鞘内注射IL-18中和抗体对大鼠吗啡耐受形成的影响

Effect of intrathecal injection of anti-rat IL-18 antibody on the development of morphine tolerance in rats

目的探讨阻断脊髓IL-18对大鼠吗啡耐受形成的影响及可能机制。方法♂SD大鼠40只,随机均分为5组(n=8)。Ⅰ组为生理盐水对照组,Ⅱ组为吗啡耐受组,Ⅲ组为IgG对照组,Ⅳ组、Ⅴ组为IL-18中和抗体(0.4μg、4μg)处理组。所有大鼠均行鞘内置管,手术5 d后确定导管位置(记为第1天)。第3～9天,各组建立吗啡耐受模型并进行相应处理。第2、10天,测定各组大鼠热刺激缩足反应潜伏期(paw withdrawal thermal latency,PWTL)及尾静脉注射吗啡后30 min的PWTL,计算30 min时最大可能镇痛效应比值(%MPE)。第10天行为学测试后,取大鼠脊髓腰膨大,采用免疫印迹法(Western blot)检测ERK与p-ERK蛋白表达水平,免疫荧光法分析星形胶质细胞标记物GFAP表达水平。结果①第2天:各组大鼠给予吗啡后30 min%MPE差异无统计学意义(P>0.05)。第10天:给予吗啡30 min后,与Ⅰ组相比,其余4组%MPE均明显降低(P<0.05)。与Ⅱ组相比,Ⅲ组、Ⅳ组差异无统计学意义(P>0.05),Ⅴ组%MPE明显升高(P<0.05)。②Western blot结果显示,与Ⅰ组相比,其余各组p-ERK表达量均升高(P<0.05)。与Ⅱ组相比,Ⅲ组、Ⅳ组p-ERK表达水平无统计学意义(P>0.05),V组明显降低(P<0.05)。③免疫组织荧光染色结果显示,与Ⅰ组相比,Ⅱ、Ⅲ、Ⅳ组大鼠腰段脊髓背角GFAP阳性荧光明显增加。而与Ⅱ组相比,Ⅴ组大鼠腰段脊髓背角GFAP免疫荧光强度明显降低。结论 IL-18中和抗体可以缓解吗啡耐受的形成,该效应可能与其抑制脊髓ERK的磷酸化,缓解背角星形胶质细胞的活化有关。

Aim To investigate the effect of blocking interleukin-18 in the spinal cord on the development of morphine tolerance in rats and its possible underlying mechanisms. Methods Forty male Sprague Dawley （SD） rats were randomly divided into 5 groups （n = 8） ： saline control group （group Ⅰ ）, morphine tolerance group （group Ⅱ ）, IgG control group （group Ⅲ）, anti-rat IL-18 antibody treatment group （group Ⅳ ： 0.4 μg and group V ： 4 μg）. Firstly all rats were implanted with intrathecal （ i. t. ） catheters. Then they were tested to ensure the position of catheters five days after surgery （recorded as the first day）. Tolerance to morphine antinociceptive effect was induced and drugs were injected from day 3 to day 9. On day 2 and 10, paw withdrawal thermal latency （PWTL）of the basic and 30 min after morphine injection via tail vein were evaluated in all rats and the percentage of maximal possible antinociceptive effect （% MPE ） of 30 rain was calculated. Lastly the spinal cord lumbar enlargement was obtained immediately after the behavioral tests on day 10 to determine the expression of ERK and p-ERK by western bolt and the immunoreactivity of GFAP by immunofluorescence. Results ① On day 2 there was no significant difference of the % MPE among five groups （P 〉0. 05）. On day 10, compared with group Ⅰ, the % MPE was declined significantly in group Ⅱ- Ⅴ. Compared with group Ⅱ, the % MPE was increased significantly in groupⅤ （ P 〈 0. 05 ） while no marked difference in group Ⅲ and Ⅵ（ P 〉 0. 05 ）. ② Compared with group Ⅰ, the expression of p-ERK was increased significantly in group Ⅱ- Ⅴ. Compared with group Ⅱ , the expression of p-ERK was decreased significantly in group V while no marked difference in group m and Ⅳ. ③ Compared with group Ⅰ , the immunoreactivity of GFAP was markedly increased in group Ⅱ, Ⅲ and Ⅳ. Compared with group Ⅱ, the immunoreactivity was decreased significantly in group Ⅴ. Conclusion Anti-rat IL-18 antibody can attenuate the development of morphine tolerance in naive rats through inhibiting the phosphorylation of ERK and activation of astroeytes in the spinal cord.