Bothrops moojeni Venom and Its Components Strongly Affect Osteoclasts' Maturation and Protein Patterns.

Fernanda D’Amélio,Hugo Vigerelli,Isabel de Fátima Correia Batista,Irina Kerkis,Eduardo Osório Frare,Daniel Carvalho Pimenta,Álvaro Rossan de Brandão Prieto-da-Silva

doi:10.3390/toxins13070459

Abstract

Osteoclasts (OCs) are important for bone maintenance, calcium balance, and tissue regeneration regulation and are involved in different inflammatory diseases. Our study aimed to evaluate the effect of Bothrops moojeni’s venom and its low and high molecular mass (HMM and LMM) fractions on human peripheral blood mononuclear cell (PBMC)-derived OCs’ in vitro differentiation. Bothrops moojeni, a Brazilian lanced-head viper, presents a rich but not well-explored, venom composition. This venom is a potent inducer of inflammation, which can be used as a tool to investigate the inflammatory process. Human PBMCs were isolated and induced to OC differentiation following routine protocol. On the fourth day of differentiation, the venom was added at different concentrations (5, 0.5, and 0.05 µg/mL). We observed a significant reduction of TRAP+ (tartrate-resistant acid phosphatase) OCs at the concentration of 5 µg/mL. We evaluated the F-actin-rich OCs structure’s integrity; disruption of its integrity reflects bone adsorption capacity. F-actin rings phalloidin staining demonstrated that venom provoked their disruption in treated OCs. HMM, fraction reduces TRAP+ OCs at a concentration of 5 µg/mL and LMM fraction at 1 µg/mL, respectively. Our results indicate morphological changes that the venom induced cause in OCs. We analyzed the pattern of soluble proteins found in the conditioned cell culture medium OCs treated with venom and its fractions using mass spectrometry (LC-MS/IT-Tof). The proteomic analyses indicate the possible pathways and molecular mechanisms involved in OC reduction after the treatment.

Highlights

Introduction distributed under the terms andBelonging to the Viperidae family, Bothrops moojeni is responsible for the most snakebite accidents, becoming a public health problem in Brazil and a group of larger importance that causes bothropic accidents [1]
RANKL and CSF-1 are necessary for inducing the expression of genes that genetically typify the OCs lineage, such as tartrate-resistant acid phosphatase (TRAP) [6,7,8,9]
We identified 120 proteins for the positive control, 53 proteins for the negative control, 103 proteins for the group treated with B. moojeni crude venom, 22 proteins for the high molecular mass (HMM)

Summary

Introduction

Introduction distributed under the terms andBelonging to the Viperidae family, Bothrops moojeni is responsible for the most snakebite accidents, becoming a public health problem in Brazil and a group of larger importance that causes bothropic accidents [1]. Being the most complex venom of the animal kingdom, snake venom contains actives molecules with potential pharmacological effects [2]. Moojeni venom, in particular, is characterized as possessing activated proteolytic, coagulant, and hemorrhagic factors, composed of metalloproteases (SVMPs—snake venom metalloproteases), serine proteases, phospholipases, and L-amine oxidase acid [3]. The regulation of OCs formation and differentiation is orchestrated by cytokines and factors, such as RANKL (ligand cytokine for nuclear activation of the kB factor); colony stimulating factor one (CSF-1); TNF-α (tumor necrosis factor-alpha); interleukin (IL) 1, IL 6, IL 11, and IL 17; M-CSF (macrophage colony-stimulating factor); and prostaglandin, among others [5]. RANKL and CSF-1 are necessary for inducing the expression of genes that genetically typify the OCs lineage, such as tartrate-resistant acid phosphatase (TRAP) [6,7,8,9]

Methods

Results

Conclusion