NF-κB信号通路在丝状支原体山羊亚种感染中的作用

摘要

To analyze the molecular mechanism of the nuclear factor kappa B (NF-κB) signaling pathway in goat alveolar epithelial cells infected by Mycoplasma mycoides subsp,capri (Mmc),the cells infected by Mmc were collected at 4 h,8 h,12 h and 24 h post-infection,and the expression level of NF-κBp65 mRNA was then detected by real-time quantitative PCR.After incubating with the different concentrations of NF-κB inhibitor BAY11-7082 for 1 h,the cells were then infected with mycoplasma and harvested at 24 h post-infection,and detected for interleukin-8 (IL-8) and tumor necrosis factor α (TNF-α) by real-time quantitative PCR.Meanwhile the changes in Caspase-3 activity,H2O2 and NO concentrations were measured using a kit.The pathogenicity of mycoplasma to cells was measured using plate counts.The results showed that the expression of NF-κBp65 mRNA was significantly enhanced by Mmc at 8 h,12 h,and 24 h post-infection (P ＜ 0.01).At 24 h post-infection,the levels of IL-8 mRNA,TNF-α mRNA,Caspase-3 activity,and the concentrations of H2O2 and NO were enhanced by Mmc (P ＜ 0.01),but which were significantly decreased by BAY11-7082 (5 μmol/L and 10 μmol/L) (P ＜ 0.05).In summary,the NF-κB signaling pathway plays an important role in the pathogenesis of Mmc.%为分析细胞核转录因子κB(Nuclear factor kappa B,NF-κB)信号通路在丝状支原体山羊亚种(Mmc)感染山羊肺泡上皮细胞中的分子作用机制,将Mmc感染细胞,于4h、8h、12h和24 h收集细胞,利用实时定量PCR检测NF-κBp65 mRNA的表达水平.采用不同浓度的NF-κB抑制剂BAY11-7082与细胞孵育1h,然后用Mmc感染细胞,于24 h收集细胞,利用实时定量PCR检测白细胞介素-8(IL-8)和肿瘤坏死因子α(TNF-α)mRNA的表达水平,用试剂盒检测Caspase-3活性、H202和NO浓度变化,利用平板计数检测Mmc对细胞的致病力变化.结果显示,Mmc于8h、12 h和24 h等3个时间点显著提高细胞NF-κBp65 mRNA表达水平(P ＜0.01);Mmc显著提高细胞IL-8 mRNA水平、TNF-α mRNA水平、Caspase-3的活性、H2O2和NO浓度(P＜0.01),而BAY11-7082(浓度为5μmol/L和10 μmol/L)能够显著降低Mmc介导的细胞IL-8 mRNA水平、TNF-αmRNA水平、Caspase-3的活性、H202和NO浓度的升高(P＜0.05),且可显著降低细胞中Mmc存活量(P＜0.05).综上可知,NF-κB信号通路在Mmc致病机制中发挥重要作用.