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目的:探索神经结扎(spinal nerve ligation,SNL)模型大鼠脊髓内神经元型一氧化氮合酶(neuronal nitric oxide synthase,n NOS)的磷酸化是否对神经病理性疼痛具有调节作用,研究n NOS磷酸化在神经病理性疼痛中的作用机制。方法 :选用雄性SPF级SD大鼠60只,随机分为4组。假手术组:只暴露脊神经,不结扎;实验组:制作SNL模型,鞘内注射钙依赖性蛋白激酶Ⅱ(phosphorylated calcium/calmodulin dependent kinaseⅡ,p-Ca MKⅡ)抑制剂KN93;阴性对照组:制作SNL模型,鞘内注射DMSO;模型组:制作SNL模型,不给药。于术前1 d、术后1~5 d以及鞘内给药后1~4 h测定机械痛阈值。采用Western blot检测腰段脊髓组织p-Ca MKⅡ、n NOS与p-n NOS的表达水平,利用免疫共沉淀以及免疫荧光实验检测n NOS与其接头蛋白CAPON是否具有相互作用。结果:SNL可导致大鼠机械疼痛阈值降低(P<0.01),脊髓组织p-Ca MKⅡ的表达增加(P<0.05),p-n NOS的表达下降(P<0.05),髓鞘内注射KN93可反转神经结扎所致的上述趋势;n NOS与其接头蛋白CAPON在大鼠体内存在相互作用,n NOS的磷酸化能降低其与CAPON的相互作用强度。结论:n NOS的磷酸化参与了SNL诱导的神经病理性疼痛的维持,p-Ca MKⅡ通过对n NOS的磷酸化,降低n NOS与其接头蛋白CAPON在体内的相互作用强度,针对n NOS的磷酸化信号途径的治疗可为神经病理性疼痛的治疗提供新的视野。
OBJECTIVE: To investigate whether the phosphorylation of neuronal nitric oxide synthase (nNOS) in the spinal cord of rats with spinal nerve ligation (SNL) modulates the neuropathic pain and investigate whether nNOS phosphorylation The mechanism of action in neuropathic pain. Methods: Sixty male Sprague-Dawley rats were randomly divided into 4 groups. In the sham-operation group, only the spinal nerves were exposed without ligation. In the experimental group, SNL model was made and intrathecal KN93 inhibitor of p-Ca MKII was injected intrathecally. Negative control group: SNL Model, intrathecal injection of DMSO; model group: SNL model was made without administration. The mechanical pain threshold was measured at 1 d before surgery, 1 to 5 days after operation and 1 to 4 h after intrathecal administration. Western blot was used to detect the expression of p-CaNKII, nNOS and p-nNOS in lumbar spinal cord tissues. Immunoprecipitation and immunofluorescence assay were used to detect whether nNOS interacted with its CAPON. Results: SNL could decrease the threshold of mechanical pain in rats (P <0.01), increase the expression of p-Ca MKⅡ in spinal cord (P <0.05) and decrease the expression of pn NOS (P <0.05) NNOS and its adapter protein CAPON exist interactions in rats, nNOS phosphorylation can reduce the intensity of its interaction with CAPON. CONCLUSIONS: nNOS phosphorylation is involved in the maintenance of SNL-induced neuropathic pain. P-CaKKII, through its phosphorylation of nNOS, decreases the intensity of interaction between nNOS and CAPON in vivo, The treatment of signaling pathways can provide new horizons for the treatment of neuropathic pain.