鞘内注射NF-κ B抑制剂对骨癌痛大鼠的抗伤害效应

ANTI-NOCICEPTIVE EFFECT OF INTRATHECAL INJECTION OF NF-κ B INHIBITOR ON BONE CANCER PAIN IN RATS

目的：研究鞘内注射（it） NF-κ B活化抑制剂二硫代氨基甲酸吡咯烷（pyrrolidme dithiocarbamate,PDTC）对骨癌痛大鼠机械痛敏及脊髓NF-κB表达的影响,探讨NF-κ B在大鼠骨癌痛中的作用.方法：按照本实验室建立的大鼠胫骨癌痛模型建模.雌性SD大鼠96只,体重150～180 g,按照数字表随机化的原则将大鼠分为4组（n=24）：正常对照组（N组）、骨癌痛组（BP组）、生理盐水组（S组）和NF-κB抑制剂组（PDTC组）.BP组于大鼠左侧胫骨干骺端骨髓腔内注射5μlWalker256（1×10^5）乳腺癌细胞制备骨癌痛模型,N组仅左胫骨上端注入5 μl Hank＇s液.于接种后9d鞘内注射生理盐水10 μl或PDTC 20 μg/10μl（生理盐水溶解）,2次/d,共3d.于接种前、后2、4、6、9和12d、鞘内给药后1、3、6、12、24 h采用von Frey丝测定大鼠术侧后爪机械缩足反射阈值（paw withdrawal threshold,PWT）,PWT测定结束后处死大鼠,取L4～6脊髓,采用real time-PCR法测定NF-κ B p65 mRNA表达,免疫组化法测定NF-κ B p65蛋白表达.结果：接种后6dBP组大鼠的PWT值与基础值相比开始降低,且差异有统计学意义（P＜0.05）,随着接种时间的延长其下降更加显著.鞘内注射PDTC可显著提高骨癌痛大鼠的PWT值,且与没注射PDTC前PWT值比,差异有统计学意义（P＜0.05）.与接种后6d时相比较,骨癌痛大鼠脊髓NF-κ B p65 mRNA和蛋白质的表达在接种后9d （P＜0.01）和12d（P＜0.01）时均显著上调,而鞘内注射PDTC可显著降低大鼠脊髓NF-κ B p65 mRNA和蛋白质的表达（P＜0.01）.结论：鞘内注射PDTC可减轻大鼠骨癌痛引起的痛敏,NF-κ B的激活可诱导或促进大鼠的胫骨癌痛.

Objective ： To investigate the effect of intrathecal （i.t.） pyrrolidine dithiocar-bamate （PDTC, a NF- κ B inhibitor） on the mechanical hyperalgesia and the expression of NF- κ B in the dorsal horn of spinal cord in rats with bone cancer pain, and evaluate the role of NF- κ B in the mechanism of bone cancer pain. Methods： The rat tibial cancer pain model was established in our laboratory. 96 female SD rats weighing 150- 180 g were randomly divided into 4 groups （n = 24）： normal control group （group N）, bone cancer pain group （group BP）, saline group （group S） and NF- κ B inhibitor group （group PDTC）. Bone cancer was induced by intra-tibial inoculation of 1 × 10^5 Walker 256 breast cancer cells in group BP. Group N only received 5 μl Hank＇s liquid via injection through the upper left tibia. At day 9 after the inoculation, 10 μl normal saline or 20 μg/10 μl PDTC （dissolved in normal saline） was administered twice a day for three days. Paw withdrawal threshold （PWT） to mechanical stimulation was measured with yon Frey filaments beforeinoculation and 2, 4, 6, 9,12 d after inoculation as well as 1, 3, 6, 12, 24 h after intrathecal administration. Rats were sacrificed after PWT measurement and L4- 6 spinal cords were collected to determine the expression of NF- κ B p65 mRNA by real time-PCR and the expression of NF- κ B p65 protein by immunohistochemistry. Results： The PWT significantly decreased in group BP at day 6 after inoculation compared to baseline （P 〈 0.05）. It further decreased with the prolonging of inoculation. The PWT value significantly increased after intrathecal injection of PDTC compared to the value before the injection （P 〈 0.05）. Compared to 6 d after inoculation, the expression levels ofNF- κ B p65 mRNA and protein significantly increased at 9 d （P 〈 0.01） and 12 d （P 〈 0.01） after inoculation, indicating that intrathecal injection of PDTC could obviously inhibit the activation of NF- κ B p65 in the spinal cord induced by bone cancer pain （P 〈 0.01）. Conclusion： Intrathecal injection of PDTC can alleviate the mechanical hyperalgesia induced by bone cancer pain. Activation of NF- κ B may induce or contribute to bone cancer pain.