The Mechanism Research Of Neuropeptide Y Regulating Activities Of Both Osteoblastic And Osteoclastic Differentiation Linked To Wnt Signaling Pathways

The physiological process of fracture healing and the pathological processes such as delayed union or nonunion of bone are commonly observed in fracture patients.However,the underlying mechanism is still unclear.Recent studies have shown that canonical and non-canonical Wnt signaling pathways are essential to bone healing,while neuropeptides secreted by the peripheral nervous system also play an important regulatory role during bone healing.Based on the facts above,our research was going to answer the questions below:1.Whether neuropeptides regulate bone healing through Wnt signaling pathways.2.What is the detailed molecular mechanism of this regulation.3.What is the biological effects of this regulation.Part 1:To explore the expression of Wnt signaling pathways through bone healing model and the regulating effect of some neuropeptides.Objective:To screen some neuropeptides that may regulate canonical and noncanonical Wnt pathways and investigate their biological functions.Methods:Firstly,mice fracture models were built to collect bone callus 1 week post-operatively.Neuropeptides in that including calcitonin gene related peptide?CGRP?,substance P?SP?,vasoactive intestinal peptide?VIP?,and neuropeptide Y?NPY?creatinine were analyzed,and gene expression of canonical Wnt3a and noncanonical Wnt5a protein were also tested.Secondly,the bone marrow mesenchymal stem cells were obtained in vitro to test the effect of several neuropeptides on canonical and noncanonical Wnt pathway.Results:The expression of CGRP,SP,VIP and NPY were different in the process of fracture healing,and Wnt3a and Wnt5a were also highly expressed.CGRP and SP had only effect on Wnt pathway by activating canonical Wnt pathway;VIP had no obvious effect on Wnt pathway;NPY could activate the expression of Wnt5a,and Wnt3a as well.Further vitro test showed that NPY could not promote the genetic transcription of?-catenin which is the core protein of canonical Wnt signaling pathway,it was opposite to JNK and NLK.NPY was able to promote the gene expression of ALP and RunX2,which are the symbol protein of osteoblastic differentiation,in a high concentration(10^-8mol/L,10^-1010 mol/L),while it could promote the gene expression of RANK,a symbol protein of osteoclastic differentiation,in a low concentration(10^-12mol/L).Conclution:NPY may activate both canonical and noncanonical Wnt signaling pathways during fracture healing.NPY may contribute to both osteoblastic and osteoclastic differentiation,which may be a concentration-dependent process.Part 2:To study the regulating effect of NPY on Wnt signaling pathways by establishing siRNA transfection model to inhibit?-catenin and NLK.Objective:To explore the regulatory effect of NPY in canonical and non-canonical Wnt signaling pathways.Methods:The expression of?-catenin,NLK and JNK were detected in MC3T3-E1 cells and RAW264.7 cells before and after the transfection of siRNA and the intervention of NPY.NPY was added to MC3T3-E1cells and RAW264.7 cells respectively with different concentration of 10^-1010 mol/L and10^-1212 mol/L.?-catenin and GSK-3?and their downstream product Tcf1 were tested.CaMK?and PKC,which are the symbolic protein of canonical Wnt/Ca²⁺signaling pathway,and JNK,which is the symbolic protein of non-canonical Wnt/PCP pathway,were identified with the intervention of NPY.Results:NPY did not suppress the expression of?-catenin itself,while activated canonical Wnt signaling pathway and the gene transcription of Tcf1 through decreasing the percentage of phospho-?-catenin in MC3T3-E1 and RAW264.7 cells.This effect did not attenuate in?-catenin?-/-?cells.NPY alone did not change the content of p-GSK-3?and?-catenin,while could increase the transcription of downstream target genes to activate canonical Wnt signaling pathway through promoting nuclear transportation of?-catenin.NPY could activate Wnt/Ca²⁺pathway in MC3T3-E1 cells via CaMK?and PKC and this effect failed to recur in NLK?-/-?cells,it could also activate Wnt/PCP pathway in RAW264.7 cells and this effect failed to recur in JNK?-/-?cells.Conclusion:NPY can activate canonical and non-canonical Wnt signaling pathways in MC3T3-E1 and RAW264.7 cells of mice,which means NPY may play an important role in fracture healing.Part 3:NPY promotes osteoblastic differentiation in MC3T3-E1 cells and osteoclastic differentiation in RAW264.7 cells via Wnt signaling pathways.Objective:To study the specific biological effects of NPY regulation canonical and non-canonical Wnt signaling pathway during the procedure of fracture healing.Method:The effects of NPY on the proliferation and osteogenic differentiation in MC3T3-E1 cells were studied first.The expression of osteogenic genes such as RunX2 and alkaline phosphatase?ALP?were observed under the intervention of si RNA transfection.The expression of NF-?B receptor activator?RANK?and NF-?B in RAW264.7 cells were studied during its osteoclastic differentiation process.Results:NPY could regulate the proliferation,apoptosis,and osteoblastic differentiation of MC3T3-E1 cells,and promote the osteoclastic differentiation of RAW264.7 cells.NPY promoted osteoblastic differentiation of MC3T3-E1 cells by activating Wnt/Ca²⁺pathway and promoted osteoclastic differentiation of RAW264.7cells by activating Wnt/PCP pathway.Conclusion:NPY can promote proliferation of MC3T3-E1 cells and decrease its apoptosis.NPY may accelerate fracture healing via activating canonical and non-canonical Wnt signaling pathway.Summarizing the results of this study,we can draw a conclusion that NPY secreted by peripheral nerve can promote bone healing via activating canonical Wnt/?-catenin signaling pathway.NPY can also promote osteoblastic differentiation and osteoclastic differentiation via activating non-canonical Wnt/Ca2⁺and Wnt/PCP signaling pathways respectively.Further more,the aforementioned biological effects of NPY have a feature of concentration dependence.The results of this study further reveal the mechanism of NPY regulating bone metabolism,and provide us new ideas and scientific basis for clinical treatment of fracture,bone defect and metabolic diseases of bone.