探讨CB2受体逆向激动剂JTE-907对哮喘模型大鼠中性粒细胞迁移的作用

Effects of CB2 Receptor Reverse Agonist JTE-907 on Neutrophil Migration in Asthmatic Rats

目的探讨CB2在哮喘中的作用,并通过观察JTE-907(CB2受体反向激动剂)对中性粒细胞(neutrophil,NEU)迁移影响来确定CB2受体活化的潜在治疗机制。方法将30只大鼠随机分为3个实验组:正常对照组,模型组和JTE-907组(JTE-907-L,腹腔注射20 mg/kg),每组10只大鼠。除正常对照组外,其他组大鼠建立NEU性哮喘模型。从大鼠、健康人类志愿者的外周血中分离NEU。采用Western blot分析NEU中CB2、CXC趋化因子受体1(CXC chemokine receptor 1,CXCR1)、CXCR2蛋白表达,酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)试剂盒检测支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)和外周血白细胞介素8(interleukin 8,IL-8)水平,Transwell迁移试验检测NEU迁移能力。采用支气管上皮细胞(BEAS-2B)和NEU共培养及跨上皮迁移模型,进一步探讨JTE-907对NEU的作用机制。结果JTE-907治疗能够改善NEU哮喘大鼠的支气管气道炎症,并减少BALF和外周血嗜酸性粒细胞和NEU的数量。此外,JTE-907治疗可显著提高NEU中CB2蛋白表达,降低CXCR1和CXCR2表达(P均<0.05)。同样,JTE-907治疗降低了外周血和BALF中IL-8的水平以及NEU迁移。体外实验中,JTE-907治疗以剂量依赖性方式减少NEU迁移,以及降低fMLP诱导NEU中p-磷脂酰肌醇3-激酶(phosphorylated phosphatidylinositol 3-kinase,p-PI3K)和蛋白激酶B(protein kinase B,Akt)表达水平增加,并降低了聚合F-肌动蛋白的表达。结论JTE-907对NEU哮喘大鼠有一定保护作用,其可能通过下调NEU内PI3K通路,减少哮喘NEU迁移。

Objective To explore the role of CB2 in asthma, and determine the potential therapeutic mechanism of CB2 receptor activation by observing the effects of JTE-907(CB2 receptor reverse agonist) on the migration of neutrophil(NEU). Methods A total of 30 rats were randomly divided into 3 experimental groups: normal control group, model group and JTE-907 group(intraperitoneal injection of 20 mg/kg) with 10 in each. Except for the normal control group, NEU asthma models were established at other two groups. NEU were isolated from the peripheral blood of the rats and healthy human volunteers. The protein expressions of CB2, CXC chemokine receptor 1(CXCR1), CXCR2 in NEU were analyzed by Western blot, bronchoalveolar lavage fluid(BALF) and the level of interleukin 8(IL-8) in peripheral blood were detected by enzyme-linked immunosorbent assay(ELISA), and the migration ability of NEU was detected by Transwell migration test. Further investigations on the mechanism of JTE-907 on NEU were carried out by bronchial epithelial cell(BEAS-2 B) and NEU co-culture and transepithelial migration model. Results JTE-907 treatment could improve bronchial airway inflammation in NEU asthmatic rats, and reduce the number of BALF and peripheral blood eosinophils and NEU. In addition, JTE-907 treatment could significantly increase the expression of CB2 protein in NEU, and reduce the expression of CXCR1 and CXCR2(all P<0.05). Similarly, JTE-907 treatment reduced the level of IL-8 in the peripheral blood and BALF as well as the migration of NEU. In vitro, JTE-907 treatment reduced the migration of NEU in a dose-dependent manner, decreased the expression of phosphorylated phosphatidylinositol 3-kinase(p-PI3 K) and protein kinase B(Akt) in NEU induced by fMLP, and decreased the expression of polymerized F-actin. Conclusion JTE-907 has a certain protective effects on NEU asthmatic rats. It may reduce the migration of asthmatic NEU by down regulating PI3 K pathway.