【摘要】 目的探讨下调miR-203对颞叶癫痫大鼠海马胶质细胞活化和炎症反应的影响。方法将46只成年SD大鼠随机分为假手术组(n=10)、模型组(n=12)、阴性对照组(n=12)和miR-203低表达组(n=12)。立体定向辅助下,将海人酸注入大鼠左侧海马CA3区建立颞叶癫痫模型,miR-203低表达组和阴性对照组大鼠左侧海马CA3区分别注射携带miR-203 inhibitor的腺相关病毒和空腺病毒载体,7 d后取左侧海马组织,PCR法检测miR-203水平,ELISA法检测炎症因子[白细胞介素-1β(IL-1β)、IL-6和肿瘤坏死因子α(TNF-α)]水平,免疫印迹法检测肌细胞增强因子2c(MEF2C)、核因子-κB(NF-κB)p65蛋白表达,TUNEL法检测神经元凋亡,GFAP/CD11b/c免疫荧光染色分析胶质细胞活化。结果模型组大鼠海马组织miR-203、NF-κB p65蛋白表达量、炎症因子(IL-1β、IL-6和TNF-α)水平明显增高(P<0.05),MEF2C蛋白表达量明显降低(P<0.05),活化的胶质细胞数量、神经元凋亡数量明显增多(P<0.05)。双荧光素酶报告基因实验显示,MEF2C是miR-203的靶基因。miR-203表达低表达组大鼠海马组织miR-203表达量、NF-κB p65蛋白表达量、炎症因子(IL-1β、IL-6和TNF-α)水平明显降低(P<0.05),MEF2C蛋白表达量明显增高(P<0.05),活化的胶质细胞数量、神经元凋亡数量明显减少(P<0.05)。结论下调miR-203,靶向调控MEF2C/NF-κB信号通路,抑制颞叶癫痫大鼠海马胶质细胞的活化、神经元凋亡和炎症反应。更多还原

【Abstract】 Objective To explore the effect of down-regulation of miR-203 on the glial cell activation and inflammation in hippocampal tissues of temporal lobe epilepsy rats. Methods Forty-six adult SD rats were randomly divided into sham operation group(n=10), model group(n=12), negative control group(n=12) and miR-203 low expression group(n=12). Under stereotactic assistance,kainic acid was injected into the left hippocampus CA3 to establish the temporal lobe epilepsy model. Adeno-associated viral(AAV)integrated with miR-203 inhibitor and AAV vector were injected into the left hippocampal CA3 of the rats in the miR-203 low expression group and the negative control group, respectively. Seven days after the intervention, the left hippocampal tissues were isolated, and the level of miR-203 was detected by PCR, and the inflammatory factors including interleukin-1β(IL-1β), IL-6 and tumor necrosis factor α(TNF-α) were detected by ELISA, the expression levels of myocyte enhancer factor 2 c(MEF2 C), nuclear factor-κB(NF-κB) p65 protein were detected by immunoblotting method, neuronal apoptosis was detected by TUNEL method, and glial cell activation was detected by GFAP/CD11 b/c immunofluorescence staining. Results In the hippocampal tissues of rats in model group, the expression levels of miR-203 and NF-κB p65 protein, and the levels of inflammatory factors(IL-1β, IL-6 and TNF-α) were significantly increased(P<0.05), and the expression level of MEF2 C protein was significantly decreased(P<0.05), the numbers of activated glial cells and apoptic neurons were significantly increased(P<0.05). The dual luciferase reporter gene experiment showed that MEF2 C was the target gene of miR-203. In the hippocampal tissues of rats in miR-203 low expression group, the expression levels of miR-203 and NF-κB p65 protein, and the levels of inflammatory factors(IL-1β, IL-6 and TNF-α) were significantly reduced(P<0.05),the protein expression level of MEF2 C was significantly increased(P<0.05), and the numbers of activated glial cells and apoptic neurons were significantly reduced(P<0.05). Conclusions Down-regulation of miR-203 inhibits the activation of hippocampal glial cells,neuronal apoptosis and inflammatory response in temporal lobe epilepsy rats, which may be by targeted regulation of the MEF2 C/NF-κB signaling pathway.更多还原