Tat-LK15介导siRNA干扰大鼠脊髓背角nNOS表达对神经病理性疼痛的治疗作用

Therapeutic effect of Tat-LK15-mediated the interference of siRNA with nNOS expression in the spinal dorsal horn on neuropathic pain in rats

目的探讨细胞穿透肽Tat-LK15介导siRNA干扰大鼠脊髓背角神经元型一氧化氮合酶(nNOS)表达对神经病理性疼痛的治疗效应。方法采用Tat-LK15介导FAM-siRNA转染原代大鼠脊髓背角神经元细胞(RN-dsc),于倒置荧光显微镜观察其转染效果。健康雄性SD大鼠50只,随机分为5组(n=10):正常组、假手术组、神经病理性疼痛组(SNL组)、Tat-LK15-nNOS siRNA组(TS组)和Tat-LK15-NC siRNA组(TN组)。采用脊神经结扎法(SNL)制作神经病理性疼痛模型,正常组不予任何处理,假手术组仅暴露脊神经;SNL组、TS组、TN组构建SNL模型同时鞘内置管,于第7天开始每天分别鞘内注射10μl生理盐水、10μl含5μg siRNA的Tat-LK15-nNOS siRNA和Tat-LK15-NCsiRNA,持续7d。建模前1d及建模后3、7、10、14d测定各组大鼠机械缩足反应阈值(PWMT)及热缩足反应潜伏期(PWTL)。第14天测定结束后取L4-6节段脊髓,采用q-PCR及Western blotting检测脊髓背角nNOS表达水平。结果倒置荧光显微镜观察显示,Tat-LK15可成功介导siRNA转染原代大鼠脊髓背角神经元。与假手术组比较,SNL组建模后第3天起PWMT降低、PWTL缩短,脊髓背角nNOS mRNA及蛋白表达上调(P<0.01),正常组上述指标与假手术组比较差异无统计学意义;与SNL组比较,TS组第10、14天时PWMT增高、PWTL延长,脊髓背角nNOS mRNA及蛋白表达降低(P<0.01),TN组上述指标与SNL组比较差异无统计学意义。结论 Tat-LK15可成功介导nNOS siRNA转染脊髓背角神经元,沉默nNOS基因的过度表达,对SNL大鼠神经病理性疼痛具有一定的治疗作用。

Objective To investigate the therapeutic effect of cell penetrating peptide Tat-LK15 mediating small interfering RNA （siRNA） interference with the expression of neuronal nitric oxide synthase （nNOS） in rat spinal dorsal horn on neuropathic pain. Methods The transfection reagent, Tat-LK15, was used to mediate the transfection of rat spinal dorsal horn （SDH） neuronal cells with carboxyfluorescein （FAM）, and then the transfection effect was observed under inverted fluorescence microscope. Fifty healthy male SD rats were randomly divided into 5 groups （n=10）： control group, sham operation group （sham group）, neuropathic pain group （SNL group）, Tat-LK15-nNOS siRNA group （TS group） and Tat-LK15-NC siRNA group （TN group）. Neuropathic pain was induced by spinal nerve ligation （SNL）, rats in control group did not receive operation and only the spinal nerve was exposed in sham group. Groups SNL, TS and TN were made into the models by SNL and implanted intrathecal catheter, intrathecal administration was performed from the 7th day after model establishment, and 10μl normal saline, 10μl TS complex （including 5μg siRNA） and 10μl TN （including 5μg siRNA） were injected intrathecally each day for 7 days. Paw withdrawal mechanical threshold （PWMT） and paw withdrawal thermal latency （PWTL） were measured at 1 day before （baseline） and 3, 7, 10 and 14 days after model establishment. Then animals were sacrificed on the 14th day after the operation and the lumbar segment （L4-6） of the spinal cord was removed to detect the expressions of nNOS mRNA and protein using q-PCR and Western blotting analysis. Results Tat-LK15 effectively mediated FAM-siRNA into SDH neuronal cells. Compared with sham group, SNL significantly decreased PWMT and PWTL and increased expressions of nNOS mRNA and protein from the 3rd day （P〈0.01）, but there was no significant difference between the sham and control group. Tat-LK15-nNOS siRNA complex significantly increased PWMT and PWTL and down-regulated nNOS mRNA and protein expressions in TS group compared with SNL group on the days 10 and 14. There was no significant difference between TN and SNL group. Conclusion Tat-LK15 not only can mediate successful nNOS siRNA transfection and inhibit the expression of nNOS, but also effectively relieve SNL-induced neuropathic pain in rats.