Chinese Herbal Medicine Guilu Erxian Glue Inhibits Osteoclast Formation and Activity via Mc3t3-Derived Extracellular Vesicles in vitro.

ABSTRACT

Background: Osteoporosis is a systemic bone disease characterized by decreased bone density and quality, destruction of bone microstructure, and increased bone fragility. Extracellular vesicles are lipid bilayer nanoparticles that participate in intercellular communication. Extracellular vesicles are becoming popular in the study of osteoporosis and the bone cell microenvironment. Extracellular vesicles can transmit cell signals and regulate bone homeostasis. Our previous studies revealed that the Chinese herbal medicine Guilu Erxian Glue promotes type I collagen synthesis and osteoprotegerin secretion by osteoblasts in rats, reverses the imbalance of bone homeostasis, and alleviates osteoporosis. Objective: We investigated how osteoblast-derived extracellular vesicles treated with Guilu Erxian Glue affected osteoclasts in vitro. Methods: We quantified osteoclast differentiation of RAW 264.7 using TRAP staining, cell apoptosis using flow cytometry, extracellular vesicle uptake by fluorescence tracing, bone absorption functions by bone resorption lacuna , and transcription of key genes by quantitative real-time PCR. Results: Fluorescently labeled mouse preosteoblastic MC3T3-E1 cells secreted nanoscale substances less than 1 µm in diameter. Mouse macrophage RAW 264.7 cells adsorbed these nanoparticles and PKH26-labeled extracellular vesicles derived from MC3T3-E1 cells on the cell membrane surface. Extracellular vesicles from MC3T3-E1 cells treated with Guilu Erxian Glue inhibited the differentiation of osteoclasts induced by receptor activator of nuclear factor-κB ligand and macrophage colony-stimulating factor and reduced the number of lacunae formed by osteoclasts in vitro compared with controls. Extracellular vesicles from MC3T3-E1 cells treated with Guilu Erxian Glue downregulated the relative messenger RNA expression of c-Fos, cathepsin K, nuclear factor of activated T cells 1, and tartrate-resistant acid phosphatase in osteoclasts, which may be part of the mechanism by which they regulate osteoclasts. Conclusions: Our results demonstrate that extracellular vesicles are essential for signal exchange between osteoblasts and osteoclasts. Although we do not know how Guilu Erxian Glue affects the signaling molecules carried by extracellular vesicles, we have shown for the first time, to our knowledge, that Guilu Erxian Glue can inhibit osteoclast differentiation and function via osteoblast-derived extracellular vesicles. Our findings are conducive to providing a new target for the development of osteoporosis drugs.