大麻素受体-1拮抗剂抑制炎性痛大鼠的吗啡耐受效应

Cannabinoid receptor-1 antagonist inhibits morphine tolerance in rats with inflammatory pain

目的观察吗啡对炎性痛治疗时产生耐受效应的模型大鼠脊髓背角大麻素受体-1(CB1)蛋白表达变化,探讨CB1受体拮抗剂(AM251)对炎性痛大鼠吗啡耐受效应的干预作用。方法 40只成功行鞘内置管术的成年雄性Sprague-Dawley大鼠,随机分入对照组、0.9%氯化钠溶液+完全弗氏佐剂(CFA)组、吗啡+CFA组、吗啡+AM251+CFA组,每组10只。对照组大鼠于右后足底皮下注射0.9%氯化钠溶液100μL,其余3组大鼠于右后足底皮下注射CFA100μL,建立CFA模型。于置管后第8天(CFA模型建立后第4天),对照组和0.9%氯化钠溶液+CFA组予0.9%氯化钠溶液10μL;吗啡+CFA组鞘内予吗啡10μg/10μL;吗啡+AM251+CFA组鞘内联合予吗啡10μg+AM2515nmol,共10μL。连续给药7d。于置管后第15天(CFA模型建立后给药第8天)将大鼠处死。采用热刺激缩足潜伏期(PWTL)评价痛行为学,采用免疫蛋白印迹法测定大鼠L2至L4脊髓背角组织中CB1的表达。结果 0.9%氯化钠溶液+CFA组、吗啡+CFA组和吗啡+AM251+CFA组CFA模型建立后鞘内给药前的PWTL值均较同组CFA模型建立前显著降低(P值均<0.001)。0.9%氯化钠溶液+CFA组CFA模型建立后鞘内给药第1、3、5、7天的PWTL值的差异无统计学意义(P值均>0.05);吗啡+CFA组在CFA模型建立后鞘内给药第1、3、5天的PWTL值显著高于0.9%氯化钠溶液+CFA组同时间点(P值均<0.001),且呈逐渐下降趋势,到CFA模型建立后鞘内给药第7天与0.9%氯化钠溶液+CFA组的差异无统计学意义(P>0.05)。吗啡+AM251+CFA组在CFA模型建立后鞘内给药第1、3、5、7天的PWTL值仍维持在较高水平,但各时间点间的差异均无统计学意义(P值均>0.05)。CFA模型建立后鞘内给药第7天,在吗啡+CFA组大鼠脊髓背角中的CB1相对表达量显著高于对照组同时间点(P<0.001)及0.9%氯化钠溶液+CFA组同时间点(P<0.05)。结论 CB1拮抗剂AM251可抑制吗啡耐受形成。

Objective To observe changes of cannabinoid receptor-1 （CB1） protein expression in spinal dorsal horn of morphine tolerant rats with inflammatory pain so as to explore the effect of CB1 antagonist in the process of morphine tolerance. Methods Forty male adult Spragueawley rats with intrathecal catheters implanted successfully were randomly divided into four groups（n = 10）. In control group, 100 μL normal saline were injected by right metapodium and 3 days later 10μL saline were given again. In CFA group, 100μ L complete Freund adjuvant （CFA） were injected by right metapodium and 3 days later 10 μL saline were given. In Mor group, 100 μL CFA were injected by right metapodium and 3 days later 10μL morphine were given. In AM251 group, 100 μL CFA were injected by right metapodium and 3 days later 10 pg morphine and 5 nmol（10 pL） AM251 were given for 7 d. Then the rats were sacrificed at the 8th day. Heat hyperalgesia was evaluated by paw withdrawal thermal latency （PWTL）. The expression of CB1 in spinal L2 -- L4 was detected by Western blotting. Results The PWTL after CFA administration were significantly lower than before CFA administration in the CFA group, Mor group and AM251 group （all P〈0.001）. There was no significant difference in PWTL 1, 3, 5, or 7 d after CFA administration in the CFA group （all P〈0.05）. The PWTL in Mor group was significantly higher than that in CFA group 1, 2,3 and 5 d after CFA administration （all P〈0. 001）, and the difference was not statistically significanton day 7 after CFA administration between the two groups （P〈0.05）. The PWTL in the AM251 group maintained at a high level on day 1, 3, 5, and 7 after CFA administration and the differences of PWTL at each time point were not statistically significant （all P〈0.05）. The expression of CB1 in Mer group was significantly higher than those in control group （P〈0. 001） and CFA group （P〈0.05） after development of morphine tolerance. Conclusion CB1 antagonist AM251 can inhibit the development of morphine tolerance.