| Background:Spinal fusion technique has been one of the most commonly surgical methods in spinal surgery,which can restore the normal sequence of the spine by reconstructing the stability of the spine.It is widely used in the treatment of spinal diseases such as spinal instability,degeneration,tumors,trauma,infection and tethered spinal cord.With the advent of aging in China,the incidence of spinal degeneration and other diseases has increased,and the number of patients requiring spinal surgery has also increased significantly.However,serious complications after spinal fusion include fusion failure,pseudoarticular formation,leading to long-term pain,deformity,spinal instability,and even compression of the spinal cord.In patients with osteoporosis and hormones,the incidence of fusion failure can be as high as 34.3%.Osteoclasts are important regulatory cells in osteogenesis and bone balance,and are the core cells involved in osteogenesis,bone remodeling and bone regeneration in cartilage.In the early stages of bone regeneration and bone remodeling,osteoclasts first reach the target area and absorb bone trabeculae;then osteoblasts are recruited to secrete the bone matrix and form new bone trabeculae.Osteoclast functional activation may be a key cellular event in the early initiation and development of bone regeneration.Therefore,it is of great scientific significance and medical value to study the related factors and signaling pathways that regulate the formation and differentiation of osteoclasts.It has been found that calcitonin gene-related peptide,substance P,vasoactive intestinal polypeptide and neuropeptide Y（NPY）are closely related to bone regeneration.Among them,NPY nerve fibers appeared earlier in the bone regeneration area and were distributed in the area where bone metabolism was active,suggesting that it may be a key regulatory factor in early bone regeneration.However,there are few studies on the direct effect of NPY on osteoclasts,and the specific mechanism of how NPY regulates osteoclast formation and differentiation is unclear.Objective and Methods:In the first part of the experiment,we mainly studied the effects of NPY on osteoclast precursors,including the acquisition of bone marrow-derived macrophages,the induction and culture of osteoclast precursors;CCK-8 method to detect the effects of different concentrations of NPY on the proliferation of osteoclast precursors;cell scratch test and real-time quantitative PCR（q RT-PCR）method to detect the effects of different concentrations of NPY on osteoclast precursors The influence of NPY on the secretion of VEGF and PDGF by osteoclast precursor was detected by ELISA.In the second part of the experiment,we further explore the effect of NPY on the formation and differentiation of osteoclasts and its mechanism.By detecting the expression of NPY related receptors in the process of osteoclast formation and differentiation,as well as the expression of related factors（c-Fos,NFATc1,CTSK,TRAP,CTR,Akt,NF-κB,m TOR）in the process of osteoclast formation and differentiation under the intervention of NPY,and the molecular pathway The main detection methods include trap staining,ghost pen cyclic peptide staining cytoskeleton,q RT-PCR,Western blot and so on.In the third part,we used NPY in the animal body,established the model of bone graft fusion between the transverse processes of rats and added NPY for intervention,and preliminarily observed the effect of NPY on bone graft fusion by mirco CT.Results:Our study used the CCK-8 method to detect the effects of different concentrations of NPY on the osteoclast precursor proliferation ability,and found that the cell viability values of the 10-6mol/L group and the 10-8mol/L group were higher than those of the control group（0mol/L）.Especially in the 10-6mol/L group,the difference was statistically significant（p<0.05）.There was no significant difference between the other groups and the control group.Therefore,it can be seen that the NPY concentration used in our experiments is safe for the osteoclast precursor.Does not show obvious toxic effects and reduce cell viability,and at the same time,the concentration of 10-6mol/L NPY and10-8mol/L NPY promotes osteoclast precursor proliferation.From the results of the scratch test,we can see that NPY(10-6mol/L)can inhibit the migration of osteoclast precursors,especially in the early stage of the effect.After treating osteoclast precursors with NPY(10-6mol/L)for 1,3,and 5 days,q RT-PCR results showed thatβ3-integrin m RNA expression was significantly lower in the 10-6mol/L NPY group than in the control group.This indicates that NPY affects the migration of osteoclast precursors by inhibiting the expression of osteoclast precursor integrinαγβ3.Then RANKL（100ng/ml）was added to the medium to induce the osteoclast precursor to mature osteoclasts.Analysis of the number and area of??TRAP-positive osteoclasts after TRAP staining revealed that compared with the control group without NPY intervention,the number and area of??TRAP-positive cells in the NPY(10-6mol/L)group were significantly reduced（p<0.05）.),Indicating that NPY inhibits osteoclast precursor differentiation into osteoclasts.Then we conducted in-depth analysis and research on this effect.Osteoclast precursors were co-cultured with 10-6mol/L NPY,M-CSF,and RANKL,and samples were taken for q RT-PCR on days 0,1,3,and 5.Compared with the control group,with the osteoclast precursors gradually differentiated into osteoclasts.Under the effect of NPY,the expression of key fusion gene DC-STAMP was lower than that of the control group without intervention（p<0.05）.In the study,the m RNA expression level of osteoclast-specific marker molecules was measured,and it was found that the m RNA expression levels of osteoclast-specific marker molecules CTSK,CTR,and TRAP were inhibited under the intervention of 10-6mol/L NPY（p<0.05）.The q RT-PCR method and Western Blot method were used to detect the protein and m RNA expression levels of key transcription factors c-Fos and NFATc1 during osteoclast differentiation.It was found that NPY can reduce the protein and m RNA expression of c-Fos and NFATc1（p<0.05）.In order to explore the mechanism by which NPY regulates the expression of transcription factors c-Fos and NFATc1 in osteoclast differentiation,we have performed relevant studies on the signaling pathways upstream of these factors that are closely related to osteoclast differentiation,including the AKT signaling pathway,NF-κB signaling pathway and MAPKs signaling pathway.Western Blot results showed that 10-6mol/L NPY can specifically inhibit the activation of ERK/c-Fos/NFATc1 during osteoclast differentiation（p<0.05）,without affecting the pathways such as NF-κB,p38,and JNK.We have also done relevant research on which NPY receptor in osteoclasts conducts the above-mentioned effects of NPY.During the differentiation of osteoclast precursors into osteoclasts,there was no significant change in the overall expression of NPY receptor YIR.The expression of Y2R gradually increased and reached a peak on the third day after RANKL stimulation（p<0.05）.Because the peak period of osteoclast differentiation was also concentrated on the third day after RANKL stimulation,it was speculated that NPY had a significant effect on osteoclasts.The effect of differentiation is mainly mediated by the NPY receptor Y2R.Summarizing the results of the first two parts,we finally applied NPY to the bone graft fusion model of the transverse process in vivo.The results of mirco-CT showed that bone formation was seen in the control group,but the bone cortex was thinner.Compared with the control group,the NPY group showed more obvious bone fusion,the formed bone cortex was relatively thick,and some areas formed bone blocks.Through statistical analysis,it was found that the bone volume index,the number of trabeculae and the thickness of trabeculae were higher than those of the control group（p<0.05）.It was initially shown that the treatment of allograft bone with NPY promoted the bone fusion process.The reason for this result may be that NPY reduces bone resorption and indirectly enhances bone formation by inhibiting the activation of osteoclasts in the bone graft area,thereby promoting bone fusion.Conclusions:In summary,in this study,we combined in vitro cell experiments with in vivo animal experiments and found that NPY has an important regulatory effect on osteoclast formation and differentiation.High concentration of NPY(10-6mol/L)can promote osteoclast precursor proliferation,inhibit its migration,and promote its secretion of related proangiogenic factor VEGF.During osteoclast formation,NPY can reduce the expression of the key fusion gene DC-STAMP,thereby inhibiting osteoclast formation.At the same time,after adding exogenous NPY stimulation,the NPY receptor Y2R in osteoclasts receives signals and transduces the stimulation signals into osteoclasts,which affects c-Fos by reducing the phosphorylation level of key pathways ERK/MAPK/NFATc1 activation,thereby inhibiting osteoclast differentiation.NPY is also found in the rat bone graft fusion model to promote bone formation in the bone graft fusion region. |
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