| BackgroundIt has been observed clinically that an overgrowth of callus and even neurogenic Heterotopic Ossification (HO) in the muscle may occur in a patient with a bone fracture combined with Traumatic Brain Injury(TBI).In these patients the process of bone healing were frequently promoted with a large quantity of callus. Many patients who got a sensory nerve injuried often have high rate of fracture nonunion and defect nonunion. According to the reserrch, fracture nonunion can be found in the rats with periosteum mechanical receptors removed, and the normal neurons can not be found in the area of fracture nonunion., which reveal that central nervous system can regulate the born healing and the normal bone metabolism. With the further exploration of the fracture healing mechanism of patients with fracture combined with central nerve injury.Some researches found that intrathecal administration of nerve growth factor can promote the healing of tibial shaft fracture in the rat, and proved that central nervous system can regulat the born healing through the pathway of CNS-neuropeptide-bone . It can be supposed that CNS regulate the osteoblast and osteoclast metabolism via neuropeptide from peripheral nerve. Calcitonin gene-related peptide (CGRP) is play a pretty significant role of the bone healing, as one of the most important neuroeptide.some reserchers reveal that CGRP affect the bone reconstruction by regulating the metabolism of osteoblas, but the mechanism isn't clear so far.Many studies have carefully investigated the interaction between osteoblasts and osteoclasts Osteoprotegerin(OPG), receptor activator of NF-kB (RANK) and RANK ligand play a critical role in bone remodeling by regulating the function of osteoclasts. OPG is a member of the TNFR family and a soluble decoy receptor competitive against RANKL and soluble RANKL (sRANKL). OPG produced by osteoblasts and other cells, has been found to be a key factor in the inhibition of differentiation and activation of osteoclasts. On the other hand, RANKL, a member of the TNF family,induces osteoclasts differentiation, maturation and activates mature osteoclasts through binding with their receptor RANK expressed on the surface. While sRANKL cleaved from RANKL behaves similarly to RANKL as a soluble factor. The increase of RANKL expression and RANKL release leads to bone absorption and loss. The expression of OPG and RANKL regulate activation of osteoclasts, and therefore deeply affects bone remodeling. The receptor activator of the NF-KB ligand (RANKL) is one of the key regulatory molecules in osteoclast formation and binds to OPG the ratio of OPG/RANKL in bone microenvironment is a key of osteoclast formation and differentiation.The calcitonin gene-related peptide(CGRP) is composed of 37 amino acids that are transcribed from the calcitonin(CT) gene by alternative splicing. CGRP is distributed throughout the central and peripheral nervous systems. In the peripheral nervous system, CGRP is found in both sensory and motor neurons and possesses multiple functions, such as neurotransmitter, vasodilator,and neuro trophic effector. Notwithstanding this, CGRP also takes part in the local regulation of bone growth and metabolism.CGRP has been previously influence proliferation of several cell types (eg.endothelial cells,Schwann cell,tracheal epithelial cells). More recently, it was demonstrated on gingival fibroblasts that CGRP mediates its mitogenic effect via activation of the mitogen-activated protein kinases (MAPK) pathway.MAPK plays a role in signal transduction associated with cell proliferation, differentiation and the production of cytokines. There are three well-characterized MAPK subfamilies in mammalian cells:extracellular-signal regulated protein kinase (ERK), the P38 mitogen-activated protein kinases(P38 MAPK) and the c. Jun N-terminal kinase(JNK).Previous studies have found that CGRP is involved in repairing 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.objective:To investigate the effect of calcitonin gene-related peptide(CGRP) on the proliferation and apoptosis of MG-63 cells by MTT and cell-counting analyzing the expression of OPG/RANKL and ERK through immunocytochemical methods and image analysis. To find the regulation mechanism between central nerve and non-neural system and to demonstrate the hypothesis based on the relationship between CGRP and ERK sigal transduction.Methods:1. The MG-63 cells come from orthopaedics laboratory of nanfang hospital.2. The MG-63 cells were revivalled and cultured in 37℃,5% CO2 incubator.3. In experiment, to study the effect of ERK inhibitor PD98059 and CGRP inhibitor CGRP8-37, two groups of MG-63 cells were treated with PD98059 and CGRP8-37 for 1 h prior to serum treatment. And the other four groups was cultured with serum from high doses, middle doses, low doses compared with contor group.4. Proliferation and apoptosis of MG-63 cells were observed by MTT and cell-counting. 5. The expression of the OPG/RANKL and ERK were observed through immunocytochemical methods and image analysis.6. All data are expressed as means±SD, and differences among groups were analyzed by one-way ANOVA followed by LSD test or Durmett test. A p value of <0.05 was considered statistically significant.Results1. CGRP increased MG-63 cells proliferationMTT experiment showed that CGRP could promote the proliferation of MG-63 cells and the effect was dose-dependent. The promotion effect was to the most after treated 48h at the 10-8 concentration level.2. Effect of CGRP8-37, PD98059 on CGRP-induced MG-63 cells proliferationMG-63 cells were pretreated for one hour with CGRP8-37, PD98059, followed by 24-hour CGRP treatment and MTT assay. The CGRP induced MG-63 cells activity was attenuated by the addition of CGRP 8-37, PD980593. CGRP induced expression and production of OPG,ERK expression was significantly increased by CGRP except RANKL The promotion effect was to the most at the concentration level.4. Effect of CGRP8-37, PD98059 on CGRP-induced the OPG,ERK expression and production of RANKL in MG-63 cells.MG-63 cell were treated with the antagonist of CGRP receptor(CGRP 8-37)or PD98059 at a concentration of 10-8 for 1 h prior to the addition of 10-8CGRP. Either CGRP 8-37 or PD98059 significantly inhibited the CGRP-increased OPG,ERK expression and its production except RANKL.Conclusions1. Exposure of MG-63 cells to different concentration level CGRP induced proliferation through the CGRP receptor and ERK pathway.CGRP8-37 and PD98059 can inhibite this effect.2. CGRP can upregulate the expression of OPG and downregulate he expression of RANKL.3. CGRP triggers the proliferation of MG-63 cells by CGRP receptor and ERK pathway to regulate the ratio of OPG/RANKL. |
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