摘要
目的:探究犀角地黄汤合银翘散(XDY)对流感病毒引起的炎症反应中巨噬细胞焦亡的作用及其机制。方法:将巨噬细胞J774A.1分为正常对照组(正常培养)、模型组(流感病毒PR8感染24 h)、XDY组(PR8感染+XDY作用24 h)、H2O2对照组(H2O2作用24 h)和H2O2+XDY组(H2O2+XDY作用24 h)。用乳酸脱氢酶(LDH)试剂盒检测各组细胞LDH的释放量,流式细胞术检测由caspase-1介导的细胞焦亡率,Western blot检测核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)和gasdermin D N端片段(GSDMD-N)蛋白表达量,ELISA检测白细胞介素1β(IL-1β)分泌水平,激光共聚焦显微镜观察活性氧簇(ROS)与线粒体的共定位和释放情况。结果:流感病毒感染及H2O2作用后,巨噬细胞LDH释放和GSDMD-N蛋白表达增多,caspase-1介导的细胞焦亡率升高,同时NLRP3蛋白表达、IL-1β分泌和线粒体ROS(mtROS)生成亦增多;与模型组和H2O2对照组相比,XDY可显著减少mtROS生成、NLRP3和GSDMD-N蛋白表达及IL-1β和LDH分泌,降低caspase-1介导的细胞焦亡率。结论:犀角地黄汤合银翘散通过干预mtROS-NLRP3炎症小体通路抑制巨噬细胞焦亡,这可能是其抗流感病毒引起的过度炎症反应的机制之一。
AIM:To investigate the effect of Xijiao-Dihuang decoction combined with Yinqiao powder(XDY)on the inflammation caused by influenza virus and its mechanism.METHODS:Macrophage J774A.1 was divided into normal control group,model group(infected with influenza virus PR8 for 24 h),XDY intervention group(PR8+XDY),H2O2 control group,and H2O2+XDY group.Lactate dehydrogenase(LDH)kit was used to quantity the release of LDH,and the pyroptosis mediated by caspase-1 was detected by flow cytometry.The protein expression of nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)and gasdermin D N-terminal fragment(GSDMD-N)was deter‐mined by Western blot.The secretion of interleukin-1β(IL-1β)in the supernatant was measured by ELISA,and immuno‐fluorescence was applied to observe the localization of reactive oxygen species(ROS)in mitochondria.RESULTS:After influenza virus infection and H2O2 treatment,the release of LDH in the macrophages,the protein expression of GSDMD-N and NLRP3,the caspase-1-mediated pyroptotic rate,the secretion of IL-1β,and the production of mitochondrial ROS(mtROS)were all increased.Compared with model group and H2O2 control group,XDY significantly reduced the secre‐tion of IL-1β,the production of mtROS,the release of LDH,the protein expression of NLRP3 and GSDMD-N,and the rate of caspase-1-mediated cell death.CONCLUSION:The inhibition of macrophage pyroptosis by interfering with mtROS-NLRP3 inflammasome pathway may be one of the mechanisms of XDY against excessive inflammatory response caused by influenza virus.
作者
赵梦繁
苏日娜
毛钦
马璐瑶
刘天怡
吴珺
游雷鸣
郝钰
ZHAO Meng-fan;SU Ri-na;MAO Qin;MA Lu-yao;LIU Tian-yi;WU Jun;YOU Lei-ming;HAO Yu(Department of Immunology and Microbiology,School of Life Science,Beijing University of Chinese Medicine,Beijing 102488,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2021年第6期1049-1054,共6页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81573723)。
