Estudo da influência da secreção cutânea do anfíbio Rhinella diptycha (Rhinella schneideri) sobre o processo de formação in vitro de osteoclastos

Abstract

Osteoclasts (OCs) are multinucleated cells, specialized in reabsorbing calcified bone matrix. OCs are derived from hematopoietic precursor cells and can be generated in vitro by stimulation of peripheral blood mononuclear cells (PBMC) with the cytokines M-CSF and RANKL. Abnormal activation of OCs occurs in pathologies such as osteoporosis, rheumatoid arthritis, osteoarthritis, periodontal disease, Paget's disease, metastatic cancers and others, causing morbidity and promoting physical limitations. Chronic inflammation leads to the secretion of many pro-inflammatory cytokines and RANKL among them. These cytokines are primarily responsible for osteoclast activation and subsequent bone destruction. Poisoning, caused by different animal species (bees, frogs, scorpions, snakes, etc.), is also known to induce a local and acute inflammatory process. The effect of the venoms of these animals on the differentiation, maturation and degeneration of OCs is unknown. To improve the understanding of the physiology of OCs, and a possible interference in their formation and function processes, we used a model of differentiation from human PBMCs. PBMCs were induced for osteogenic differentiation using the routine protocol. Then we treated the PBMCs with the cutaneous secretion of the amphibian Rhinella dypticha (Rhinella schneideri) to evaluate its effect on the OC differentiation process, which can be divided into three stages, pre-osteclasts, OCs and mature multinucleated OCs. When we used amphibian skin secretion at concentrations of 25 ng/mL and 2.5 ng/mL, we observed a statistically significant decrease in the viability of these cells. Additionally, we showed that the lower concentrations of 0.25 ng/mL and 0.125 ng/mL, although not toxic, induced a decrease in the formation of OCs, which was evidenced by a positive Tartrate-Resistant Acid Phosphatase (TRAP), which is a marker of OCs. mature. With the labeling with Phalloidin conjugated, we saw that the concentration of 0.25 ng/mL affected the formation of multinucleated OCs due to the decrease in the number of

cells with 2 and 3 - 5 nuclei. The results obtained suggest the presence of molecules in the cutaneous secretion of Rhinella diptycha that influence the differentiation of OCs in vitro, decreasing the formation of TRAP-positive OCs – mature and affecting the process of formation of multinucleated OCs with 2 to 5 nuclei.