Ⅱ型大麻素受体选择性抑制剂在钛颗粒诱导炎症性骨溶解中的作用

Role of cannabinoid receptor 2 selective antagonist in titanium particles-induced inflammatory osteolysis

目的观察Ⅱ型大麻素受体：（eannabinoid receptor 2, CB2）选择性抑制剂（AM630）对磨损颗粒诱导炎症性骨溶解的治疗作用。方法雌性cBALB／c小鼠45只，其中15只作为颅骨供体，其余采用随机数字表法分为空白组、对照组和药物治疗组，每组10只。采用钛颗粒诱导的小鼠air—pouch骨溶解模型，药物组建模前2d经腹膜下注射CB2抑制剂-AM630（200μg·kg-1·d-1），持续至建模后2周。小鼠处死取材，大体观察植入骨片周围炎症变化；HE染色观察细胞浸润程度；形态计量学检测囊壁厚度；抗酒石酸酸性磷酸酶（tartrate—resistant acid phosphatase staining, TRAP）染色检测成熟破骨细胞；定量RT—PCR检测CB2、IL-1β、TNF-α、核因子NF—KB配体的受体（receptor activator of NF - κB ligand, RANKL）和核因子NF—κB配体（receptor activator of NF—κB，RANK）mRNA含量。结果标本大体观察，对照组植入骨片周围有红肿、渗出等明显的炎症变化，药物治疗后，炎症改变明显减轻。HE染色结果表明，对照组囊壁内大量巨噬细胞浸润；空白组及药物组囊壁内细胞少，多为成纤维细胞；对照组囊壁较厚（192．2±19．4）μm，空白组和药物组囊壁厚度分别为（88．5±14．7）¨m、（122．1±15．2）Ixm，与对照组比较差异有统计学意义（P〈0．05）；对照组植入骨片周围TRAP深染区域较多，AM630治疗后深染区域明显减少；定量RT—PCR结果表明，钛颗粒刺激能够上调CB2、IL-1β、TNF-α、RANKL和RANKmRNA表达水平，药物组上述基因mRNA水平较低，与对照组比较差异有统计学意义（P〈0．05）。结论CB2选择性抑制剂能降低磨损颗粒引起的炎症反应，抑制破骨细胞活化。CB2有望成为治疗人工关节无菌性松动的新靶点。

Objective To explore the therapeutic effect of selective antagonist-AM630 of cannabionid receptor 2 （CB2） in treatment of the titanium particles-induced inflammatory osteolysis. Methods Forty-five female BALB/c mice, 6-8 weeks old, were involved in the study, of which 15 mice were used as skull donors and the rest experimental animals were randomly divided into three groups, ie, black group, control group and treatment group, 10 mice per group. The mice model with air-pouch osteolysis induced by the titanium particles were established. The mice in the treatment group were injected with CB2 selective antagonist-AM630 （200 μg·kg^-1·d^-1 ） intraperitoneally from two days before establishment of the air-pouch osteolysis model to two weeks after establishment of the model. Then, the mice were sacrificed and the pouch tissues were collected for molecular and histological analyses. The pouch membrane thickness and cell infiltration were tested by using computerized image analysis system and HE staining respectively. Osteoclast-like ceils in the pouch membrane were determined by using tartrate-resistant acid phosphatase （TRAP） staining. Quantitative real-time polynlerase chain reaction （RT-PCR） was employed to detect the mRNA levels of CB2, IL-1β, TNF-α, receptor activator of NF-κB ligand （[IANKI,） and receptor activator of NF-κB （RANK）. Results There exhibiled apparent erythematous and oedematous changes in the control group, which was mitigated around the bone implants with AM630 trcatment. Quantitative image analysis of the histological sections revealed significant difference of the pouch membrane thickness among three groups, （ 192.2±19.4） μm in control group, （ 88.5±14.7 ）μm in blank group and （ 122.1±15.2 ）μm in treatment group （ F = 101.74, P 〈 0.05 ）. Intensive TRAP staining was identified ,nueh in the control group but markedly reduced after AM630 treatment in the pouch tissues. RT-PCR showed thai titanium particle stimulation could enhance the expressions of CB2, IL-1β、TNF-α, RANKL and RANK gene in the air pouch tissues. However, the mRNA levels of these genes were markedly reduced after AM630 treatment, with statistical difference compared with control group （P 〈0.05 ）. Conclusions CB2 selective antagonisl AM630 can inhibit the process of titanium particlesstimulated inflammatory reaction and osteoelast activation. Therefore, CB2 represents a new suitable therapeutic candidate tot the prevention anti treatment of aseptic loosening of the artificial joint.