br Materials and methods br Results

Materials and methods
Results
Discussion NPY is one of the most common neuropeptides in mammals, and it is abundant in the central and peripheral nervous system. Recent studies have indicated that the neuropeptides SP and NPY can regulate many biological activities in BMSCs and MC3T3 Wnt agonist 1 in vitro. For instance, NPY and SP promoted the osteogenic differentiation of BMSCs and MC3T3 cells (Liu et al., 2016; Fu et al., 2014), and SP enhanced the proliferation and inhibited apoptosis of BMSCs (Fu et al., 2015; Mei et al., 2013). However, the impact of NPY on BMSC proliferation and apoptosis, as well as the putative underlying mechanisms, has not been fully investigated yet. In the present report, we hypothesized that NPY may serve as a proliferative and protective agent in BMSCs and that this activity is likely mediated through the Y1 receptor and activation of the Wnt/β-catenin pathway, which might help illuminate the mechanisms of neuronal involvement in bone repair. Although there is sufficient evidence showing that NPY promotes cell proliferation, including in postnatal neuronal precursor cells (Hansel et al., 2001) and adipocyte precursor cells (Yang et al., 2008), its potential effect on BMSCs has only recently been reported; however, there are divergent findings among studies as to whether NPY promotes BMSC proliferation (Lee et al., 2010; Igura et al., 2011). Similar to our results, treatment with 10M NPY was shown to stimulate BMSC proliferation, which was more pronounced in YngBMSCs (Igura et al., 2011). In contrast, Lee indirectly showed that NPY may inhibit the proliferative ability of BMSCs because purified BMSCs isolated from Y1−/− mice formed significantly more CFU-Fs than their wild-type counterparts (Lee et al., 2010). This could be explained by different cell signal transduction mechanisms (e.g., other NPY receptors) in BMSCs isolated from Y1−/− mice compared with BMSCs from wild-type mice. We showed that NPY had a proliferative effect on BMSCs, as demonstrated by the increased percentage of cells in the S and G2/M phases and the increased expression of the downstream gene p53 and the upstream gene cyclin D1. Previous studies have shown that p53 triggers both G1 arrest and apoptosis in bone marrow cells (Spronck et al., 2007; Chen et al., 2014) and inhibits cyclin D1 promoter activity (Guo et al., 1789), the latter of which is a key regulator of the G1-to-S phase cell cycle transition. We showed that NPY may act downstream of p53 and upstream of cyclin D1 and increase the proliferative ability of BMSCs. Cell apoptosis and proliferation are basic cellular phenomena that are essential for maintaining a dynamic balance in the number of cells in an organism. Therefore, we further studied the role of NPY in BMSC apoptosis. Serum deprivation has been established as a stable model for apoptosis (Liang et al., 2013; Berlier et al., 2015), similar to the lack of nutrition provided at the site of a fracture. The present study is the first to examine the effects of NPY on serum deprivation-induced apoptosis in BMSCs. The anti-apoptotic effect of NPY in BMSCs was accompanied by attenuated activation of pro-apoptotic molecules, such as caspase-3, caspase-8, and Bax. Previous studies have demonstrated that overexpression of Bax and caspase-3 accelerates programed cell death (Oltvai et al., 1993; An et al., 2011). Moreover, it was shown that caspase-3 is an important effector protease that, when cleaved, acts as the ultimate enforcer of caspases during apoptosis (Wang et al., 2015; Roy, 2000). We confirmed that NPY can reduce the expression of cleaved caspase-3 at both the gene and protein level and decrease the percentage of early and late apoptotic cells. Thus, NPY has an important protective role against drug- or chemical-induced neuronal necrosis and apoptotic cell death (Santos-Carvalho et al., 2013; Alvaro et al., 2008; Goncalves et al., 2012), primarily via the Y2 receptors, at a concentration of 10M.