The in vitro effects of diallyl trisulfide on osteoclast formation and function in RAW 264.7 murine macrophages

Abstract

Osteoclasts are large multinucleated cells that resorb or degrade bone. Over-active osteoclasts result in fragile bones that fracture easily, causing bone degenerative disorders such as osteoporosis. Inhibiting osteoclast activity represents a viable strategy to combat bone degeneration. When the receptor activator nuclear factor-kappa B ligand (RANKL) interacts with the receptor activator nuclear factor-kappa B (RANK), found on osteoclast precursors, precursors of osteoclasts develop into mature osteoclasts. Reactive oxygen species (ROS), nuclear factor kappa B (NF-kappaB) and mitogen-activated protein kinase (MAPK) pathways are crucial for osteoclast differentiation and activation in a process known as osteoclastogenesis. Diallyl trisulfide (DATS) is an organosulfur compound produced in garlic which has shown powerful anti-cancer effects in vitro including decreased expression of MAPK proteins. However, the effects of DATS on osteoclast differentiation, stimulation, and function are unknown. As a result, this study aimed to investigate how DATS influences osteoclast differentiation and function by utilising RAW 264.7 murine macrophages.

In this study, various methods were carried out to establish the effects of DATS on osteoclast formation and function in RAW 264.7 murine macrophages. Using a resazurin assay, 1-5 µM of DATS was shown not to affect the viability of the RAW 264.7 murine macrophages. RAW 264.7 murine macrophages were then differentiated into osteoclasts using RANKL and stained for tartrate-resistant acid phosphatase (TRAP). It was found that DATS significantly decreased the number of TRAP-positive osteoclasts formed after exposure to a DATS concentration of 2 µM. The effects of DATS on MAPK expression were then investigated through western blotting. It was shown that DATS significantly decreased the expression of crucial MAPKs, c-Jun N-terminal kinase (JNK) at 2 minutes of RANKL exposure, potentially inhibiting c-Jun phosphorylation and impeding nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) expression crucial for osteoclastogenesis. While ERK protein expression remained unchanged, the significant reduction in P38 protein expression at 5 minutes of RANKL exposure could diminish NFATc1 levels, thereby reducing osteoclast formation. The effects of DATS on the expression of oxidative stress markers were investigated in RAW 264.7 murine macrophages through western blotting. It was shown that DATS exhibited a significant increase in the expression of the antioxidative protein, nuclear factor erythroid 2-related factor 2 (NRF2) while significantly decreasing the expression of oxidative stress marker nicotinamide adenine dinucleotide phosphate oxidase-1 (NOX1), suggesting a potential reduction in ROS-mediated MAPK activation and mature osteoclast formation.

This study represents a significant milestone in elucidating the impact of DATS on osteoclastogenesis using RAW 267.4 murine macrophages. The findings underscore the novel role of DATS in influencing osteoclast formation and key proteins essential for osteoclastogenesis. Notably, DATS was shown to be a potent inhibitor of osteoclastogenesis by decreasing the number of TRAP-positive osteoclasts, targeting MAPK signalling and ROS pathways. The comprehensive investigation conducted in this study provides valuable insights into the intricate interplay between DATS and osteoclastogenesis, offering a promising avenue for future research and therapeutic interventions in the realm of bone degenerative diseases.