MiR-146a高表达对神经胶质细胞BV2炎性反应的影响

Effect of High MiR-146a Expression on the Inflammatory Reaction in BV2 Cells

目的观察高表达MiR-146a对细菌脂多糖(LPS)诱导后神经胶质细胞BV2炎性反应的影响。方法采用LPS刺激接受MiR-146a模拟物转染后的BV2细胞,Real-time PCR检测MiR-146a转染效率,ELISA检测促炎症因子白细胞介素-6(IL-6)和肿瘤坏死因子α(TNFα)的表达水平,Real-time PCR和Western blot法检测TLR4信号通路上肿瘤坏死因子受体相关因子6(TRAF6)和白细胞介素-1受体相关激酶1(IRAK1)的表达水平。结果与正常组比较,采用50 nmol/L的转染浓度对BV2细胞进行MiR-146a模拟物转染,可明显增加细胞内MiR-146a含量(t=5.846,P=0.0021);LPS刺激导致BV2细胞激活,可明显增加细胞内IRAK1和TRAF6表达,也可明显诱发细胞分泌更多的IL-6和TNFα;而与单纯采用LPS刺激相比,用MiR-146a模拟物转染后再接受LPS刺激作用可明显降低IL-6(t=5.200,P=0.0003)和TNFα(t=9.812,P<0.0001)的表达水平,同时明显降低细胞内TRAF6分子在基因(t=5.353,P=0.0007)和蛋白(t=6.980,P=0.0009)水平的表达,而非IRAK1的表达。结论 MiR-146a可能是通过调控TLR4信号通路中TRAF6分子的表达,负反馈抑制BV2细胞的炎性反应。

Objective To explore the effect of MiR-146 a regulator function on the inflammatory response in neuroglia cell（ microglia）. Methods BV2 cells were transfected by MiR-146 a mimics,and then stimulated by lipopolysaccharide（ LPS）. MiR-146 a expression was measured by real-time polymerase chain reaction（ real-time PCR）. Interleukin（ IL）-6 and tumor necrosis factor α（ TNFα） were measured by enzymelinked immunosorbent assay（ ELISA）. Furthermore,IL-1 receptor-associated kinase 1（ IRAK1） and TNF receptor-associated factor 6（ TRAF6） were detected by PCR and Western blotting. Results Compared to the normal control group,MiR-146 a expression was significantly elevated by transfection with MiR-146 a mimics（ t =5. 846,P = 0. 0021）. The expression levels of IRAK1,TRAF6,TNFα,and IL-6 significantly increased in the LPS-stimulated BV2 cells compared to the non-stimulated BV2. The enhancement of MiR-146 a resulted insignificantly decreased IL-6（ t = 5. 200, P = 0. 0003） and TNFα（ t = 9. 812, P〈0. 0001） secretion. The mRNA（ t = 5. 353,P = 0. 0007） and protein（ t = 6. 980,P = 0. 0009） levels of TRAF6,but not IRAK1,also significantly decreased. Conclusion MiR-146 a may negatively suppress the inflammatory response of BV2 cells by regulating the expression of IRAF6 molecules in the TLR4 signaling pathway.