诱导型一氧化氮合酶反义核酸在脊髓损伤后细胞凋亡和p-p38 MAPK信号转导通路中的作用

Role of antisense oligodeoxynucleotide of inducible nitric oxide synthase in the apoptosis and p-p38 MAPK signal transduction in rat neurons after spinal cord injury

目的研究一氧化氮合酶(NOS)反义核酸(ASODN)对脊髓损伤后NOS表达、细胞凋亡、信号转导通路的影响。方法设计并合成诱导型NOS反义寡脱氧核苷酸(ASODN-iNOS)及诱导型NOS无义寡脱氧核苷酸(NSODN-iNOS),微量注入大鼠蛛网膜下腔并制备成脊髓压迫伤动物模型。伤后6 h分别用RT-PCR检测iNOS mRNA表达及用Western blotting检测组织中磷酸化丝裂原激活蛋白激酶(p-p38 MAPK)的变化,用双标染色流式细胞术检测伤后72 h细胞凋亡情况。损伤对照组鞘内注射不含核酸的溶液成分。结果脊髓损伤后组织中存在iNOS的表达和p-p38 MAPK变化;损伤后局部组织中存在明显的细胞凋亡;ASODN-iNOS可以抑制iNOS的表达,并可以显著抑制损伤后p-p38 MAPK的表达和细胞凋亡的发生,与损伤对照组相比差异有统计学意义(P<0.01)。结论ASODN-iNOS可以抑制脊髓损伤后iNOS的表达和细胞凋亡,ASODN的这种作用可能与p-p38 MAPK信号转导通路有关。

Objective To study the role of antisense oligodeoxynucleotides (ASODN) of inducible nitric oxide synthase (iNOS) on the apoptosis and p-p38 mitogen-activated protein kinase (p-p38 MAPK) signal transduction of the neurons after spinal cord injury (SCI). Methods Antisense and non-sense oligodeoxynucleotides of iNOS were designed and synthesized, and injected into the subarachnoid space 12 h before the compressive injury was given to the rat spinal cord. Reverse transcription-PCR and flow-cytometry were used to examine the iNOS mRNA expression and the apoptosis of the neurons within the injured spinal cord. The changes of p-p38 MAPK signal transduction pathway were detected by Western blotting. Results Obvious increase in iNOS expression and up-regulated p-p38 MAPK were detected after SCI, and neuronal apoptosis was observed in the injured spinal cord. ASODN-iNOS treatment resulted in decreased expression levels of iNOS mRNA and p-p38 MAPK, and the neuronal apoptosis was alleviated. Conclusion ASODN- iNOS inhibits iNOS expression and neuronal apoptosis following SCI which may be related to the p-p38 MAPK signal transduction pathway.