降钙素基因相关肽对下颌骨骨折愈合的影响及其信号转导通路的实验研究
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摘要
神经系统在不同水平参与骨折修复和改建以及正常骨代谢的调控。神经系统与其它系统或局部因子相互作用的机制非常复杂,其调控作用最终通过分布在骨组织局部的末梢神经释放神经肽等信号分子,影响成骨细胞和破骨细胞功能来实现[1-5]。降钙素基因相关肽(Calcitonin gene-related peptide CGRP)主要来源于感觉神经,具有多种生理功能。近年研究表明:CGRP参与骨折愈合的调控,并可能是通过影响成骨细胞功能实现的,但CGRP调控骨折愈合的分子机制及其对成骨细胞作用的胞内信号转导通路的研究国内外鲜见报道。
我们在以往的研究中从组织学水平探讨了下牙槽神经(Inferior alveolar nerve IAN)对下颌骨骨折愈合的影响及其可能机制[6、7]。本研究通过建立失IAN和保留IAN兔下颌骨骨折动物模型,运用骨组织形态计量学(Bone histomorphometry)技术从细胞水平探讨IAN和CGRP对下颌骨骨折愈合的影响;通过原代培养兔颅骨成骨细胞,在分子水平上阐明hCGRP作用于兔成骨细胞的信号转导通路,以进一步探讨感觉神经调控骨折愈合的分子机制。
目的:探讨CGRP对兔下颌骨骨折愈合的影响及其作用于兔成骨细胞的胞内信号转导通路,以进一步探讨感觉神经调控骨折愈合的分子机制。
方法:选用中国大白兔105只,随机分为三组,实验组(35只)为给予LY303870(P物质受体拮抗剂)保留IAN下颌骨骨折组,对照组(70只)为失IAN下颌骨骨折组(对照组1,35只)和保留IAN下颌骨骨折组(对照组2,35只);分别在术后第7、14、21、28天处死动物,制取冰冻切片和未脱钙骨切片两种标本。冰冻切片用于HE染色观察骨痂愈合方式,免疫组化染色观察P物质(Substance P SP)和CGRP在新生骨痂中的表达;四环素双标的未脱钙骨切片用于Goldner’s Masson Trichrome Stain染色和骨组织形态计量学分析。用二次酶消化法原代培养兔颅骨成骨细胞,按hCGRP终浓度分为6组,第1组和第2组为对照组,分别为不含hCGRP的DMEM液和10-9mol/L hCGRP+10-6mol/L hCGRP(8-37)的DMEM液,第3、4、5和6组分别为含hCGRP终浓度为10-10mol/L、10-9mol/L、10-8mol/L和10-7mol/L的DMEM液。用激光扫描共聚焦显微镜技术监测胞内游离Ca2+浓度变化,放免法检测胞内cAMP浓度,凝胶迁移试验(Electrophoretic mobility shift assay EMSA)检测激活转录因子4(Activiting transcrigtion factor 4 ATF4)活性,Western Blot检测ATF4、骨钙素(Osteocalcin OC)、核因子-κB受体活化因子配体(Receptor activator of nuclear factor-κB ligand RANKL)和骨保护素(Osteoprotegerin OPG)的表达,Northern Blot检测OC、RANKL和OPG mRNA的表达。结果:1. SP和CGRP在新生骨痂中的表达:与保留神经组相比,在骨折愈合不同阶段SP和CGRP在失神经组骨痂中的表达显著减少(p<0.01)。保留神经组在术后第7天,SP和CGRP呈阳性表达;术后第14天,SP持续阳性表达,CGRP呈高表达并达峰值;术后第21天, SP表达减弱,CGRP仍呈阳性表达;术后第28天,SP表达显著增强,CGRP表达显著减弱。
2. CGRP对骨折愈合的影响:成骨细胞形成表面和成骨细胞指数在实验组和对照组中差异无统计学意义;实验组破骨细胞吸收表面和破骨细胞指数明显低于对照组1,对照组1明显低于对照组2,各组间差异有统计学意义;类骨质表面对照组1高于实验组和对照组2,差异有统计学意义,实验组和对照组2之间差异无统计学意义;矿化骨痂体积实验组高于对照组1和对照组2,实验组与对照组1之间差异有统计学意义,与对照组2差异无统计学意义;骨形成率实验组高于对照组1和对照组2,实验组与对照组1差异有统计学意义,实验组和对照组2之间差异无统计学意义。
3. hCGRP对兔成骨细胞内游离Ca2+浓度的影响:hCGRP不能改变兔成骨细胞内游离Ca2+浓度,各组间差异无统计学意义。
4. hCGRP对兔成骨细胞内cAMP浓度的影响:hCGRP可促进兔成骨细胞内cAMP浓度升高,并且呈剂量依赖性,在hCGRP终浓度为10-9mol/L时达到峰值,hCGRP(8-37)可以抑制其促进作用。
5. hCGRP对兔成骨细胞内ATF4活性的影响:hCGRP可增强兔成骨细胞内ATF4活性,并且呈剂量依赖性,在hCGRP10-9mol/L时达到峰值,hCGRP(8-37)可以抑制其促进作用。
6. hCGRP对兔成骨细胞内ATF4蛋白表达的影响:hCGRP可促进兔成骨细胞内ATF4蛋白表达,并且呈剂量依赖性,在hCGRP终浓度为10-9mol/L时达到峰值,hCGRP(8-37)可以抑制其促进作用。
7. hCGRP对兔成骨细胞内OC及其mRNA表达的影响:hCGRP可促进和上调兔成骨细胞内OC及其mRNA表达,并且呈剂量依赖性,在hCGRP10-9mol/L时达到峰值,hCGRP(8-37)可以抑制其促进作用。
8. hCGRP对兔成骨细胞内RANKL及其mRNA表达的影响:hCGRP可抑制和下调兔成骨细胞内RANKL及其mRNA表达,并且呈剂量依赖性,在hCGRP终浓度为10-9mol/L时抑制作用最为明显,hCGRP(8-37)可以阻断其抑制作用。
9. hCGRP对兔成骨细胞内OPG及其mRNA表达的影响:hCGRP可促进和上调兔成骨细胞内OPG及其mRNA表达,并且呈剂量依赖性,在hCGRP10-9mol/L时达到峰值,hCGRP(8-37)可以抑制其促进作用。
10.采用Pearson相关系数描述每两个指标之间(cAMP、ATF4、OC、OPG、RANKL和RANKL/OPG)的相关程度,cAMP与其它指标的相关系数在0.713～0.988之间,ATF4与其它指标的相关系数在0.777～0.975之间,OC与其它指标的相关系数在0.803～0.988之间, OPG与其它指标的相关系数在0.712～0.988之间;RANKL与其它指标的相关系数在0.777～0.982之间, RANKL/OPG与其它指标的相关系数在0.717～0.975之间。经相关系数的假设检验均有统计学意义(p<0.01)。可认为cAMP、ATF4、OC、OPG、RANKL和RANKL/OPG的变化趋势相同。
结论:1. IAN损伤影响骨痂愈合的质量,可能是通过影响矿化骨痂的形成和骨改建来实现;
2.骨折愈合过程中CGRP和SP在骨痂中表达是动态变化的,并且与骨折愈合过程相适应,提示神经系统对骨折愈合调控是通过分布在骨组织局部的末梢神经分泌神经肽实现的;
3. CGRP对骨折愈合的调控作用可能是通过影响成骨细胞功能及破骨细胞分化和功能实现的;
4. hCGRP对兔成骨细胞功能影响可能的细胞信号转导通路是:hCGRP与成骨细胞上特异性受体CRLR结合,激活腺苷酸环化酶活性,促使胞内第二信使cAMP生成,通过cAMP信号系统促使胞内ATF4积聚并使ATF4磷酸化,启动成骨细胞分化相关基因OC/RANKL/OPG的表达;
5. CGRP可以通过调控成骨细胞合成RANKL和OPG来影响破骨细胞分化,实现对骨折修复和改建的调控。
The nervous system involves in the regulation of the process of repair and remodeling of fracture and bone metabolism at different levels. The mechanism, of which systemic and local factors cooperate with nervous system, is quite complicated. However, the regulation achieves finally through the effect of neural signals on function of osteoblasts and osteoclasts in bone, which are released from the nerve terminals distributed in bone[1-5]. Calcitonin gene-related peptide (CGRP) secrected by sensory nerve has many kinds of physiological activities. Previous studies have found that CGRP is involved in repair and remodeling of fracture through influencing functions of osteoblasts, and little is known about the molecular mechanism and cell sigal transduction of CGRP’s modulating the healing process of fracture at the present time.
In our previous studies, we explored the effect of inferior alveolar nerve(IAN) amputation on the healing of mandibular fracture and its possible mechanism at the level of tissue[6,7]. To explore the molecular mechanism of the nervous system’s modulating fracture healing, in this study we will explore further the effect of CGRP on mandibular fracture healing at the level of cell with bone histomorphometry through the foundation of animal model of mandibular fracture with or without IAN amputation, and clarify cell signal transduction of osteoblasts at the level of molecule by the primary rabbit osteoblast cultures of neonatal calvaria.
Purpose
The aims of the study were to explore the possible mechanism of CGRP’s modulating the healing of rabbit mandibular fracture and cell signal transduction of rabbit cultured osteoblast acted by hCGRP, and so that to explore further the molecular mechanism of sensory nerve’s modulating fracture healing.
Methods
105 adult China white rabbits were randomly divided into two groups (experimental and control groups). They all suffered from a fracture in the left mandible. Animals in experimental group were injected by LY303870, an antagonist to Substance P receptor, without IAN amputation. The control group was divided into two groups, and half of them with or without IAN amputation. The rabbits were sacrified on 7, 14, 21 and 28 day after operation, and the specimens were collected and stained with hematoxylin-eosin and for immunochemistry to observe the process of fracture healing and the expression of SP and CGRP in bone calllus, and stained with Goldner’s Masson Trichrome stain and the double marks of tetracycline to analyse the newly callus quantitively with bone histomorphometry. Osteoblast cultures were isolated from newly-born rabbit calvaria by sequential enzymatic digestion. The cultured osteoblasts were divided into six groups according to the final concentration of CGRP. The control group was divided into two groups. One is DMEM without CGRP or CGRP(8-37), and the other is DMEM with 10-9mol/L CGRP and 10-6mol/L CGRP(8-37) together. The other four groups are DMEM with 10-10mol/L, 10-9mol/L, 10-8mol/L and 10-7mol/L CGRP respectively and without CGRP(8-37). The detection of cAMP accumulation in cultured cells was performed using radioimmunology assay and the change of cyto free Ca2+ concentration was monitored by laser scanning confocal microscopy. The activity of activiting transcription factor 4(ATF4) was measured in electrophoretic mobility shift assay, and the expression and their mRNA of ATF4, osteocalcin, receptor activator of nulear factor-κB ligand and osteoprotegin in cultured cells were detected by Western Blot and Northern Blot respectively.
Results:
The expression of SP and CGRP in cullus:
Immunoreactivities of SP in callus occurred strong in the group without IAN amputation on Day 7 after operation, and became stronger on Day 14 and less on Day 21 after operation, and became stronger again on Day 28 after operation. Immunoreactivities of CGRP in callus occurred strong in the group without IAN amptation, became the strongest on Day 14 and less stronger gradually on Day 21 and 28 after operation. Immonoreactivities of SP and CGRP in callus occurred weakly at early stage and became stronger at late one in the group with IAN amputation. There were significant differences in the expression of SP and CGRP in callus between two groups.
The effect of CGRP on the healing of mandibular fracture: there were no significant differences in ob?s/BS and ob?Nb/BPm among three groups. On the contrary, there were significant differences among three groups in oc?s.BS, N?oc/BS, BFR, OS and MLV by analysing the callus using bone morphometry.
The effect of hCGRP on free Ca2+ concentration in rabbit cultured osteoblasts: hCGRP can’t increase cyto free Ca2+ concentration in cultured osteoblasts. There were no signifficant differences among three groups.
The effect of hCGRP on cAMP concentration in rabbit cultured osteoblasts: hCGRP can stimulate the accumulation of cAMP in cultured osteoblasts. And it displayed a dosage-effect relationship, which can be blocked by CGRP(8-37).
The effect of hCGRP on ATF4 activity in cultured osteoblasts: hCGRP can promot the activities of ATF4 in cultured osteoblasts. It displayed a dosage-effect relationship, which can be blocked by CGRP(8-37).
The effect of hCGRP on the expression of ATF4 in cultured osteoblasts: hCGRP can enhance the expression of ATF4 in cultured osteoblasts. It displayed a dosage-effect relationship, which can be blocked by CGRP(8-37).
The effect of hCGRP on the expression of ATF4 in cultured osteoblasts: hCGRP can promote the expression of ATF4 in cultured osteoblasts. It displayed a dosage-effect relationship, which can be blocked by CGRP(8-37).
The effect of hCGRP on the expression of OPG and its mRNA in cultured osteoblasts: hCGRP can promote the expression of OPG and up-regulate its mRNA in cultured osteoblasts. It displayed a dosage-effect relationship, which can be blocked by CGRP(8-37).
The effect of hCGRP on the expression of RANKL and its mRNA in cultured osteoblasts: hCGRP can inhibit the expression of RANKL and down-regulate its mRNA in cultured osteoblasts. It displayed a dosage-effect relationship, which can be blocked by CGRP(8-37).
All the obtained data in vitro experiments were evaluated by the statistical analysis using Pearson’s correlation coefficient to describe the degree of corelation in each index. And there was significant difference in corelation among cAMP, ATF4, OC, OPG and RANKL.
Conlusion:
1. The injury of IAN can influence the quality of newly callus through affecting the mineralization and remodeling of bone callus.
2. The expression of SP and CGRP in callus is dynamic, which is coherent with the healing process of fracture. It suggests that nervous system’s modulating the healing process of fracture may achieve by neuropeptides releasing from the nerve ending distributed in bone.
3. The involvement of CGRP in regulation of the healing process of fracture may achieve through affecting the function of osteoblasts and the function and differentiation of osteoclasts.
4. It may be one of the cell signal transductions of rabbit cultured osteoblast that the hCGRP’s binding to CRLR can activate AC and promote the accumulation of cAMP, as a result, PKA may be activated and stimulate phosphorylation and accumulation of ATF4, and at last induce osteoblast-specific gene expressioin.
5. In order to modulate the repair and remodeling of fracture, CGRP may affect the production of OPG and RNAKL in osteoblasts and accordingly influence the differentiation of osteoclasts.
我们在以往的研究中从组织学水平探讨了下牙槽神经(Inferior alveolar nerve IAN)对下颌骨骨折愈合的影响及其可能机制[6、7]。本研究通过建立失IAN和保留IAN兔下颌骨骨折动物模型,运用骨组织形态计量学(Bone histomorphometry)技术从细胞水平探讨IAN和CGRP对下颌骨骨折愈合的影响;通过原代培养兔颅骨成骨细胞,在分子水平上阐明hCGRP作用于兔成骨细胞的信号转导通路,以进一步探讨感觉神经调控骨折愈合的分子机制。
目的:探讨CGRP对兔下颌骨骨折愈合的影响及其作用于兔成骨细胞的胞内信号转导通路,以进一步探讨感觉神经调控骨折愈合的分子机制。
方法:选用中国大白兔105只,随机分为三组,实验组(35只)为给予LY303870(P物质受体拮抗剂)保留IAN下颌骨骨折组,对照组(70只)为失IAN下颌骨骨折组(对照组1,35只)和保留IAN下颌骨骨折组(对照组2,35只);分别在术后第7、14、21、28天处死动物,制取冰冻切片和未脱钙骨切片两种标本。冰冻切片用于HE染色观察骨痂愈合方式,免疫组化染色观察P物质(Substance P SP)和CGRP在新生骨痂中的表达;四环素双标的未脱钙骨切片用于Goldner’s Masson Trichrome Stain染色和骨组织形态计量学分析。用二次酶消化法原代培养兔颅骨成骨细胞,按hCGRP终浓度分为6组,第1组和第2组为对照组,分别为不含hCGRP的DMEM液和10-9mol/L hCGRP+10-6mol/L hCGRP(8-37)的DMEM液,第3、4、5和6组分别为含hCGRP终浓度为10-10mol/L、10-9mol/L、10-8mol/L和10-7mol/L的DMEM液。用激光扫描共聚焦显微镜技术监测胞内游离Ca2+浓度变化,放免法检测胞内cAMP浓度,凝胶迁移试验(Electrophoretic mobility shift assay EMSA)检测激活转录因子4(Activiting transcrigtion factor 4 ATF4)活性,Western Blot检测ATF4、骨钙素(Osteocalcin OC)、核因子-κB受体活化因子配体(Receptor activator of nuclear factor-κB ligand RANKL)和骨保护素(Osteoprotegerin OPG)的表达,Northern Blot检测OC、RANKL和OPG mRNA的表达。结果:1. SP和CGRP在新生骨痂中的表达:与保留神经组相比,在骨折愈合不同阶段SP和CGRP在失神经组骨痂中的表达显著减少(p<0.01)。保留神经组在术后第7天,SP和CGRP呈阳性表达;术后第14天,SP持续阳性表达,CGRP呈高表达并达峰值;术后第21天, SP表达减弱,CGRP仍呈阳性表达;术后第28天,SP表达显著增强,CGRP表达显著减弱。
2. CGRP对骨折愈合的影响:成骨细胞形成表面和成骨细胞指数在实验组和对照组中差异无统计学意义;实验组破骨细胞吸收表面和破骨细胞指数明显低于对照组1,对照组1明显低于对照组2,各组间差异有统计学意义;类骨质表面对照组1高于实验组和对照组2,差异有统计学意义,实验组和对照组2之间差异无统计学意义;矿化骨痂体积实验组高于对照组1和对照组2,实验组与对照组1之间差异有统计学意义,与对照组2差异无统计学意义;骨形成率实验组高于对照组1和对照组2,实验组与对照组1差异有统计学意义,实验组和对照组2之间差异无统计学意义。
3. hCGRP对兔成骨细胞内游离Ca2+浓度的影响:hCGRP不能改变兔成骨细胞内游离Ca2+浓度,各组间差异无统计学意义。
4. hCGRP对兔成骨细胞内cAMP浓度的影响:hCGRP可促进兔成骨细胞内cAMP浓度升高,并且呈剂量依赖性,在hCGRP终浓度为10-9mol/L时达到峰值,hCGRP(8-37)可以抑制其促进作用。
5. hCGRP对兔成骨细胞内ATF4活性的影响:hCGRP可增强兔成骨细胞内ATF4活性,并且呈剂量依赖性,在hCGRP10-9mol/L时达到峰值,hCGRP(8-37)可以抑制其促进作用。
6. hCGRP对兔成骨细胞内ATF4蛋白表达的影响:hCGRP可促进兔成骨细胞内ATF4蛋白表达,并且呈剂量依赖性,在hCGRP终浓度为10-9mol/L时达到峰值,hCGRP(8-37)可以抑制其促进作用。
7. hCGRP对兔成骨细胞内OC及其mRNA表达的影响:hCGRP可促进和上调兔成骨细胞内OC及其mRNA表达,并且呈剂量依赖性,在hCGRP10-9mol/L时达到峰值,hCGRP(8-37)可以抑制其促进作用。
8. hCGRP对兔成骨细胞内RANKL及其mRNA表达的影响:hCGRP可抑制和下调兔成骨细胞内RANKL及其mRNA表达,并且呈剂量依赖性,在hCGRP终浓度为10-9mol/L时抑制作用最为明显,hCGRP(8-37)可以阻断其抑制作用。
9. hCGRP对兔成骨细胞内OPG及其mRNA表达的影响:hCGRP可促进和上调兔成骨细胞内OPG及其mRNA表达,并且呈剂量依赖性,在hCGRP10-9mol/L时达到峰值,hCGRP(8-37)可以抑制其促进作用。
10.采用Pearson相关系数描述每两个指标之间(cAMP、ATF4、OC、OPG、RANKL和RANKL/OPG)的相关程度,cAMP与其它指标的相关系数在0.713～0.988之间,ATF4与其它指标的相关系数在0.777～0.975之间,OC与其它指标的相关系数在0.803～0.988之间, OPG与其它指标的相关系数在0.712～0.988之间;RANKL与其它指标的相关系数在0.777～0.982之间, RANKL/OPG与其它指标的相关系数在0.717～0.975之间。经相关系数的假设检验均有统计学意义(p<0.01)。可认为cAMP、ATF4、OC、OPG、RANKL和RANKL/OPG的变化趋势相同。
结论:1. IAN损伤影响骨痂愈合的质量,可能是通过影响矿化骨痂的形成和骨改建来实现;
2.骨折愈合过程中CGRP和SP在骨痂中表达是动态变化的,并且与骨折愈合过程相适应,提示神经系统对骨折愈合调控是通过分布在骨组织局部的末梢神经分泌神经肽实现的;
3. CGRP对骨折愈合的调控作用可能是通过影响成骨细胞功能及破骨细胞分化和功能实现的;
4. hCGRP对兔成骨细胞功能影响可能的细胞信号转导通路是:hCGRP与成骨细胞上特异性受体CRLR结合,激活腺苷酸环化酶活性,促使胞内第二信使cAMP生成,通过cAMP信号系统促使胞内ATF4积聚并使ATF4磷酸化,启动成骨细胞分化相关基因OC/RANKL/OPG的表达;
5. CGRP可以通过调控成骨细胞合成RANKL和OPG来影响破骨细胞分化,实现对骨折修复和改建的调控。
The nervous system involves in the regulation of the process of repair and remodeling of fracture and bone metabolism at different levels. The mechanism, of which systemic and local factors cooperate with nervous system, is quite complicated. However, the regulation achieves finally through the effect of neural signals on function of osteoblasts and osteoclasts in bone, which are released from the nerve terminals distributed in bone[1-5]. Calcitonin gene-related peptide (CGRP) secrected by sensory nerve has many kinds of physiological activities. Previous studies have found that CGRP is involved in repair and remodeling of fracture through influencing functions of osteoblasts, and little is known about the molecular mechanism and cell sigal transduction of CGRP’s modulating the healing process of fracture at the present time.
In our previous studies, we explored the effect of inferior alveolar nerve(IAN) amputation on the healing of mandibular fracture and its possible mechanism at the level of tissue[6,7]. To explore the molecular mechanism of the nervous system’s modulating fracture healing, in this study we will explore further the effect of CGRP on mandibular fracture healing at the level of cell with bone histomorphometry through the foundation of animal model of mandibular fracture with or without IAN amputation, and clarify cell signal transduction of osteoblasts at the level of molecule by the primary rabbit osteoblast cultures of neonatal calvaria.
Purpose
The aims of the study were to explore the possible mechanism of CGRP’s modulating the healing of rabbit mandibular fracture and cell signal transduction of rabbit cultured osteoblast acted by hCGRP, and so that to explore further the molecular mechanism of sensory nerve’s modulating fracture healing.
Methods
105 adult China white rabbits were randomly divided into two groups (experimental and control groups). They all suffered from a fracture in the left mandible. Animals in experimental group were injected by LY303870, an antagonist to Substance P receptor, without IAN amputation. The control group was divided into two groups, and half of them with or without IAN amputation. The rabbits were sacrified on 7, 14, 21 and 28 day after operation, and the specimens were collected and stained with hematoxylin-eosin and for immunochemistry to observe the process of fracture healing and the expression of SP and CGRP in bone calllus, and stained with Goldner’s Masson Trichrome stain and the double marks of tetracycline to analyse the newly callus quantitively with bone histomorphometry. Osteoblast cultures were isolated from newly-born rabbit calvaria by sequential enzymatic digestion. The cultured osteoblasts were divided into six groups according to the final concentration of CGRP. The control group was divided into two groups. One is DMEM without CGRP or CGRP(8-37), and the other is DMEM with 10-9mol/L CGRP and 10-6mol/L CGRP(8-37) together. The other four groups are DMEM with 10-10mol/L, 10-9mol/L, 10-8mol/L and 10-7mol/L CGRP respectively and without CGRP(8-37). The detection of cAMP accumulation in cultured cells was performed using radioimmunology assay and the change of cyto free Ca2+ concentration was monitored by laser scanning confocal microscopy. The activity of activiting transcription factor 4(ATF4) was measured in electrophoretic mobility shift assay, and the expression and their mRNA of ATF4, osteocalcin, receptor activator of nulear factor-κB ligand and osteoprotegin in cultured cells were detected by Western Blot and Northern Blot respectively.
Results:
The expression of SP and CGRP in cullus:
Immunoreactivities of SP in callus occurred strong in the group without IAN amputation on Day 7 after operation, and became stronger on Day 14 and less on Day 21 after operation, and became stronger again on Day 28 after operation. Immunoreactivities of CGRP in callus occurred strong in the group without IAN amptation, became the strongest on Day 14 and less stronger gradually on Day 21 and 28 after operation. Immonoreactivities of SP and CGRP in callus occurred weakly at early stage and became stronger at late one in the group with IAN amputation. There were significant differences in the expression of SP and CGRP in callus between two groups.
The effect of CGRP on the healing of mandibular fracture: there were no significant differences in ob?s/BS and ob?Nb/BPm among three groups. On the contrary, there were significant differences among three groups in oc?s.BS, N?oc/BS, BFR, OS and MLV by analysing the callus using bone morphometry.
The effect of hCGRP on free Ca2+ concentration in rabbit cultured osteoblasts: hCGRP can’t increase cyto free Ca2+ concentration in cultured osteoblasts. There were no signifficant differences among three groups.
The effect of hCGRP on cAMP concentration in rabbit cultured osteoblasts: hCGRP can stimulate the accumulation of cAMP in cultured osteoblasts. And it displayed a dosage-effect relationship, which can be blocked by CGRP(8-37).
The effect of hCGRP on ATF4 activity in cultured osteoblasts: hCGRP can promot the activities of ATF4 in cultured osteoblasts. It displayed a dosage-effect relationship, which can be blocked by CGRP(8-37).
The effect of hCGRP on the expression of ATF4 in cultured osteoblasts: hCGRP can enhance the expression of ATF4 in cultured osteoblasts. It displayed a dosage-effect relationship, which can be blocked by CGRP(8-37).
The effect of hCGRP on the expression of ATF4 in cultured osteoblasts: hCGRP can promote the expression of ATF4 in cultured osteoblasts. It displayed a dosage-effect relationship, which can be blocked by CGRP(8-37).
The effect of hCGRP on the expression of OPG and its mRNA in cultured osteoblasts: hCGRP can promote the expression of OPG and up-regulate its mRNA in cultured osteoblasts. It displayed a dosage-effect relationship, which can be blocked by CGRP(8-37).
The effect of hCGRP on the expression of RANKL and its mRNA in cultured osteoblasts: hCGRP can inhibit the expression of RANKL and down-regulate its mRNA in cultured osteoblasts. It displayed a dosage-effect relationship, which can be blocked by CGRP(8-37).
All the obtained data in vitro experiments were evaluated by the statistical analysis using Pearson’s correlation coefficient to describe the degree of corelation in each index. And there was significant difference in corelation among cAMP, ATF4, OC, OPG and RANKL.
Conlusion:
1. The injury of IAN can influence the quality of newly callus through affecting the mineralization and remodeling of bone callus.
2. The expression of SP and CGRP in callus is dynamic, which is coherent with the healing process of fracture. It suggests that nervous system’s modulating the healing process of fracture may achieve by neuropeptides releasing from the nerve ending distributed in bone.
3. The involvement of CGRP in regulation of the healing process of fracture may achieve through affecting the function of osteoblasts and the function and differentiation of osteoclasts.
4. It may be one of the cell signal transductions of rabbit cultured osteoblast that the hCGRP’s binding to CRLR can activate AC and promote the accumulation of cAMP, as a result, PKA may be activated and stimulate phosphorylation and accumulation of ATF4, and at last induce osteoblast-specific gene expressioin.
5. In order to modulate the repair and remodeling of fracture, CGRP may affect the production of OPG and RNAKL in osteoblasts and accordingly influence the differentiation of osteoclasts.
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