芬太尼对慢性吗啡耐受大鼠中脑导水管周围灰质μ受体与β-arrestin2表达的影响

Effect of fentanyl on expression of mu-receptor and β-arrestin 2 in periaqueductal gray of rats tolerant to morphine

目的观察芬太尼对慢性吗啡耐受大鼠中脑导水管周围灰质μ受体与β—arrestin2表达的影响。方法健康成年雄性SD大鼠40只，体重（230±20）g，随机分为5组（n=8）：NS组（对照组）：皮下注射生理盐水1ml／kg2次，间隔30min；M组（慢性吗啡耐受组）：皮下注射吗啡10mg／kg，30min后皮下注射生理盐水1ml／kg；MF1、MF2、MF3组：吗啡用药同M组，注射吗啡30min后分别皮下注射芬太尼3、6、12μg／kg。各实验组每日给药2次，连续9d，给药后进行痛阈测定。断头处死大鼠，取中脑导水管周围灰质组织（PAG），逆转录聚合酶链反应（RT—PCR）方法测定μ受体、p-arrestin2的mRNA表达，免疫印迹（Westernblot）方法测定斗受体、β—arrestin2的蛋白表达。结果注射吗啡后，M组大鼠痛阈值明显高于NS组（P〈0．01），连续给药9天后，痛阈值降至给药前水平。与M组比较，MF2组与MF3组大鼠痛阈值下降趋缓，第7、9天，MF2组与MF3组痛阈值均高于M组（P〈0．05或0．01），MF1组差异无统计学意义（P〉0．05）。与NS组比较，M组μ受体mRNA及其蛋白表达显著下调（P〈0．01）；与M组比较，MF2组和MF3组μ受体mRNA及其蛋白表达上调（P〈0．05或0．01），MF1组差异无统计学意义（P〉0．05）。M组β-arrestin2mRNA及其蛋白表达明显低于NS组（P〈0．01）；MF2组和MF3组β—arrestin2mRNA及其蛋白表达高于M组（P〈0．05或0．01），而MFl组差异无统计学意义（P〉0．05）。结论芬太尼6、12μg/kg通过上调慢性吗啡耐受大鼠PAG部位μ受体与IS-arrestin2表达，可增强吗啡镇痛作用，部分延缓慢性吗啡耐受产生。

Objective To investigate the effect of fentanyl upon the expression of mu-receptor and β-arrestin 2 in peri-aqueductal gray of morphine-tolerant rats. Methods Forty male SD rats weighing （230 ± 20） g were randomly divided into 5 groups of eight animals each： group NS, group M, group MF1, group MF2 and group MF3. Rats in group NS received only subcutaneous normal saline lml/kg twice a day for 9 consecutive days; group M received subcutaneous morphine 10 mg/kg followed by NS 1 ml/kg twice a day for 9 consecutive days; In groups MF1, MF2 and MF3, morphine 10 mg · kg-1 was injected subcutaneously followed by fentanyl 3, 6, 12p, g/kg respectively. All animals were sacrificed at Day 9 after measurement of pain threshold. Periaqueductal gray was removed for determination of the expression of mRNA （RT-PCR） and protein （Western-blot） of mu-receptor and β-arrestin 2. Results Compared with group NS, TFL of group M was significantly elevated after the first morphine injection （ P 〈 0. 01 ）. But TFL of group M returned to the baseline value after chronic morphine treatment. Compared with group M, TFL increased in groups MF2 and MF3 at Days 7 and 9 （ P 〈 0. 05 or 0. 01 ）. However, TFL of group MF1 was negative （ P 〉 0. 05 ）. The expression of mu-receptor mRNA and protein was significantly lower in group M than in group NS （P 〈 0. 01 ）. Compared with group M, the expressions of mu-receptor mRNA and protein were significantly elevated in group MF2 and MF3 （P 〈0. 05 or 0. 01 ） but there was no significant change in group MF1 （P 〉 0. 05）. The expression of β-arrestin 2 mRNA and protein significantly decreased in group M as compared with group NS （P 〈0. 01 ）. Compared with group M, the expressions of β- arrestin 2 mRNA and protein were significantly elevated in group MF2 and MF3 （ P 〈 0. 05 or 0. 01 ）, but there was no significant change in group MF1 （P 〉 0.05 ）. Conclusion Fentayl at 6 and 12μg/kg can partly inhibit morphine tolerance through an increased expression of mu-receptor and β-arrestin 2 in periaqueductal gray of morphine-tolerant rats.