Department of Orthopaedic Surgery, First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China.
Chin Med J (Engl). 2011 Feb;124(4):586-90.
The cannabinoid receptor-2 (CB2) is important for bone remodeling. In this study, we investigated the effects of CB2 selective antagonist (AM630) on receptor activator of nuclear factor kappa B (RANK) ligand (RANKL) induced osteoclast differentiation and the underlying signaling pathway using a monocyte-macrophage cell line-RAW264.7.
RAW264.7 was cultured with RANKL for 6 days and then treated with AM630 for 24 hours. Mature osteoclasts were measured by tartrate-resistant acid phosphatase (TRAP) staining using a commercial kit. Total ribonucleic acid (RNA) was isolated and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was done to examine the expression of RANK, cathepsin K (CPK) and nuclear factor kappa B (NF-κB). The extracellular signal-regulated kinase (ERK), phosphorylation of ERK (P-ERK) and NF-κB production were tested by Western blotting. The effect of AM630 on RAW264.7 viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay.
AM630 did not affect the viability of RAW264.7. However, this CB2 selective antagonist markedly inhibited osteoclast formation and the inhibition rate was dose-dependent. The dose of ≥ 100 nmol/L could reduce TRAP positive cells to the levels that were significantly lower than the control. AM630 suppressed the expression of genes associated with osteoclast differentiation and activation, such as RANK and CPK. An analysis of a signaling pathway showed that AM630 inhibited the RANKL-induced activation of ERK, but not NF-κB.
AM630 could inhibit the osteoclastogenesis from RAW264.7 induced with RANKL.
大麻素受体 2（CB2）对于骨重塑很重要。在这项研究中，我们使用单核细胞-巨噬细胞系 RAW264.7 研究了 CB2 选择性拮抗剂（AM630）对核因子 kappa B 受体激活剂（RANK）配体（RANKL）诱导的破骨细胞分化的影响及其潜在的信号通路。
RAW264.7 用 RANKL 培养 6 天，然后用 AM630 处理 24 小时。通过使用商业试剂盒用抗酒石酸酸性磷酸酶（TRAP）染色来测量成熟的破骨细胞。分离总核糖核酸（RNA），并进行实时逆转录聚合酶链反应（RT-PCR）以检查 RANK、组织蛋白酶 K（CPK）和核因子 kappa B（NF-κB）的表达。通过 Western 印迹测试细胞外信号调节激酶（ERK）、ERK 的磷酸化（P-ERK）和 NF-κB 的产生。使用 3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑溴化物（MTT）测定法确定 AM630 对 RAW264.7 活力的影响。
AM630 不影响 RAW264.7 的活力。然而，这种 CB2 选择性拮抗剂显着抑制破骨细胞形成，且抑制作用呈剂量依赖性。剂量≥100nmol/L 可将 TRAP 阳性细胞减少至明显低于对照的水平。AM630 抑制与破骨细胞分化和激活相关的基因的表达，如 RANK 和 CPK。信号通路分析表明，AM630 抑制了 RANKL 诱导的 ERK 激活，但不抑制 NF-κB。
AM630 可抑制 RANKL 诱导的 RAW264.7 破骨细胞生成。
