect of Bothrops moojeni's venom and its fractions of human peripheral blood mononuclear cells derived OCs in vitro differentiation. After the induction of OCs differentiation, on day 4 the venom was added at different concentrations (5, 0.5, and 0.05 µg/mL), and the reduction of tartrate-resistant acid phosphatase positive (TRAP+) osteoclasts, which was more prominent at the concentration of 5 µg / mL were observed. In order to evaluate the effect of major venom’s components (metalloproteases and serinoproteases) on TRAP+ OCs, the EDTA, and aprotinin were used to inhibit the catalytic activity of both proteases. The results suggest that proteases were not crucial for the reduction of TRAP+ OCs. Phalloidin staining was used for morphological analyzes of F-actin rings integrity. Venom provoked F-actin ring disruption in treated versus control OCs. We obtain high molecular weight (HW) and low molecular weight (LW) venom fractions. Both fractions induced the reduction of TRAP+ OCs (HW fraction at a concentration of 5 µg / mL and LW fraction at -1 µg / mL, respectively). We performed a secretome analysis of OCs treated with venom and its fractions using mass spectrometry (LC-MS/IT-Tof). The data obtained demonstrate possible pathways and mechanisms involved in OCs reduction after the treatment. Example giving is catabolic mechanisms for HW and proteins correlated with genetic modifications for LW. New experiments are in progress, aiming to discover the molecules that possibly interfering in the osteoclasts differentiation.
Highlights
Bothrops moojeni, a Brazilian lanced-head viper, presents a rich, but not well explored, venom composition
Osteoclasts (OC) are extremely important for bone maintenance, calcium physiology, and balance of tissue regeneration being involved in such diseases as osteoporosis and rheumatoid arthritis
The effect of B.moojeni venom in OCs differentiation model was evaluated using phenotypic assays based on characteristics of mature OCs, such as number of tartrate-resistant acid phosphatase (TRAP)+ cells, Facting rings integrity and OCs multinuclearity
Summary
A Brazilian lanced-head viper, presents a rich, but not well explored, venom composition. The goal of our study was to evaluate the effect of Bothrops moojeni's venom and its fractions of human peripheral blood mononuclear cells derived OCs in vitro differentiation.After the induction of OCs differentiation, on day 4 the venom was added at different concentrations (5, 0.5, and 0.05 μ g/mL), and the reduction of tartrate-resistant acid phosphatase positive (TRAP+) osteoclasts, which was more prominent at the concentration of 5 μ g/mL were observed. Venom provoked F-actin ring disruption in treated versus control OCs. We obtain high molecular weight (HW) and low molecular weight (LW) venom fractions. The goal of our study was to evaluate the effect of Bothrops moojeni's venom and its fractions of human peripheral blood mononuclear cells derived OCs in vitro differentiation
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