腹膜腔给予SAHA通过上调大麻素受体1的表达改善大鼠骨癌痛的研究

Suberoylanilide hydroxamic acid promotes up-regulation of cannabinoid receptor 1 and attenuates cancer-induced bone pain of rats

目的:观察腹膜腔给予辛二酰苯胺异羟肟酸(suberoylanilide hydroxamic acid,SAHA)对于骨癌痛(cancer-induced bone pain,CIBP)模型大鼠脊髓背角内大麻素受体1(cannabinoid-like receptor 1,CB1R)表达的影响。方法:将健康雌性SD大鼠随机分为4组:Sham+saline组、CIBP 7 d+saline组、CIBP 14 d+saline组、CIBP 14d+SAHA组。后三组动物胫骨内注射Walker 256乳腺癌肿瘤细胞制作骨癌痛模型。CIBP+SAHA组术后第1 d开始每日连续腹膜腔给予50 mg/kg SAHA至第14 d。利用机械刺激法连续观察各组大鼠的痛行为变化。应用免疫组织化学染色和Western Blot方法观察大鼠腰膨大节段脊髓背角内CB1R的表达情况。结果:自术后第7 d开始,与假手术组相比,CIBP组大鼠机械性缩足阈值(paw withdrawal threshold,PWT)明显下降(P<0.01),这种变化一直持续到至少术后第14 d。从术后第1~14 d连续腹膜腔内给予SAHA能明显缓解大鼠机械性痛敏(P<0.05)。免疫荧光组织化学染色显示:CB1R主要表达于脊髓背角浅层,CIBP组大鼠脊髓内CB1R表达上调,而腹膜腔内给予SAHA后可进一步促进脊髓背角内CB1R的上调。Western Blot结果显示:与对照组相比,造模后第7d和14 d脊髓背角内CB1R表达上调(P<0.05)。与骨癌痛相比,从术后第1~14 d连续腹膜腔给予SAHA能明显促进脊髓背角内CB1R的表达(P<0.05)。结论:在骨癌痛状态下,大鼠脊髓背角内CB1R表达增加,可能与体内内源性镇痛系统的激活有关,但此时与对照组相比,上调的CB1R不足以发挥镇痛效果;而腹膜腔内给予SAHA后,脊髓背角内CB1R受体表达进一步上调,从而发挥其镇痛效果。

Objective ： To observe the effect of intraperitoneal administration of suberoylanilide hydroxamic acid （ SA- HA） on the cancer-induced bone pain （CIBP） by regulating the expression of cannabinoid receptor I （CB1R） in spinal dorsal horn （ SDH ） at the lumbar 4-5 level. Methods ： Female Sprague-Dawley rats were randomly divided into four groups： Sham ＋ saline group, CIBP 7 d ＋saline group, CIBP 14 d ＋ saline group, CIBP 14 d ＋ SAHA group. Walker 256 breast tumor cells were injected into the right tibia to establish a rat model of CIBP. Sham group received same amount of heat-killed tumor cells injection. In SAHA treatment group, SAHA with the dosage of 50 mg/kg was injected i. p. from post-operation day 1 to day 14. Von Frey filament test was used to investigate the mechanical allodynia, hnmuno- fluorescent histochemistry and Western Blot were applied to observe and analyze the expression level of cannabinoid recep- tor 1 in spinal dorsal horn at lumbar 4-5 segments ipsilateral to cancer inoculation. Results ： Compared with sham group, the paw withdrawal threshold （PWT） was significantly decreased on day 7 （P 〈0.01 ） and this decrease lasted to day 14 in CIBP group, and the mechanical allodynia was obviously attenuated by intraperitoneal, administration of SAHA. Immunofluorescent histochemical staining showed that the highest density of CB1R-immunopositive punctuates were observed in the superfacial layer of SDH . The expression level of CB1 in SDH had aroused after tumor cell injection compared with sham group. Western Blot results fuether revealed a significant difference between the CIBP group and sham group. The expression of CB1 in SDH of CIBP group was increased on post operational day 7 （P 〈 0.05） and more significantly upregulated on day 14 （ P 〈 0.05 ）, while intraperitoneal administration of SAHA greatly promoted this up-regulation （ P 〈 0.05）. Conclusions： In the pathological state of CIBP, the expression of CBI in SDH was increased, which might be associated with endogenous analgesic system. However the increased level of CBlfailed to exhibit the analgesic effect compared with sham group. While intraperitoneal SAHA could promote the increasing level of CBI, so as to perform its analgesic effect.