Ⅱ型大麻素受体选择性抑制剂对钛颗粒诱导破骨细胞活化的作用

Inhibitory effects of AM630 on inflammatory osteolysis induced by titanium particles

目的观察Ⅱ型大麻素受体（CB2）选择性抑制剂AM630对钛颗粒（Ti）诱导炎性破骨细胞（OC）活化的影响。方法实验分5组，即空白组、核因子-κB（NF-κB）受体活化因子配体（RANKL）组、Ti组、R＋Ti组、药物组。采用噻唑蓝（MTT）法检测0．1g／LTi颗粒和不同浓度AM630（50、100、200nmol／L）处理小鼠单核／巨噬细胞株RAW264．7后24、48、72h细胞增殖活性；以0．1g／LTi颗粒和（或）50μg／LRANKL诱导RAW264．7，6d时加入AM630再培养24h，以抗酒石酸酸性磷酸酶（TRAP）染色检测成熟OC，以逆转录-聚合酶链反应（RT-PCR）检测CB2、NF-κB受体活化因子（RANK）、肌酸磷酸激酶（CPK）基因mRNA含量，酶联免疫吸附试验（EusA）检测炎性因子白细胞介素（IL）-1β和肿瘤坏死因子（TNF）-α表达水平。结果MTF结果表明0．1g／LTi颗粒和（或）AM630（50、100、200nmol／L）对RAW264．7细胞增殖能力无影响。TRAP染色，RANKL组、R＋Ti颗粒组均可见大量的紫红色多核细胞；Ti组、药物组阳性细胞较少，统计分析结果表明AM630≥100nmol／L时，TRAP阳性细胞数量与RANKL组[（18L80±14．23）／孔]比较差异有统计学意义（P〈0．05）。定量RT-PCR结果显示R＋Ti组CB2、RANK以及CPKmRNA含量分别为11．26±1．39、9．68±0．91和9．93±0．56；药物组（100nmol／LAM630）上述基因mRNA含量为4．73±0．55、3．85±0．45和5．42±0．87，差异有统计学意义（P〈0．05）。ELISA结果显示，Ti颗粒加入24h后IL-1β、TNF-α的含量分别为（176．9±9．2）、（159．7±8．2）ng/L；与药物组[IL-1β：（105．5±7．0）ng／L，TNF-α：（75．9±8．1）．g／L]比较，差异有统计学意义（P〈0．05）。结论Ⅱ型大麻素受体选择性抑制剂AM630能够抑制Ti颗粒引起的炎性破骨细胞活化。’

Objective To explore the effect of cannabinoid receptor 2 （CB2） selective antagonist （AM630） on the regulation of osteoclast differentiation from a murine macrophage cell line （ RAW264. 7 ） stimulated with titanium .（Ti） particles and the receptor activator of nuclear factor-KB （NF-κB） ligand （RANKL）. Methods The effect of AM630 and Ti particles on RAW264. 7 cell viability was examined by using the methyl thiazol tetrazolium （MTT） assay. Osteoclast formation was measured by tartrate resistant acid phosphatase （TRAP） staining using a commercial kit. The mRNA levels of CB2, creatine phosphokinase（CPK） and NF-κB receptor activation factor（RANK） were detected by using quantitative real-time reverse transcription-polymerase chain reaction （RT-PCR）. Enzyme-linked immunosorbent assay （ELISA） was performed to determine interleukin （IL）-1β and tumor necrosis factor （TNF）-or. Results AM630 （0- 200 nmol/L） did not affect viability of RAW264. 7 cells cultured with Ti particles. The mature osteoclasts were obtained from RAW264. 7 cells stimulated with RANKL and Ti particles for 6 days and detected by TRAP staining. AM630 at a doses of ≥100 nmol/L significantly reduced the number of TRAP-positive cells as compared with controls. After culture for 6 days, mRNA levels of CB2, RANK and CPK were significantly higher in the media with RAN KL and Ti particles than in those without RANKL and Ti particles. When 100 nmol/L AM630 in combination with RANKL and Ti particles was added to the culture system, mRNA levels of CB2, RANK and CPK were significantly decreased. The concentrations of IL-1β and TNF-α in RAW264. 7 cell cultures were measured by using ELISA. After culture for 24 h, IL1β and TNF-α concen- trations were significantly increased in the media with Ti particles as compared with those in the media withou Ti particles. The cytokine concentration was further increased with culture time. Conclusion AM630 could significantly inhibit Ti particle-induced inflammatory osteoclastogenesis.