| Background:With the coming of aging population,the brittle fracture caused by osteoporosis is increasing year by year,which has caused a heavy economic burden to the family and society.Furthermore,tumor resection,cancer,severe trauma or congenital diseases can lead to huge segmental bone defects,it has caused a lot of difficulties for clinical work.Synthetic bone graft substitutes combined with bioactive molecules,such as peptides,growth factors and small molecules,can improve the delivery of bone precursor cells,as well as subsequent bone formation and metabolism.Bioactive molecules are required in patients with larger bone defects(>2 cm).For this purpose,growth factors,peptides and small molecules are currently being evaluated at the pre-clinical and clinical levels.Based on previous findings,combining Neuropeptide Y(NPY)with the appropriate corresponding bone scaffold may bring about effective clinical translations and novel insights into the future of bone fracture.Neuropeptide Y(NPY)is a 36-amino acid peptide that is a neurotransmitter located in the brain and peripheral nervous system.NPY is an essential skeletal neurotrophic factor and plays a principal role in promoting fracture healing,improving knee osteoarthritis,and treating osteoporotic brittle fracture.Furthermore,NPY can promote bone metabolism through direct and indirect activation of multiple pathways in osteocytes,osteoblasts and osteoclasts.Additionally,NPY enhances osteoblast mediated extracellular osteogenesis and inhibits osteoclast mediated bone resorption and remodeling,which are key factors of fracture healing.Furthermore,NPY plays an important role in articular cartilage repair and bone metabolism.Studies have found that there are abundant neuropeptides in synovium,articular cartilage,meniscus and subchondral bone.NPY is an important sympathetic neuropeptide,and its neurotrophic effect on hip and knee joints is attracting more and more attention.NPY is considered as a potential therapeutic target for osteoarthritis(OA).Additionally,NPY is an important neurotrophic factor in the treatment of postmenopausal osteoporosis(PMO).In human and rodent models,NPY regulates bone mass balance through NPY receptor Y1(NPY1R)and NPY receptor Y2(NPY2R).Mice lacking the anxiolytic factor NPY showed more anxious behavior and elevated corticosterone levels.Furthermore,due to the inhibition of osteoblast activity,the bone loss of NPY silenced mice was 3-fold greater than that of wild-type mice under 6-week restraint and cold stress.The stress-protective NPY pathway acts specifically through NPY2R.In other words,NPY can promote bone formation under stress.However,the molecular mechanism by which NPY regulates the osteogenesis of MC3T3-E1 remains unclear,which needs further study.In conclusion,it is of great significance to investigate the osteogenic molecular biological mechanism of NPY in promoting bone defect healing after fracture.In this study,we established a C57BL/6 mouse tibial fracture model to investigate the distribution and expression of NPY and osteogenic related genes in the early stage of fracture in vivo.It was suggested that NPY was involved in the early inflammatory reaction of fracture and may play a role in bone induction.In vitro,we synthesized NPY small interfering RNA(siRNA)and NPY overexpression plasmid,and transfected them into mouse pre-osteoblast MC3T3-E1 cells.We further detected the expression of osteogenic related genes and proteins,and revealed the molecular biological mechanism of NPY gene promoting osteogenesis by up regulating Runx2 and osterix gene expression.PARTⅠEffect and mechanism of neuropeptide Y on fracture healing of miceObjective:1.To investigate the serum expression of NPY gene in a C57BL/6 mouse model of tibial closed fracture.2.To detect the gene and protein expression of osteogenic related factors neuropeptide Y(NPY),neuropeptide Y1 receptor(NPY1R),Runx2,Osterix at different time points of fracture healing in mice.3.To analyze the biological effect of NPY on fracture healing in mice.Methods:1.Model establishmentEighteen SPF C57BL/6 male mice were selected as experimental subjects.The unilateral tibial fracture was artificially broken without fixation under isoflurane induced anesthesia.The mice were kept and monitored in containers with free access to distilled water and food.2.Dynamic observation of callus formation and remodeling in the process of fracture in miceThree mices were randomly taken out under under isoflurane induced anesthesia and tested by IVIS(?)SpectrumCT at 0,1,7 and 14 days after surgery,respectively,to observe the callus growth at the fracture site.3.The expression of serum NPY in the fracture healing Blood was randomly collected from three inner canthus of mice at 1,7,and 14 days,and the concentration of NPY in serum was detected by ELISA.4.The expression of osteogenic genes in the fracture healingThree mices were randomly killed at 1,7,and 14 days after modeling.At 1,7 and 14 days,the fracture tissue were taken to extract RNA.The mRNA expressions of NPY,NPY1R,Runx2 and osterix were detected by RT-PCR.The fracture tissue was extracted and paraffin-embedded sections were performed for hematoxylin eosin staining(HE)or fixed in 70%ethanol for immunohistochemical staining(IHC)of NPY,NPY1R,Runx2 and osterix,respectively.The relative protein was detected by AI image analysis system(alpathwell)after immunohistochemical staining(IHC).Results:1.Successfully established the model of closed tibial fractureThrough gross observation,we found that on the 1st day after the operation,the mice did not move a great amount and may have shown a little pain,but it did not affect their eating and water intake.On the 2nd day after operation,the mice could move gradually.The walking function of mice could be initially established at 14 days,and sometimes both limbs of mice could stand at the same time.It is suggested that there is bony connection in fracture.2.Macroscopic and microscopic observation showed that the tibial fracture healed well in the animal modelFrom a macroscopic point of view,we used IVIS(?)Spectrum CT to monitor the situation in 14 days after operation.CT showed that the fracture line was indistinct and callus formed at the fracture end,indicating that the modeling was successful.From the microscopic point of view,HE staining showed that primary ossification center and new bone trabecular structure were found at the healing site at 14 days.3.Changing trend of serum NPY concentration in C57BL/6 mice after operationELISA was intended to explore the expression of NPY in the serum of mice.NPY concentrations ranged from 925.35pg/ml-1055.2pg/ml at different time points(1,7 and 14 days after modeling).The concentration of NPY had a transient decrease and then gradually increased in the 14 days,but the overall difference was not statistically significant(P=0.152).4.Expression levels of osteogenic related genes and proteins during fracture healing in miceRT-PCR was intended to explore the mRNA expression level of osteogenesis related genes in the process of fracture healing in mice.NPY mRNA was highly expressed in blood callus,moderately expressed in cartilage callus,and lowly expressed in bone callus.The expression of NPY mRNA was decreased by～0.27-fold at day 7 and 0.09-fold at day 14,in comparison with the day 1(P<0.001).There was no significant difference in NPY1R mRNA expression(P=0.133).The expression of NPY1R mRNA was increased by～1.6-fold at day 7 and 3.8-fold at day 14(P=0.059).The expression of Runx2 mRNA was elevated by～7.9-fold at day 7 and～65.6-fold at day 14,in comparison with the day 1.The expression of Osterix mRNA was elevated by～124.0-fold at 7 days and～775.1-fold at day 14,in comparison with the day 1.Immunohistochemical staining and AI image analysis system were used to analyze osteogenic related proteins in the process of fracture healing in mice.NPY protein was highly expressed in the early stage of fracture.The expression of NPY protein was decreased by～0.37-fold at day 7 and 0.55-fold at day 14,in comparison with the day 1(P<0.05).The expression of NPY1R protein was decreased by～0.08-fold at 7 days and～0.21-fold at day 14,in comparison with the day 1(P<0.001).The expression of Runx2 protein was decreased by～0.15-fold at day 7 and～0.19-fold at day 14,in comparison with the day 1(P<0.001).The expression of osterix protein was decreased by～0.12-fold at day 7 and～0.10-fold at day 14,in comparison with the day 1.Conclusions:1.NPY gene and protein are involved in the early inflammatory response of fracture and may play a role in osteoinduction.2.The expression of osteogenic factors NPY,NPY1R,Runx2 and osterix is specific in time and space.PART ⅡExpression and Biological function of NPY in the Mouse Pre-osteoblast Cell Line MC3T3-E1Objective:1.To investigate the mRNA and protein expression after overexpression or interference of NPY in mouse pre-osteoblast cell line MC3T3-E1 in vitro.2.To detect the effect of NPY in osteogenic differentiation of MC3T3-E1 cells in vitro.Methods:1.Construction of three NPY siRNA sequences.The siRNA1,siRNA2 and siRNA3 sequences targeting NPY gene were constructed.They were transfected into pre-osteoblast cell line MC3T3-E1 by rfect siRNA transfection reagent respectively.Reverse transcription quantitative(RT-q)PCR was intended to explore the expression of NPY mRNA in pre-osteoblast cell line MC3T3-E1.To verify and screen out the NPY siRNA with the best interference efficiency.2.Construction of NPY overexpression plasmid and sequencing verification Overexpression plasmid targeting NPY gene were constructed.After sequencing verification,rfect DNA transfection reagent was used to transfect NPY overexpression plasmid and control plasmid vector into MC3T3-E1 cells to verify NPY expression,3.The expression of NPY mRNA in MC3T3-E1 cells of different intervention groupsNPY siRNA,scrambled siRNA,NPY overexpression plasmid and empty vector plasmid were transfected into MC3T3-E1 cells respectively.Reverse transcription quantitative(RT-q)PCR method was perforned to observe the NPY mRNA expression of MC3T3-E1 cells in the groups at 4,7 days.4.The expression of NPY protein in MC3T3-E1 cells of different intervention groupsNPY siRNA and NPY overexpression plasmid were transfected into MC3T3-E1 cells.Western blot method was performed to evaluate NPY protein expression in the groups at 4,7 days.5.Observe the changes of MC3T3-E1 cells staining after overexpression or interference of NPY.MC3T3-E1 cells were resuscitated and cultured in osteogenic induction medium.NPY siRNA and NPY overexpression plasmid were added respectively.At 14 days,alizarin red staining,ALP staining and-catenin immunofluorescence staining were performed respectively for microscopic observation.Results:1.Screening of NPY-siRNA sequences with the highest interference efficiencyThe siRNA 1,siRNA2,siRNA3 containing the target gene NPY were successfully constructed.NPY siRNA3 possessed the highest interference efficiency,up to 75%.As a result,siRNA3 was used for subsequent experimentation.2.Construction of NPY overexpression plasmidsAfter the construction of NPY overexpression plasmid,the sequence of NPY was confirmed by sequencing.3.Effects of NPY overexpression or interference on NPY gene expressionOverexpression of NPY dramatically elevated the expression of NPY mRNA in pre-osteoblast cell line MC3T3-E1 by～3.9-fold and-4.3-fold at 4,7 days,respectively,compared with the control group(P<0.001).NPY siRNA decreased the expression of NPY mRNA by～0.75-fold at 4 days(P<0.05)and～0.55-fold at 7 days,compared with the control group.4.Effects of NPY overexpression or interference on NPY protein expressionOverexpression of NPY significantly increased the expression of NPY protein by～1.2-fold and 1.5-fold at 4,7 days,respectively,compared with the control group(P<0.05).NPY siRNA decreased the expression of NPY protein by～0.45-fold and 0.35-fold at 4,7 days,respectively,compared with the control group(P<0.05).5.Effects of overexpression or interference of NPY on osteoblast differentiationAlizarin red staining showed that there were more purplish red calcium nodules in MC3T3-E1 cells after overexpression of NPY,and less purplish red calcium nodules after interference of NPY.ALP staining showed that there were more yellow calcium deposits and more blue granules in the cells after overexpression of NPY,and less blue granules after interference of NPY.Immunofluorescence staining of β-catenin showed that there was more green fluorescence in the cytoplasm after overexpression of NPY,and less green fluorescence in the cytoplasm after interference of NPY.Conclusions:1.The interference of NPY can inhibit the osteogenic differentiation of MC3T3-E1 cells.2.Overexpression of NPY promotes osteogenic differentiation of MC3T3-E1 cells.PART ⅢNPY upregulates Runx2 and Osterix and enhances osteogenesis in the Mouse Pre-osteoblast Cell Line MC3T3-E1Objective:To explore the biological mechanism of NPY upregulating the expression of osteoblast specific transcription factors Runx2 and osterix to promote the osteogenesis of MC3T3-E1 cells in vitro.Methods:1.The expression of Runx2,osterix,ALP and OCN mRNA after overexpression or interference of NPY in MC3T3-E1 cellsNPY siRNA and NPY overexpression plasmid were transfected into MC3T3-E1 cells.The mRNA expressions of Runx2,osterix,ALP and OCN were observed by reverse transcriptional quantitative(RT-q)PCR at 4,7 days.2.The expression of Runx2,osterix,ALP and OCN proteins after overexpression or interference of NPY in MC3T3-E1 cells NPY siRNA and NPY overexpression plasmid were transfected into MC3T3-E1 cells.The expression levels of Runx2,osterix,ALP and OCN proteins in the groups were investigated by Western blot at 4,7 days.Results:1.NPY upregulated Runx2 and osterix expression in MC3T3-E1 cells.1.1 Effects of overexpression or interference of NPY on Runx2 and osterix mRNA expressionOverexpression of NPY significantly increased the expression of Runx2 and Osterix mRNA in MC3T3-E1 cells.Runx2 mRNA was increased by～5.4-fold and 6-fold at 4,7 days,respectively,compared with the control group.Osterix mRNA was increased～2.7-fold and～3-fold on at 4,7 days,respectively,compared with the control group.NPY siRNA decreased the expression of Runx2 and osterix mRNA.Runx2 mRNA was decreased by～0.50-fold and～0.35-fold at 4,7 days,respectively,compared with the control group.Osterix mRNA was decreased by 0.80-fold(P<0.001)and 0.55-fold at 4,7 days,respectively,compared with the control group.1.2 Effects of overexpression or interference of NPY on Runx2 and osterix proteins expressionOverexpression of NPY significantly increased the expression of Runx2 and Osterix proteins in MC3T3-E1 cells.Runx2 protein expression was increased by～1.6-fold and～1.2-fold at 4,7 days,respectively,compared with the control group.Osterix protein expression increased by～1.6-fold and～2.1-fold at 4,7 days,respectively,compared with the control group(P<0.05).NPY siRNA decreased the expression of Runx2 and osterix proteins.Runx2 protein expression was decreased by～0.40-fold and～0.55-fold at 4,7 days,respectively,compared with the control group.Osterix protein expression was decreased by～0.6-fold and～0.55-fold at 4,7 days,respectively,compared with the control group(P<0.05).2.NPY is critical for osteoblastic differentiation.2.1 Effects of overexpression or interference of NPY on ALP and OCN mRNA expressionOverexpression of NPY significantly promoted the expression of ALP and OCN mRNA.At 4,7 days,ALP mRNA was increased by～3.7-fold and 4.9-fold,and OCN mRNA was increased～4.0-fold and～4.3-fold,respectively,compared with the control group(P<0.001).NPY siRNA decreased the expression of ALP and OCN mRNA.At 4,7 days,ALP mRNA was decreased by～0.56-fold and～0.26-fold,and OCN mRNA was decreased by～0.60-fold and～0.35-fold,respectively,compared with the control group.The differences were statistically significant at 4 days(P<0.05).2.2 Effects of overexpression or interference of NPY on ALP and OCN proteins expressionOverexpression of NPY significantly promoted the expression of ALP and OCN proteins.At 4,7 days,OCN protein expression was increased by～1.4-fold and～1.6-fold,and ALP protein expression was increased by～1.6-fold,respectively,compared with the control group(P<0.05).NPY siRNA decreased the expression of ALP and OCN proteins.At 4,7 days,ALP protein expression was decreased by～0.60-fold at 4,7 days,respectively.OCN protein expression was decreased by-0.64-fold and～0.78-fold at 4,7 days,respectively(P<0.05).Conclusions:1.NPY may promote the osteogenesis of MC3T3-E1 cells by upregulating the expression of Runx2 and osterix genes.2.NPY is expected to be a target for the treatment of huge bone defects in traumatic fractures.Innovations and LimitationsInnovations:(1)The study systematically described the mRNA and protein expression of NPY,NPY1R,Runx2 and osterix in different cells at the early and middle stages of fracture healing in mice,and partially revealed that NPY participated in the early inflammatory reaction of fracture,which may play the role of bone induction.(2)In the study,we successfully screened out the most efficient NPY siRNA and prepared the NPY overexpression plasmid,which verified the expression and biological function of NPY in the mouse pre-osteoblast cell Line MC3T3-E1.(3)The study revealed that NPY may promote the osteogenesis of MC3T3-E1 cells by upregulating the expression of Runx2 and osterix genes.Limitations:(1)In vitro cytological experiments,we only studied a single osteoblast line,and then we can add human primary osteoblast to further elaborate the biological effects of NPY.(2)In vivo animal studies we only established the traumatic fracture model,but we did not establish the osteoporotic brittle fracture model.In the future,we can further study the role of NPY in different fractures and elaborate the related mechanism. |
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