Pharmacological Study on Neuronal Regulation of Osteoclast Formation

在本研究中，为了证明交感神经在活体骨代谢中的生理作用，我们用RT-PCR分析了束缚应激(30min)和脑室内(i.c.v.)注射IPS(50 ng/只)对小鼠颅骨COX-2和IL-6mRNA表达的影响LP(I.C.V.)众所周知，它会增加外周交感神经系统的流出。神经毒素6-羟基多巴胺(6-OHDA100 mg/kg/d，ip，连续3d)或β阻滞剂心得安(25 mg/kg，ip)可增加COX-2和IL-6mRNAs的表达。同样，束缚应激可诱导小鼠颅骨中IL-6mRNA的表达。这种诱导作用不受6-OHDA的影响，但可被心得安抑制。此外，异丙肾上腺素(isp；100μM)处理颅骨可增加器官培养系统中前列腺素E_2和IL-6的合成。这些发现表明，束缚应激和i.c.v增加了基因表达。脂多糖注射介导的…更多的是通过激活小鼠颅骨中的交感神经纤维和β肾上腺素受体，并表明在体内交感神经系统的激活调节骨骼代谢。其次，在小鼠骨髓培养体系中，阐明了isp对破骨细胞形成的作用方式，并研究了降钙素基因相关肽(CGRP)对isp诱导破骨细胞形成的影响。小鼠骨髓细胞经异丙肾上腺素处理后，可产生抗酒石酸酸性磷酸酶阳性的多核细胞，能够在牙本质切片上挖掘吸收陷窝，并导致骨髓细胞核因子-骨保护素B受体激活剂(RANKL)的增加和骨保护素(κ)的产生减少。OPG明显抑制破骨细胞的形成，提示RANKL-RANK系统参与了破骨细胞的形成。CGRP可抑制isp或可溶性RANKL诱导的破骨细胞的形成，但对isp处理的骨髓细胞产生的RANKL或OPG无影响，提示CGRP通过干扰经isp处理的骨髓细胞产生的RANKL的作用而抑制破骨细胞的形成，而不影响RANKL或OPG的产生。由于编码降钙素受体样受体的mRNA已被报道在成熟的破骨细胞前体、前破骨细胞和成熟的破骨细胞中表达。这一体外数据提示交感神经和感觉神经在体内破骨细胞形成过程中的生理相互作用。较少

In the present study, to prove the physiological role of sympathetic nerves in bone metabolism in vivo, we examined by RT-PCR analysis the effects of a restraint stress (30 min) and intracerebroventricular (i.c.v.) injection of IPS (50 ng/mouse) on COX-2 and IL-6 mRNAs expression in mouse calvaria. LPS (i.c.v.) is well known to increase the outflow of the peripheral sympathetic nervous system. The expression of COX-2 and IL-6 mRNAs were increased by the treatment with the neurotoxin 6-hydroxydopamine (6-OHDA, 100 mg/kg/day, i.p., for 3 days) or β-blocker, propranolol (25 mg/kg, i.p.). Similarly, a restraint stress induced the expression of IL-6 mRNA in mouse calvaria. The induction was not influenced by 6-OHDA, but inhibited by propranolol. In addition, the treatment of calvaria with isoprenaline (Isp ; 100 μM) increased PGE2 and IL-6 synthesis in the organ culture system. These findings show that gene expressions increased by a restraint stress and i.c.v. injection of LPS was mediated … More by the activation of sympathetic nerve fibers and β-adrenoceptor in mouse calvaria and suggests that in vivo activation of the sympathetic nervous system modulates bone metabolism. Secondly, to elucidate the mode of action of Isp on osteoclast formation and to characterize the effect of the calcitonin gene-related peptide (CGRP) on osteoclast formation induced by Isp in a mouse bone marrow culture system. Treatment of mouse bone marrow cells with Isp generated tartrate-resistant acid phosphatase-positive multinuclear cells capable of excavating resorptive pits on dentine slices, and caused an increase in receptor activator of NF-κB ligand (RANKL) and a decrease in osteoprotegerin (OPG) production by the marrow cells. The osteoclast formation was significantly inhibited by OPG, suggesting the involvement of the RANKL-RANK system. CGRP inhibited the osteoclast formation caused by Isp or soluble RANKL but had no influence on RANKL or OPG production by the bone marrow cells treated with Isp, suggesting that CGRP inhibited the osteoclast formation by interfering with the action of RANKL produced by the Isp-treated bone marrow cells without affecting RANKL or OPG production. Since the mRNA encoding the calcitonin receptor-like receptor has been reported to express in mature osteoclast precursors, pre-osteoclasts, and mature osteoclast. This in vitro data suggest the physiological interaction of sympathetic and sensory nerves in osteoclastogenesis in vivo. Less