Li Xiao-Qian, Lv Huang-Wei, Wang Zhi-Lin, Tan Wen-Fei, Fang Bo, Ma Hong
J Neuroinflammation. 2015 Feb 7;12:25. doi: 10.1186/s12974-015-0246-3.
Spinal cord ischemia reperfusion (IR) injury causes inflammation and subsequently increases blood-spinal cord barrier leakage and Toll-like receptor 4 (TLR4) pathway activation. MicroRNAs (miRs) effectively regulate numerous target mRNAs during ischemia. However, their roles during IR injury are poorly understood. We investigated miRs involvement, particularly miR-27a, in TLR4 pathway-mediated inflammatory responses after IR.
We used a genomics approach to examine changed miRs of rats that had undergone 14 minutes of ischemia, followed by 24 or 72 hours of reperfusion. Quantitative RT-PCR was used to identify and confirm the miRs involved in regulating TLR4 pathway activation. We scanned miR databases for potential miR targets and confirmed these targets by quantitative RT-PCR. The miR mimic and anti-miR oligonucleotides (AMOs) were intrathecally injected at 12-hour intervals beginning three days before the ischemia. The effects of miRs on the TLR4 pathway and downstream cytokines were analyzed by PCR, western blotting, and ELISA. Double immunofluorescence staining was perfumed to determine the relationship between the targets and TLR4. Blood-spinal cord barrier (BSCB) permeability was examined using Evans blue (EB) dye.
A microarray analysis revealed that at 24 hours post-injury, three miRs were upregulated (>2.0 fold) and 15 miRs were downregulated (<0.5 fold), and at 72 hours, four miRs were upregulated and 14 were downregulated compared to their levels in sham-operated controls. We focused on miR-27a, which is predicted to contain sequences complementary to the 3'-untranslated region (UTR) of Toll-like receptor adaptor molecule 2 (TICAM-2). Double immunostaining indicated that TLR4 activation correlated with changes in TICAM-2 expression. Compared to the rats in the IR and negative control groups, intrathecal infusion of the miR-27a mimic attenuated IR-induced TLR4 activation and inflammatory damage to the BSCB, which was shown as decreased EB extravasation and lower levels of nuclear factor kappa-B (NF-κB) and lnterleukin (IL)-1β at 24 and 72 hours after reperfusion, whereas pretreatment with miR-27a AMO aggravated these injuries.
We present the first evidence that miRs play an important role in spinal cord IR injury. We identified TICAM-2 as a novel target of miR-27a. miR-27a upregulation attenuates IR-induced inflammatory damage to the BSCB by negatively regulating TICAM-2 of the TLR4 signaling pathway and inhibiting the NF-κB/IL-1β pathway. These results provide new therapeutic targets for IR injury treatment.
脊髓缺血再灌注（IR）损伤会引发炎症，进而增加血脊髓屏障渗漏以及Toll样受体4（TLR4）通路的激活。微小RNA（miRs）在缺血过程中可有效调控众多靶mRNA。然而，它们在IR损伤中的作用却鲜为人知。我们研究了miRs，特别是miR - 27a，在IR后TLR4通路介导的炎症反应中的作用。
我们采用基因组学方法检测经历14分钟缺血，随后再灌注24或72小时的大鼠中miRs的变化。定量逆转录聚合酶链反应（RT - PCR）用于鉴定和确认参与调节TLR4通路激活的miRs。我们在miR数据库中搜索潜在的miR靶标，并通过定量RT - PCR确认这些靶标。在缺血前三天开始，每隔12小时鞘内注射miR模拟物和抗miR寡核苷酸（AMOs）。通过PCR、蛋白质印迹法和酶联免疫吸附测定（ELISA）分析miRs对TLR4通路和下游细胞因子的影响。进行双重免疫荧光染色以确定靶标与TLR4之间的关系。使用伊文思蓝（EB）染料检测血脊髓屏障（BSCB）通透性。
微阵列分析显示，与假手术对照组相比，损伤后24小时，3个miRs上调（>2.0倍），15个miRs下调（<0.5倍）；72小时时，4个miRs上调，14个miRs下调。我们聚焦于miR - 27a，其预测含有与Toll样受体衔接分子2（TICAM - 2）的3'非翻译区（UTR）互补的序列。双重免疫染色表明TLR4激活与TICAM - 2表达变化相关。与IR组和阴性对照组大鼠相比，鞘内注入miR - 27a模拟物可减轻IR诱导的TLR4激活和对BSCB的炎症损伤，表现为再灌注后24和72小时伊文思蓝外渗减少以及核因子κB（NF - κB）和白细胞介素（IL）- 1β水平降低，而用miR - 27a AMO预处理则加重了这些损伤。
我们首次证明miRs在脊髓IR损伤中起重要作用。我们鉴定出TICAM - 2是miR - 27a的新靶标。miR - 27a上调通过负调控TLR4信号通路的TICAM - 2并抑制NF - κB/IL - 1β通路，减轻IR诱导的对BSCB的炎症损伤。这些结果为IR损伤治疗提供了新的治疗靶点。
