鞘内联合给予T10与MK-801对神经病理性痛模型大鼠脊髓背角NR1与CREB蛋白磷酸化的影响  

The impacts of intrathecal administration of triptolide and MK-801 on NR2B subunit and CREB phosphorylation in spinal dorsal horn of neuropathic pain model rats

作　　者：王健 WANG Jian(Ambulatorium in 94750 Army Hospital of PLA,Longyan 366200,China)

机构地区：[1]中国人民解放军94750部队医院门诊所

出　　处：《延安大学学报（医学科学版）》2019年第2期1-6,共6页Journal of Yan'an University:Medical Science Edition

摘　　要：目的 研究鞘内联合给予雷公藤内酯醇(triptolide,T10)与MK-801对神经病理性痛模型大鼠痛行为及脊髓背角N-甲基-D-门冬氨酸(N-methyl-D-aspartic acid,NMDA)受体NR2B亚单位与环磷腺苷效应原件结合蛋白(cAMP-response element binding protein,CREB)磷酸化水平的影响。方法 选取健康雄性SD大鼠36只,随机分为6组:正常对照组(normal),假手术-生理盐水组(sham-saline),L5脊神经结扎(spinal nerve ligation;SNL)-生理盐水组(SNL-saline),SNL-T10单纯给药组(SNL-T10),SNL-MK-801单纯给药组(SNL-MK-801),SNL-T10+MK-801联合给药组(SNL-T10+MK-801)。模型组和给药组均采用L5SNL构建慢性神经病理性痛模型,生理盐水、T10(10μg/kg)和MK-801(30μg/kg)从术后第1d连续鞘内注射至第7d,1次/d。利用Von-Frey丝刺激法连续观察造模后大鼠的机械性缩足阈值(paw withdrawl threshold,PWT);应用免疫荧光组织化学染色方法观察腰膨大节段脊髓背角内pCREB蛋白的表达;Western blot方法定量检测NR2B和CREB的磷酸化水平。结果 术后第1d起,SNL组大鼠术侧后爪PWT明显下降(P<0.01),且在3d后基本保持平稳;鞘内单独给予T10或MK-801干预后第7d术侧后爪的MWT明显提高,与给药前相比有显著性差异(P<0.05);SNL-T10+MK-801联合给药组大鼠第7d术侧后爪的PWT提高幅度较SNL-T10和MK-801组明显,与给药前相比差异更加显著(P<0.01);停止给药后,SNL-T10+MK-801联合给药组镇痛疗效较SNL-T10以及MK-801组持续时间长。免疫荧光组织化学染色显示:pCREB主要表达于神经元内。Western blot检测结果显示:与对照组相比,SNL后第7d脊髓背角内pCREB蛋白的表达明显上调,SNL-T10+MK-801联合给药组大鼠脊髓背角内pNR2B和pCREB蛋白的表达水平要依次低于单独SNL-T10、SNL-MK-801给药组和SNL-saline组。结论 鞘内联合给予T10和MK-801能够有效缓解神经病理性痛模型大鼠的机械性痛行为,并且降低了二者的给药剂量,其机制可能与抑制脊髓背角神经元Objective To investigate the effects of intrathecal triptolide combined with MK-801 on the neuropathic pain modal rats by inhibiting the expression of spinal neuronal N-methyl-D-aspartic acid (NMDA) receptor 2B subunit and cAMP response element bound protein (CREB) phosphorylation after spinal nerve ligation (SNL). Methods 36 rats were divided into 6 groups randomly:Normal control group (normal),sham-saline group(sham-saline),L5 spinal nerve ligation-saline group (SNL-saline),SNL-triptolide group (SNL-T10),SNL-MK-801 group (SNL-MK-801),and co-administration of T10 and MK-801 group (SNL-T10+MK-801) after SNL. The neuropathic pain modal rats were set up by L5 spinl nerve ligation both in modal group and administration group.Saline,Triptolide (10 μg/kg) and MK-801 (30 μg/kg) was injected intrathecally once a day starting from the post operation day 1 until the post operation day 7.Von-Frey filament was used to investigate the paw withdrawal threshold (PWT),immunofluorescent histochemistry were applied to locate the expression of pCREB in spinal dorsal horn;Western Blot was qualitatively used to analyze the phosphorylation level of NR2B and CREB after SNL. Results From day 1 after SNL operation,the rat PWL in SNL-saline group was significantly decreased compared with sham groups ( P <0.01) and keep at low level after operation 3 d.The mechanical allodynia of modal rats were detected to obviously increased after intrathecal administration of T10 or MK-801 from post operation day 1 to day 7 ( P <0.05),and combined group was significant than single injection groups ( P <0.05),which also present more apparent than model groups ( P <0.01);in addition,the lasting time of anti-nociceptive effects was longer than SNL-T10 and SNL-MK-801 group after stopping drugs treatment.Immunofluorescent histochemical staining showed the co-expression of pCREB and NeuN.Western Blot results revealed that the expressions of pNR2B and pCREB in spinal dorsal horn were significantly increased on post operational day 7 compared with the sham g

关 键 词：神经病理性痛 雷公藤内酯醇 MK-801 N-甲基-D-门冬氨酸 PCREB 大鼠 

分 类 号：R965[医药卫生—药理学]