小檗碱对流感病毒感染肺泡巨噬细胞炎性细胞因子的影响及其分子机制研究  

The effect and molecular mechanism of berberine on inflammatory cytokines in NR8383 cells infected by influenza virus

作　　者：吴莹[1] 金叶智[1] 张舒[1] 闫玥捷[1] 路广琳[1] 郝钰[1] 

机构地区：[1]北京中医药大学医学病原系,北京100029

出　　处：《中国免疫学杂志》2012年第2期125-131,共7页Chinese Journal of Immunology

基　　金：国家自然科学基金(No.30772871);北京中医药大学创新团队"经方现代应用关键科学问题的基础研究"资助项目(No.2011-CXD-04)

摘　　要：目的:探讨小檗碱对流感病毒感染大鼠肺泡巨噬细胞(NR8383)后TNF-α、MCP-1转录、表达的影响及与TLR7介导的MyD88依赖性信号通路的关系。方法:流感病毒感染NR8383细胞1小时后,加入含小檗碱的培养基(终浓度5μg/ml),药物作用后6、12、24小时,ELISA检测细胞上清中TNF-α、MCP-1的含量;24小时,Real time PCR检测细胞内TNF-α、MCP-1的mRNA水平,RT-PCR检测TLR7、MyD88、NF-κB P65 mRNA水平;4、6、24小时,免疫组化法检测NF-κB P65核转位情况,并做半定量分析;24小时,Western blot检测NF-κB P65表达水平。结果:小檗碱抑制了流感病毒感染NR8383细胞后TNF-α、MCP-1的转录和表达(P<0.05),降低了TLR7、MyD88、NF-κB P65 mRNA水平(P<0.05、P<0.05、P<0.01),抑制了流感病毒感染后NF-κB P65的核转位及表达(P<0.01)。结论:小檗碱通过抑制TLR7介导的MyD88依赖性信号通路,抑制了NF-κB P65的核转位及表达,从而减少流感病毒感染巨噬细胞后炎性细胞因子TNF-α、MCP-1的产生,在流感治疗中发挥抗炎作用。Objective：To investigate the effect of berberine on the transcription and expression of TNF-α and MCP-1 in NR8383 cells infected by influenza virus and the relationship with TLR7 mediated MyD88-dependent signaling pathway.Methods：After 1 h adsorption of influenza virus,the medium containing 5 μg/ml berberine was added to NR8383 cells in wells.At 6 h,12 h,24 h after berberine application,ELISA method was used to detect the concentration of tumor necrosis factor-alpha（TNF-α） and monocyte chemotactic protein-1（MCP）-1 in the supernants;at 24 h after drug application,Real time PCR was used to detect the mRNA level of TNF-α and MCP-1,and RT-PCR was used to detect the mRNA level of MyD88,NF-κB P65 and TLR7;at 4 h,6 h,24 h after drug application,immunocytochemistry was used to detect nuclear translocation of NF-κB P65 in the cells and to do semi-quantity analysis;at 24 h after wogonin application,Western blot was used to detect the expression of NF-κB P65 protein.Results：Berberine repressed the transcription and expression of TNF-α and MCP-1（P0.05）,reduced the mRNA level of TLR7,and MyD88（P0.05,P0.05,P0.01）,and inhibited the nuclear translocation and expression of NF-κB P65（P0.01）.Conclusion：Berberine inhibited the transcription and expression of TNF-α and MCP-1 by repressing the TLR7 mediated MyD88-dependent signaling pathway and the nuclear translocation and expression of NF-κB P65.Berberine exerted anti-inflammatory effects in the therapy of influenza virus infection.

关 键 词：小檗碱 流感病毒 炎性细胞因子 TLR7 MYD88 NF-ΚBP65 

分 类 号：R285.5[医药卫生—中药学]