Nrf2基因敲除小鼠脊髓损伤后脊髓内TNF—α及MMP－9的变化

Influence of nuclear factor erythroid 2-retated factor 2 genotype on tumor necrosic factor-a and metalloproteinase-9 expression in spinal cord after spinal cord injury in mice

目的探讨转录因子NF．E2相关因子2（Nri2）对小鼠脊髓损伤后脊髓内肿瘤坏死因子-α（TNF—α）及基质金属蛋白酶-9（MMP-9）表达的影响。方法分别利用野生型Nrf2（＋／＋）小鼠及Nrf2（-／-）基因敲除小鼠制作脊髓钳夹损伤模型。在小鼠脊髓损伤后48h用干湿比法测定脊髓水肿程度。在脊髓损伤后24h，用逆转录聚合酶链式反应（RT—PCR）法测定脊髓组织MMP-9mRNA含量的变化；酶联免疫吸附试验测定脊髓组织中TNF—α及MMP-9蛋白的含量；明胶酶谱法测定脊髓组织中MMP-9的酶活性。用SPSS16．0软件进行统计学分析。结果脊髓损伤后48h，Nrf2（-／-）基因敲除小鼠及Nrf2（＋／＋）小鼠的脊髓均出现明显水肿，但Nrf2（-／-）基因敲除小鼠尤为严重（P〈0．05）。脊髓损伤后24h，Nrf2（-／-）基因敲除小鼠及Nrf2（＋／＋）小鼠的脊髓内TNF—α与MMP-9的表达均增加，但Nrf2（-／-）基因敲除小鼠尤为显著（P〈0．01）。同时，Nrf2（-／-）基因敲除小鼠在脊髓损伤后24h，脊髓内MMP-9明胶酶活性较Nrf2（＋／＋）小鼠的损伤组明显增强（P〈0．01）。结论Nrf2在小鼠脊髓损伤后可能是通过减少炎性介质TNF—α及MMP-9的表达，降低MMP-9的明胶酶活性来减轻炎症反应，减轻脊髓水肿程度，起到神经保护的作用。

Objective To investigate the role of nuclear factor erythroid 2-related factor 2 （ Nrf2 ） as a key transcription factor of cytoprotection against inflammation in the spinal cord upregulation of matrix metalloproteinase-9 （MMP-9） , tumor necrosis factor-or （TNF-α） after spinal cord injury （SCI）. Methods Wild-type Nrf2 （ ＋/＋ ） and Nrf2 （ -/- ）-deficient mice were subjected to a murine SCI model induced by the application of vascular clips （ force of 10 g） to the dura after a three-level TS-T10 laminectomy. The wet/dry weight ratio was used to reflect the percentage of water content of impaired spinal cord tissue at 48 h after SCI. The mRNA levels of MMP-9 were determined using reverse-transcriptase polymerase chain reaction （RT-PCR）, and the protein levels of TNF-α and MMP-9 were detected by enzyme-linked immunosorbent assay at 24 h after SCI. Furthermore, gelatin zymography analysis was used to show MMP-9 activity of spinal cord tissue at 24 h after SCI. Software SPSS 16. 0 was used for the statistical analysis. Results After SCI, spinal cord water content , the expression of TNF-α and MMP-9 all increased in both injured Nrf2 （ ＋/＋ ） and Nrf2 （ -/- ） mice compared with their respective sham-operated mice. However, Nrf2 （ -/- ） mice were shown to have more severe spinal cord edema, more TNF-α expression, more production and activity of MMP-9 compared with their wild-type Nrf2 （ ＋/＋ ） counterparts after SCI （P 〈 0.05 ）. Conclusions The results suggest that Nrf2 plays an important protective role in limiting the spinal cord upregulation of TNF-α and MMP-9 after SCI. It may be a new therapic target of SCI.