Regulating Osteogenic Effect Of Neuropeptide Y On Bone Marrow Mesenchymal Stem Cells With The Canonical Wnt Signaling Pathway

Fracture as well as non-unions and delayed fracture healing etc. are normally-seen diseases in traumatic orthopedics. The treatments and related principles have also been difficult and heat topics in bone healing researches. Dissatisfactory curative effect leads to severe influence on the prognosis of patients. To improve the prognosis of patients function, the investigation on the biological mechanism as well as the pathophysiological features of bone metastasis and bone renewal should be regarded as the basis and premise of our studies. Deep and vast investigation on fracture regeneration and reconstruction will provide a brand new thread for clinical treatment as well as new technique.Bone sensory neurons secrete many types of neuropeptides including Calcitonin gene related peptide (CGRP), substance P (SP) and neuropeptide Y (NPY) etc. Previous researches have shown that neuropeptides have an impact on bone tissue generation and metastasis in many ways, and also play an important role in fracture healing. NPY is one of the neuropeptides that have highest content and that are most widely distributed in central neural system. NPY have a lot of biological function as a 36-amino polypeptide, and classic NPY mainly has mediation effect on food intake, cardiovascular activity, and energy regulation etc. NPY receptors are members of G protein-coupled receptor family, mainly including Y1, Y2, Y3, Y4, Y5, and Y6 subtypes. Among them, Y1 and Y2 receptors have close association with bone metastasis mediation. In central neural system, NPY functions on bone metastasis through hypothalamus Y2 receptor, while in bones, NPY functions through Y1 receptor. A great number of researches in vitro and in vivo have confirmed that NPY as well as its receptors play an important role in mediating bone regeneration, reconstruction, and bone formation, however, the biological effect of NPY on bone marrow mesenchymal stem cells (BMSCs) is still controversial, and its function mechanism is still unknown. Therefore, further investigation on the effect of NPY on BMSCs osteogenic differentiation is beneficial in understanding the function of neuropeptides on bone metastasis.Fracture healing is a complicated process in which many tissues and factors involve in and have synergistic effect on each other. Diverse proteins in broken end of fracture bone that promote fracture healing and angiogenesis have important effect on fracture healing. Bone morphogenetic protein (BMP) is one of the essential proteins among them, and it has the capability to promote bone regeneration and bone reconstruction. BMP-2 has the most obvious function on promoting osteogenic effect in BMP family. In osteogenic process, angiogenesis is the prior condition, which can not only restore blood stream in broken end of fracture bone and provide nutrition for bone growth, but also promote osteogenic effect via growth factors that mediate osteogenic cells viability. The effect of reconstruction of blood supply in bone biology has also been investigated in many studies, and its importance has also been confirmed. Vascular endothelial growth factor (VEGF) plays an important role in maintaining angiogenesis and blood supply in surrounded tissue as the most powerful angiogenesis factor in fracture healing process. Therefore, the investigation in the relation of BMP-2 and VEGF expression and NPY mediation effect on BMSCs osteogenic differentiation may be the important link in the biological process of NPY mediating fracture healing.Cell biological effect has a close association with cell signaling pathways. Classic Wnt/β-catenin signaling pathway is the key pathway in bone metastasis biology, its important role in bone biology and bone generation process has been confirmed in researches. Activating Wnt/β-catenin signaling pathway promotes bone formation and adds to bone mass. Classic Wnt/β-catenin signaling pathway can be concluded as the accumulation of β-catenin in cytoplasm. Accumulated β-catenin translocates into cell nucleolus and results in downstream Wnt-related target gene expression. At present, the effect of classic Wnt/β-catenin signaling pathway on NPY function on BMSCs osteogenic differentiation and the cooperation effect of NPY and related factors including BMP-2 and VEGF have not been reported by related articles. Therefore, whether the biological effect of NPY on BMSCs is conducted by classic Wnt/β-catenin signaling pathway is worth of studying.Based on the background above, this study focused on BMSCs, and discussed on the effect of NPY on BMSCs osteogenic differentiation and the impact of related factor expression like BMP-2 and VEGF, and investigated the related mechanism via classic Wnt/p-catenin signaling pathway. It will also further study on the molecular mechanism of the mediating effect of neuron factor on fracture healing through neuropeptides, and will provide new threads and experimental basis for the clinical apply of NPY in fracture and bone metastasis diseases. The materials, methods, contents, and conclusion of this experiment are mainly as the following few parts.Objective of Study1. The impact of NPY on rat BMSCs osteogenic differentiation.2. The impact of NPY on BMP-2 and VEGF expression in the process of rat BMSCs osteogenic differentiation.3. The relation of NPY medicating effect on rat BMSCs osteogenic differentiation and classic Wnt/β-catenin signaling pathway.Section 1 The impact of NPY on rat BMSCs osteogenic differentiation.Objective of StudyPanmyeloid adherence method was used to achieve SD rats BMSCs. Flow cytometry identifying assay was used to evaluate its cell purity, and to investigate on the impact of NPY on BMSCs osteogenic differentiation.Experimental Methods1. The harvesting, culturing, and indentifying of BMSCs80-100g male SD rats were selected in this experiment, and were killed off the neck in a clean condition according to the experimental ethics requiring animals. After cutting rat skin and subcutaneous muscle, the rats femur and tibia were removed with sterile tools under clean condition. After isolated and cultured for 48-72h, non-adherence blood cells were removed with the exchange of culture fluid. Cells were then treated with inoculation trypsin digestion and passage to 25cm2 culture flask according to a 1:2 ratio, and were cultured with DMEM medium containing fetal calf serum (10% volume fraction) and penicillin streptomycin solution (1% volume fraction). The culture medium was changed every 2-3 days, and cells were passage three times after trypsin digestion by 1:2 passages when they grew to cover the bottom, and the rat BMSCs morphology characteristics were evaluated through the light microscope and inverted microscope. Three generations of BMSCs were taken, and after washed with PBS and treated with digestive EDTA-free trypsin for three times respectively, digestive cells were treated with flow cytometry assay to identify the cells through the detection of CD29, CD34, CD44, and CD45 expression.2. Experimental groupingThe cells were divided with the following four groups:the control group (added with PBS), NPY(10’8 mol/L) group (added with the same amount of PBS), NPY(10"10 mol/L) group (added with the same amount of PBS), and NPY(10’12 mol/L) group (added with the same amount of PBS). The third generation of BMSCs were exchanged into the osteogenic induction medium containing lOmM sodium β-glycerophosphate, 100nm dexamethasone,50 μg/mL vitamin C,10% FBS, and a-MEM. After inducted with induction medium and cultured for 7 and 14 days, cells were treated with alkaline phosphatase (ALP) staining and alizarin red staining. After cultured for 7,14, and 21 days, the osteogenic markers ALP, collagen type Ⅰ, osteocalcin, and Runx2 gene were detected in each group using q-PCR assay, and ALP protein expression was detected in each group using Western blot assay, to determine the dose-specification of the NPY modulation on BMSCs osteogenic differentiation under the condition of osteogenic induction..3. Using ALP staining and alizarin red staining to detect cell osteogenic conditionThe third generation of BMSCs were taken into the osteogenic induction medium, and were treated with ALP staining after cultured for 7 days. After washed with PBS, the cells were fixed in 96-cell plate with 95% ethanol. The cells were than treated with ALP dyeing solution, washed and mounted. After cultured for 14 days, cells were treated with alizarin red staining. After washed with PBS, the cells were fixed in 24-cell plate with Methanol, and were dyed with alizarin red for 5min. Then the cells were washed several times with distilled water, and then were dried and mounted. At last, the cells were observed and pictured under microscope to determine the osteogenic induction effect.4. Using q-PCR and Western blot assay to determine the expression of osteogenic genes and proteinsAfter the total RNA of experimental groups were extracted, qPCR was used to detect the amount of ALP, collagen type I, osteocalcin, and Runx2. The results were detected on day 7, day 14, and day 21 respectively. After ALP proteins of experimental groups were extracted, qPCR was used to detect the expression of ALP protein.Experimental ResultsRats BMSCs can be successfully isolated and cultured by using bone marrow culture method in vitro. Flow cytometry identification showed CD29 (92.4% ± 1.6%), CD34 (5.1%±1.3%), CD44 (86.7% ±3.1%), CD45(9.2%±2.5%) which were consistent with BMSCs immune phenotypic characteristics. Meanwhile, different concentrations of NPY elevated the levels of osteogenic markers and ALP protein, and NPY (10-10 mol/L) had the most intense promotion effect.Experimental ConclusionNPY had dose-specific promotion effect on BMSCs osteogenic differentiation.Section2 The effect of NPY on the expression of BMP-2 and VEGF in BMSCs osteogenic differentiationObjective of StudyTo discuss the effect of NPY in different concentrations on the expression of BMP-2 and VEGF in BMSCs osteogenic differentiationExperimental Methods1. Experimental groupingThe cells were divided with the following four groups:the control group (added with PBS), NPY (10-8 mol/L) group (added with the same amount of PBS), NPY(10-10 mol/L) group (added with the same amount of PBS), and NPY(10-12 mol/L) group (added with the same amount of PBS). The third generation of BMSCs were exchanged into the osteogenic induction medium containing lOmM sodium β-glycerophosphate, 100nm dexamethasone,50 μg/mL vitamin C,10% FBS, and a-MEM. After cultured for 7,14, and 21 days, the cells were treated with DAB immunohistochemistry, and the expression of BMP-2 and VEGF gene and protein was detected by q-PCR and Western blot essay.2. The detection of BMP-2 and VEGF expressionThe third generation of BMSCs were fixed with methanol for 30mins, and ruptured with 0.1% Triton X solution for 10mins. Endogenic peroxydase was blocked using skim milk for lOmins at room temperature prior to VEGF and BMP-2 antibodies incubated in 4℃ overnight and secondary antibodies incubated at room temperature for 30mins. Then the cells were colored using diaminobenzidine (DAB), dried, and mounted. At last, the cells were observed and pictured under microscope to determine the osteogenic induction effect.3. The detection of BMP-2 and VEGF genes and proteins expression using q-PCR and Western blot essayThe total RNA of experimental groups was treated with q-PCR to determine the amount of BMP-2 and VEGF. The results were tested on day 7, day 14, and day 21 respectively for three times. After the BMP-2 and VEGF proteins were extracted, they were treated with Western blot essay to determine the expression of BMP-2 and VEGF proteins.Experimental ResultsImmunohistochemistry essay showed that after 7,14, and 21 days of NPY treatment, the coloring of BMP-2 and VEGF were positive, with the cytoplasm colored brown. The results of q-PCR and Western blot showed that after 7,14, and 21 days of NPY treatment, the expression of BMP-2 and VEGF genes and proteins were promotedExperimental ConclusionNPY enhanced the expression of BMP-2 and VEGF which is dose-specific in BMSCs osteogenic differentiation.Section 3 The discussion of classic Wnt/p-catenin signaling pathway mechanism of NPY modulating BMSCs osteogenic differentiationObjective of StudyPrimarily discuss the classic Wnt/β-catenin signaling pathway mechanism of NPY modulating BMSCs osteogenic differentiation.Experimental Methods1. Experimental groupingThe cells were divided with the following four groups:the control group (added with PBS), NPY(10-10 mol/L) group (added with the same amount of PBS), NPY (10-10mol/L)+NPYY1 receptor antagonist PD160170 group (1 μM, added with the same amount of PBS), and NPY(10-10 mol/L) group+Wnt signaling pathway antagonist DKK 1(0.2 μg/mL, added with the same amount of PBS). Expressions of osteogenic markes ALP, collagen type Ⅰ, osteocalcin, and Runx2 gene and Wnt signaling pathway related gene β-Catenin were tested by q-PCR assay, and the expressions of Wnt signaling pathway related proteins P-Catenin and P-GSK-3β were evaluated using Western blot essay.2. The test of Wnt signaling pathway and osteogenic markers genes and proteins expressions using q-PCR and Western blot essayThe total RNA of experimental groups was treated with q-PCR to determine the amount of ALP, collagen type I, osteocalcin, Runx2, BMP-2, VEGF and P-catenin. The results were tested on day 7, day 14, and day 21 respectively for three times. After the P-catenin and p-GSK-3β proteins were extracted, they were treated with Western blot essay to determine the expression of P-catenin and p-GSK-3β proteins.3. The detection of β-catenin protein using immunofluoresence essayThe third generation of BMSCs were fixed with methanol for 30mins, and ruptured with 0.1%Triton X solution for lOmins. Endogenic peroxydase was blocked using skim milk for 10mins at room temperature prior to 1:300 dilution of β-catenin antibodies incubated in 4℃ at darkness overnight and FITC green fluorescent secondary antibodies incubated at room temperature for 30mins after washed by PBS for three times. Then the cells were colored using DAPI for 15mins after the solution was sucked out. At last, the cells were observed and pictured under fluorescence microscope.Experimental Results10-10mol/L NPY significantly promoted the gene expressions of BMSCs osteogenic markers and Wnt/β-catenin signaling pathway related genes, and promoted the protein level of P-catenin and p-GSK-3β. Meanwhile, NPY promoted the translocation of β-catenin into nucleus, while NPYY1 receptor antagonist and Wnt signaling pathway inhibitor DKK1 significantly compressed the promotion effect of NPY.Experimental Conclusion10-10 mol/L NPY significantly enhanced the differentiation of BMSCs into osteoplasts, and classic Wnt/β-catenin signaling pathway may be the possible mechanism.