NMDA受体和NOS参与骨癌痛小鼠吗啡耐受的形成

NMDA receptor and NOS in morphine tolerance in mice with bone cancer

目的:制备骨癌痛模型,并在骨癌痛模型上模拟慢性吗啡耐受的产生,以探讨癌痛吗啡耐受的表现及机制。方法:选用40只成年雄性C57BL/6小鼠,其中20只接种Lewis肺癌细胞2×106个建立骨癌痛模型,于接种后的第15天将骨癌痛组再随机分为模型吗啡组与模型盐水组。另外20只正常小鼠完全随机分为正常吗啡组与正常盐水组。模型吗啡组和正常吗啡组小鼠皮下注射吗啡10 mg/kg,每天2次,连续7 d,建立慢性吗啡耐受模型,模型盐水组和正常盐水组按体质量给予等体积生理盐水。从给药的第1天开始,隔天上午给药后1 h采用机械触痛法测量小鼠的50%缩足反应阈值,以此机械触痛阈值作为疼痛指标。第7天处死小鼠分别作脊髓NMDA受体亚单位1型(NMDAR1)免疫组织化学检测和脊髓一氧化氮合酶(NOS)的定量测定。结果:第1,3,5,7天给药后1 h行为学测试结果发现,模型盐水组和正常盐水组小鼠整个过程中50%缩足反应阈值没有明显变化。模型吗啡组第1,3,5天50%缩足反应阈值与模型盐水组相比,差异有统计学意义(P<0.05);模型吗啡组第5天的50%缩足反应阈值与第1,3天比较明显变小(P<0.05);第7天模型吗啡组与模型盐水组相比,差异无统计学意义(P>0.05)。正常吗啡组第1,3,5天50%缩足反应阈值与正常盐水组相比,差异有统计学意义(P<0.05);第7天正常吗啡组与正常盐水组相比,差异无统计学意义(P>0.05)。模型吗啡组的NMDAR1染色灰度值与正常吗啡组、正常盐水组及模型盐水组相比,差异有统计学意义(P<0.05);模型盐水组的NMDAR1染色灰度值与正常盐水组相比,差异有统计学意义(P<0.05)。模型吗啡组、模型盐水组以及正常吗啡组的积分光密度(IOD)值分别与正常盐水组相比,差异均有统计学意义(P<0.05);模型吗啡组的IOD值与模型盐水组相比,差异有统计学意义(P<0.05)。模型吗啡组、模型盐水组以及正常吗啡组脊髓NOS含量与正常盐水组相比,差异均有统计学意义(P<0.05),模型吗啡组脊髓NOS含量与模型盐水组相比,差异有统计学意义(P<0.05)。结论:脊髓NMDA受体和NOS可能在癌痛模型吗啡耐受中起重要作用。

Objective To set up a mouse model with bone cancer to simulate the morphine tolerance and explore its mechanism.Methods Forty C57BL/6 male mice were divided into 4 groups： Group 1 and Group 2 were firstly set up as bone cancer pain models.Morphine（10 mg/kg） was sequentially administered subcutaneously twice daily in Group 1 and normal saline was administered in Group 2 as the control group.Similar to Group 1 and Group 2,morphine（10 mg/kg） was administered subcutaneously twice daily in Group 3 and normal saline was administered in Group 4 as the control group.To set up morphine tolerance model,we injected morphine continuously for 7 days.From Day 1 to Day 7 after the morphine injection,we measured the mice hind paw withdrawal threshold in the von Frey hair test every other day.NMDA receptor 1（NMDA1） and nitric oxide synthase（NOS） were measured on Day 7 after the morphine injection.Results The mice hind paw withdrawal threshold in the von Frey hair test in Group 1 increased on Day 1,3,and 5 after the morphine injection compared with the paw withdrawal threshold in Group 2 and had the same threshold as Group 2 on Day 7.The mice hind paw withdrawal threshold in the von Frey hair test in Group 3 increased on Day 1,3,and 5 after the morphine injection compared with the paw withdrawal threshold in Group 4 and had the same threshold as Group 4 on Day 7.The grey scales and integral optical density（IOD） of NMDAR1 and the level of NOS in the spinal dorsal horn were higher in Group 1,Group 2,and Group 3 compared with those in Group 4（P〈0.05 or P〈0.01）,and the grey scales and IOD of NMDAR1 in Group 2 was higher than that in Group 1（P〈0.05）.Conclusion NMDA receptors and NOS may play important roles in morphine tolerance in mice with bone cancer pain.