自噬在雷帕霉素逆转脊髓吗啡耐受形成机制研究

Effects of intrathecal rapamycin on the development of morphine tolerance

目的研究鞘内注射雷帕霉素对大鼠吗啡耐受形成的作用。方法选择鞘内置管成功的成年雄性SD大鼠32只,随机分为M组、C组、MR组和R组（n=8）,M组2次/d,连续7 d鞘内注射吗啡20μg;C组2次/d,连续7 d鞘内注射生理盐水;M R组2次/d,连续7 d鞘内注射吗啡20μg,并于第3天第2次注射吗啡同时鞘内注射雷帕霉素2.3μg,连续3 d;R组2次/d,连续7 d鞘内注射生理盐水,并于第3天第2次注射生理盐水前鞘内注射雷帕霉素2.3μg,连续3 d10于鞘内注射前及第1、3、5、7天,第2次鞘内注药后30 min,采用电子Von Frey测痛仪测定机械缩足反射阈值（M WT）,最后一次M WT测定结束后,随机取4只大鼠L4∽6脊髓背角,采用Western blot测定自噬标记蛋白LC3Ⅱ（LC3Ⅱ/LC3Ⅰ）及自噬调节信号相关蛋白Beclin-1的表达。结果与M组比较,MR组鞘内注射第3、5、7天后,MWT均升高（P〈0.05）,虽然MR组MWT有降低趋势,但在第7天M WT仅比第1天下降43%,表明第3天后雷帕霉素与吗啡合用能部分逆转吗啡耐受的形成。Western blot结果：与C组比较,M组、R组和MR组第7天脊髓背角Beclin-1、LC3Ⅱ与LC3Ⅱ/LC3Ⅰ表达均上调（P〈0.05）。与M组比较,R组和MR组第7天脊髓背角Beclin-1、LC3Ⅱ与LC3Ⅱ/LC3Ⅰ表达显著上调（P〈0.01）。免疫组织化学结果：与M组比较,第7天MR组脊髓背角Beclin-1表达平均光密度值明显升高（P〈0.01）。结论吗啡耐受开始形成时合用雷帕霉素可以部分逆转脊髓吗啡耐受的形成。

Objective To investigate the effects of intrathecal（ IT） rapamycin on the development of morphine tolerance. Methods 32 healthy male SD rats w ith intrathecal（ IT） catheter successfully placed w ere randomly divided into 4 groups（ n = 8） ： group Mw as given morphine 20 μg IT tw ice daily for 7 d; group C w as given normal saline（ NS） 20 μL IT tw ice daily for 7 d; group MR w as given morphine 20 μg IT tw ice daily for 7 d,and at the second injection from 3 d to 5 d,rapamycin 2. 3 μg w as added; group R w as given normal saline 20 μL IT tw ice daily for7 d,and at the second injection from 3 d to 5 d,rapamycin 2. 3 μg w as injected before the normal saline. Mechanical w ithdraw al thresholds（ MWT） to von Frey filament stimulation w ere measured before intrathecal morphine or NS w as given and at 30 min after second time of IT morphine or NS administration on 1,3,5,7 d. The rats w ere sacrificed after MWT measurement. The autophagy marker protein LC3Ⅱ（ or LC3 Ⅱ / LC3 Ⅰ） and autophagy signal pathw ay related protein Beclin-1 expression in spinal dorsal horn w as determined by Western blot. Results The analgesic effect of morphine decreased slow ly in group MR,but higher than that of group Mfrom 3 d to 7 d（ P〈0. 05）. Western blot results show ed that LC3Ⅱ,Beclin-1 expression and LC3 Ⅱ / LC3 Ⅰ ratio w ere up-regulated in group M,group MR and group R compared w ith those in group C（ P〈0. 05）. The LC3 Ⅱ,Beclin-1 expression and LC3 Ⅱ / LC3 Ⅰ ratio w ere higher in group MR and group R than those in group M（ P〈0. 01）. Immunohistochemistry results show ed that the optical density of Beclin-1 in group MR w as higher than that in group M（ P〈0. 01）. Conclusion Rapamycin could partly reverse the development of spinal morphine tolerance.