| In the peripheral nervous system,calcitonin gene-related peptide(CGRP) was found in both sensory and motor neurons and possesses multiple functions,such as neurotransmitter, vasodilator,and neurotrophic effects.Additionally,CGRP also takes part in the local regulation of bone growth and metabolism.Previous studies have found that the CGRP can increase the proliferation and differentiation of osteoblasts,and improve bone fracture healing or bone metabolism,but the detailed regulatory mechanism of CGRP in the cells or callus has not been explored.Nitric oxide(NO),as a secondary messenger molecule is involved in many biological pathways of fracture healing such as inflammation, vascularization and cytoaction of callus.It was found that CGRP and NO have alike biological action and influence each other in peripheral circulations vasodilation.Therefore, the aim of our study were to explore the possible regulatory mechanism of CGRP and NO through detecting the expression and activity of NOS in osteoblasts in vitro,and changes of the CGRP expression in the callus via an animal model of denervation in mandibular fracture.Methods:Animal experiment:To determine the effect of CGRP on bony callus in vivo,a fracture in the left mandible was created in forty-eight adult rabbits(divided into experimental and control groups randomly) and half of them with or without inferior alveolar nerve(IAN) amputation.The bony callus were collected 4,7,14,and 28 days after operations,and the expressions of CGRP and NOS in the paraffin slices were analyzed with immunohistochemical staining and Western blot,and the activity of endothelial nitric oxide synthase(eNOS) and inducible nitric oxide synthase(iNOS) in the fresh specimens were measured with the NOS detecting kit.The difference of expression and activity among experimental and control groups was analyzed by SPSS 10.0 system for windows.Cell culture:To determine the effect of CGRP on osteoblasts in vitro,osteoblasts of rabbits cultured to the fourth generation were treated with CGRP as follows:control group (group 1),treated groups(10-7-10-9 mol/L CGRP,group 2-5).After0.5,1,4,12 and 24 hours of incubation,the nitrate reductase method was applied to detect nitric oxide(NO) concentrations in cell supernatant,the NOS detecting kit was applied to detect the activity of NOS in cells,and Western blot was applied to detect the expression of eNOS and iNOS in cells.Results:In the immunohistochemical analysis of bony callus,the expression of CGRP was lower in experimental groups than control groups from 4 to 14 days(p＜0.05 or p＜0.01),and the expression of eNOS showed the same phenomena from 4 to 7 days,but the expression of iNOS didn't show any statistical difference between these tow groups.In NOS activity analysis,the activity of eNOS was lower in experimental groups than control groups from 4 to 7 days(p＜0.05 or p＜0.01),and the activity of iNOS showed the same phenomena from 7 to 14 days.In the in-vitro test,the NO concentration was higher in CGRP treated groups than the control group from 0.5 to 4 hours(p＜0.05 or p＜0.01),and the activity of eNOS in osteoblasts was higher in treated groups than the control group from 0.5 to 4 hours(p＜0.05 or p＜0.01),but we didn't find any difference of iNOS activity between these two groups at any observation point.At 12 hours the expression of eNOS was higher in treated groups than the control group(p＜0.05 or p＜0.01),but we didn't find any difference of iNOS expression between these groups.Conclusion:(1) In our study,IAN amputation obvioursly changed the expression and distribution of CGRP in the mandibular callus.The expression level of CGRP in callus can affect the volume and quality of bone callus directly.IAN transection contribute to deplete of CGRP ecpression which lead to defect of callus microstructure.(2) CGRP has a positive effect on the activity of eNOS and increases the production of NO in osteoblasts.(3) CGRP takes part in improving the fracture healing via regulating the expression and activity of NOS.
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