Senescent RAW264.7 cells exhibit increased production of nitric oxide and release inducible nitric oxide synthase in exosomes

Aging cells not only cease growing, but also secrete various proteins such as inflammatory cytokines. This secretory phenomenon is known as the senescence-associated secretory phenotype (SASP). The aim of the present study was to elucidate the effects of senescence on the differentiation of osteoclast precursors (OCPs) and corresponding SASP. RAW264.7 cells were used as OCPs and were cultured to passage (P)5, P10 and P20. Cell proliferation assays, senescence-associated beta-galactosidase staining and telomere length quantification were subsequently performed, and it was revealed that replicative senescence was induced at P20. In addition, the level of tartrate-resistant acid phosphatase activity in P20 cells treated with receptor activator of nuclear factor-kappa B ligand was significantly lower than that in P5 and P10 cells. The SASP factors interleukin-6, tumour necrosis factor-alpha and nitric oxide were significantly increased in P20 culture supernatants compared with those in P5 and P10 supernatants. Furthermore, the number of exosomes at P20 was increased compared with that at P5 and P10, and inducible nitric oxide synthase (iNOS) was expressed in exosomes at P20, but not in exosomes at P5. In conclusion, the present study revealed that senescent RAW264.7 cells exhibit increased expression of SASP factors and release iNOS in exosomes.