鞘内注射吗啡诱导脊髓背角自噬状态的改变

Intrathecal morphine induces alteration of autophagy in spinal dorsal horn

目的研究鞘内注射吗啡诱导吗啡耐受过程中大鼠脊髓背角自噬状态的变化。方法选择鞘内置管成功的成年雄性SD大鼠48只,随机分为M组和C组,每组24只。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。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VonFrey测痛仪测定机械缩足反射阈值,然后随机取6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。结果鞘内注射吗啡7 d后,吗啡镇痛作用较第1天下降60.2%,提示出现吗啡耐受形成。C组各时点LC3Ⅱ及Beclin-1均有少量表达,各时点比较差异无统计学意义;与C组比较,M组各时间点脊髓背角Beclin-1、LC3Ⅱ表达量及LC3Ⅱ/LC3Ⅰ比值均显著上调(P<0.05);M组内不同时点比较,第1天LC3Ⅱ/LC3Ⅰ比值最高(P<0.05),随后逐渐下降;第1天Beclin-1表达最高(P<0.05),随后下降,在第5天后表达量趋于稳定。结论鞘内注射吗啡可诱导脊髓背角自噬增加,Beclin-1可能参与鞘内注射吗啡诱导所致的自噬状态改变。

Objective To investigate the alteration of autophagy in spinal dorsal horn in rat model of chronic mor- phine tolerance. Methods Forty - eight healthy male SD rats with intrathecal （IT） catheter successfully placed were ran- domly divided into 2 groups （n = 24） ： Group M （morphine 20 p,g IT twice a day for 7 days） and Group C [ normal saline （NS） 20 pLL IT twice a day for 7 days]. Mechanical withdrawal thresholds （MWT） to yon Frey filament stimulation were measured before intrathecal morphine/NS was given and 30 rain after the second administration on each 1,3, 5, and 7 d. The autophagy marker protein LC3 Ⅱ （ LC3 Ⅱ/LC3 I ） and autophagy signal pathway related protein Beclin - 1 expression in spinal dorsal horn was determined by Western blot. Results Morphine analgesic effect was significantly weakened after 7 days treatment of morphine, which indicated that morphine tolerance was formed. Stable low expression of LC3 Ⅱ and Beclin - 1 in spinal dorsal horn was observed in Group C. However, LC3 Ⅱand Beclin - 1 expression, and LC3 Ⅱ/LC3 I ratio were significantly up - regulated in Group M compared with those in Group C at each time points （ P 〈 0. 05 ）. In Group M, the LC3 Ⅱ/LC3 I ratio was significantly higher on 1 d than that on 3 d, 5 d or 7 d, with gradual reduction of LC3 Ⅱ/LC3 I ratio from 1 d to 7 days. In Group M, significantly highest Beclin - 1 expression was found on 1 d （P 〈 O. 05 ）, and decreased with time - dependent manner that stabilized on 5 d. Conclusion Intratheeal morphine induces increase of autophagy in spinal dorsal horn, to which Beclin - 1 may contribute.