Neuropeptide Y stimulates osteoblastic differentiation and VEGF expression of bone marrow mesenchymal stem cells related to canonical Wnt signaling activating in vitro

• NPY exhibited a significant, concentration-dependent effect on osteoblastic differentiation of BMSCs in vitro.
• NPY stimulated BMSCs osteogenic activity which is related to activate canonical Wnt signaling pathway.
• NPY enhanced the expression of BMP-2 and the gene and protein expression in BMSCs differentiation.
• NPY enhanced BMSCs migration ability and VEGF expression.

Neuropeptide Y (NPY) is a neuropeptide secreted by sensory nerve fibers distributed in the marrow and vascular canals of bone tissue. However, the effect of NPY on the osteogenic ability of bone marrow mesenchymal stem cells (BMSCs) remains controversial and has not been thoroughly investigated. To explore the osteogenic activity and the migration and VEGF expression capabilities of BMSCs affected by NPY, as well as the underlying mechanisms, we investigated the potential relationships among NPY, osteoblastic differentiation, angiogenesis and canonical Wnt signaling in BMSCs. NPY was observed to regulate osteoblastic differentiation at concentrations ranging from 10− 8 to 10− 12 mol/L, and the effects of NPY on the levels of Wnt signaling proteins were detected using Western blotting. To unravel the underlying mechanism, BMSCs were treated with NPY after pretreatment with the NPY-1R antagonist PD160170 or the Wnt pathway antagonist DKK1, and gene expression levels of Wnt signaling molecules and osteoblastic markers were determined by qPCR. Our results indicated that NPY significantly promoted osteoblastic differentiation of BMSCs in a concentration-dependent manner and up-regulated the expression levels of proteins including β-catenin and p-GSK-3β and the mRNA level of β-catenin. Moreover, NPY promoted the translocation of β-catenin into nucleus. The effects of NPY were inhibited by PD160170 or DKK1. Additionally, NPY enhanced the ability of BMSCs to migrate and promoted the expression of vascular endothelial growth factor (VEGF) as measured by immunocytochemical staining, qPCR and Western blot. These results suggested that NPY may stimulate osteoblastic differentiation via activating canonical Wnt signaling and enhance the angiogenic capacity of BMSCs.