Nrf2基因敲除小鼠脊髓损伤后脊髓内TNF-α及MMP-9的变化

摘要

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-α(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 T8-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.%目的 探讨转录因子NF-E2相关因子2(Nrf2)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48 h用干湿比法测定脊髓水肿程度.在脊髓损伤后24 h,用逆转录聚合酶链式反应(RT-PCR)法测定脊髓组织MMP-9 mRNA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SPSS 16.0软件进行统计学分析.结果 脊髓损伤后48 h,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24 h,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24 h,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.