Background The quality and function of aging skeletal muscles gradually decline, and the exosome secreted by it have a significant impact on bone metabolism. However, the regulatory mechanisms of aging skeletal muscle exosomes on bone metabolism have not yet been clarified.
Objective To explore the effects of aging skeletal muscle-derived exosomes in regulating osteoblasts and osteoclasts.
Methods Skeletal muscle tissues of 2 months (Young) and 24 months (Old) mice were digested into single cell suspension, and exosomes were extracted from the suspension, which were divided into young skeletal muscle tissue exosomes (Young-Exo) and aging skeletal muscle tissue exosomes (Old-Exo). Exosomes were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and Western blot (WB). The extracted tissue exosomes were used to induce the osteogenic differentiation of primary osteoblasts and the differentiation of bone marrow mononuclear macrophages into osteoclasts. Tissue exosome intervention experiments were divided into control group (PBS group), Young exosome group (Young-Exo group) and Old exosome group (Old-Exo group). Alizarin red S (ARS) and alkaline phosphatase (ALP) staining were used to determine the osteogenic differentiation level. The area of osteoclasts was detected by tartrate-resistant acid phosphase (TRAP) staining. Real-time Quantitative PCR (RT-qPCR) was used to detect the mRNA relative expression levels of Alp, Opn, Ocn, Col1a1, Runx2, Ctsk, Dc-stamp, Atp6v0d2, Mmp-9 and Acp5.
Results The exosomes from the Young-Exo group and Old-Exo group were both circular vesicles with a double-layered membrane structure, approximately 110 nm in diameter, and both expressed CD9, CD63, CD81 and TSG101 exosome related marker proteins. After two groups of exosomes intervented in primary osteoblasts, the staining results showed that the amount of calcium deposition and the staining intensity of alkaline phosphatase in the Old-Exo group were reduced compared with the Young-Exo group (P<0.01), besides, the relative mRNA expression levels of osteogenic differentiation related genes Alp, Opn, Ocn, Col1a1 and Runx2 were also decreased (P<0.01). After two groups of exosomes intervented in bone marrow monocyte macrophages, the result of TRAP staining showed that the percentage of osteoclast area in Old-Exo group was increased compared with the Young-Exo group (P<0.001), in addition, the relative mRNA expression levels of osteoclast generation and function related genes Ctsk, Dc-stamp, Atp6v0d2, Mmp-9, Acp5 were increased (P<0.01).
Conclusion Compared with young skeletal muscle exosomes, aging skeletal muscle exosomes inhibit the osteogenic differentiation of primary osteoblasts, while promote the formation and function of osteoclasts.
DownLoad: