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

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NR2B/CREB的磷酸化水平密切相关。

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 group ( P <0.01).And then,co-administration of T10 and MK-801 can significantly inhibit the expression of neuronal pNR2B and pCREB compared with T10,MK-801,and SNL-saline group respectively. Conclusion Intrathecal co-administration of T10 and MK-801 could attenuate the mechanical hypersensitivity,and decreased the dosage of each.One of the mechanisms was involved in inhibiting the phosphorylation of NR2B and CREB in the neurons of spinal dorsal horn.