| Fracture and delayed fracture healing as well as non-unions etc owing to different reasons are the most major diseases in traumatic orthopedics.The treatment methods and principles are the focus of research in the field of orthopedics.As we known,Fracture healing progress involves a complex synergy between the several systems,multiple tissues structures and different cytokines.In recent years,more and more literatures have confirmed that Neuropeptide Y(NPY)plays an important role in the development of bone tissue and metabolism.NPY is a highly conserved 3 6-amino-acid neuropeptide and is one of the most abundant neuropeptides in the central nervous system and peripheral nervous system of mammals.Neuropeptide Y(NPY)exhibits a critical but still poorly understood regulatory signaling function,which is mainly through the five G protein-coupled receptors(Y1,Y2,Y4,Y5 and Y6),however the Y1 and Y2 receptors are main functional receptors.Previous studies have demonstrated that NPY regulates bone homeostasis through Y receptors expressed in bone marrow cells.Little,however,is known about the specific role of NPY on the proliferation and apoptosis of BMSCs.A fast-growing field has linked the Wnt pathway with bone development,remodeling,and repair.The best known Wnt signaling pathway involves β-catenin(commonly called the canonical pathway).It has been shown that canonical Wnt signaling is a key player in the proliferation and survival of osteoblasts as well as the proliferation and osteogenic differentiation of BMSCs.However,the extent of canonical Wnt signaling with regard to the proliferation and apoptosis of BMSCs is still poorly understood.Based on the roles of NPY and the Wnt/β-catenin pathway in regulating osteogenesis,we hypothesized that NPY enhances proliferation and decreased apoptosis in serum-deprivated BMSCs in vitro and that this activity may related to the Wnt/β-catenin pathway.First,BMSCs from SD rats were achieved by panmyeloid adherence method and the cell purity were identified by Flow cytometry,and prepared to investigate on the impact of NPY on BMSCs proliferation and apoptosis.we cultured BMSCs in the presence of NPY at final concentrations of 0(untreated control),10-8 M,10-10 M and 10-12 M,supplemented with NPY(the optimal concentration)and a Y1R antagonist(PD160170)diluted to 10-6M concentration;a mixture of NPY(the optimal concentration)and the Wnt pathway antagonist(DKK1)diluted to 0.2μg/mL,The cells were plated at a density of 6.0x103 cells/well in 96-well plates and cultured the appropriate conditions(cell proliferative experiment were incubated for 1,3,5,or 7 days with complete medium(supplemented with 10%FBS),or the cells were serum-deprivated for either 12 h or 24 h)with a range of NPY concentations(10-8 M-10-12 M).Based on the BrdU incorporation test,Cell Counting Kit-8(CCK-8),flow cytometry,quantitative polymerase chain reaction(qPCR)and Western blotting,we show that NPY significantly promoted the proliferation of BMSCs in a concentration-dependent manner,with a maximal effect observed at a concentration of 10-10M for proliferation,and 10-12M for anti-apoptosis.Furthermore,NPY significantly increased the percentage of cells in the S and G2/M phases,upregulated the expression of Cyclin D1 mRNA and downregulated the expression of P53 mRNA.In addition,NPY exhibited a protective effect after 24 h of serum starvation as illustrated by a reduction in the apoptosis rate and the expression of apoptosis markers,including the mRNA levels of caspase-3,caspase-9 and Bax;NPY also increased the mRNA and protein expression levels of canonical Wnt signaling pathway proteins,including β-catenin and c-myc,during the induced proliferative and anti-apoptotic processes.However,the proliferative and anti-apoptotic activities of NPY were partially blocked by either PD 160170(1 μM)or DKK1(0.2 μg/mL);These compounds also blocked the mRNA and protein expression of β-catenin,p-GSK-3β and C-myc.Therefore,the results of the present study demonstrated that NPY exerts a proliferative and protective effect on BMSCs in a dose-and time-dependent manner in vitro and,importantly,may be mediated via its Y1 receptor and involved in activating the canonical Wnt signaling pathway.This may provides an insight into the therapeutic application of this neuropeptide in bone fracture. |
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