摘要
目的:探究犀角地黄汤合银翘散(XDY)对自噬调节的流感病毒感染小鼠巨噬细胞线粒体活性氧(mtROS)水平的影响。方法:以流感病毒PR8感染小鼠巨噬细胞J774A.1为模型,进行下列实验:(1)分为正常组、模型组(PR8感染24 h)、XDY干预组(PR8感染及162.5 mg/L XDY干预24 h)、3-甲基腺嘌呤(3-MA)干预组(5 mmol/L 3-MA预处理3 h+PR8感染24 h)、3-MA对照组(5 mmol/L 3-MA预处理3 h再继续培养24 h)和PR8+3-MA+XDY组(5 mmol/L 3-MA预处理3 h+PR8感染及162.5 mg/L XDY干预24 h),Western blot法检测细胞微管相关蛋白1轻链3-II(LC3-II)和P62蛋白水平,ELISA检测细胞培养上清液白细胞介素1β(IL-1β)的分泌水平;(2)分为正常组、模型组和XDY干预组,免疫荧光观察细胞线粒体膜电位、自噬体与线粒体共定位;(3)分为正常组、模型组、XDY干预组、Mito-TEMPO干预组(PR8感染及500μmol/L Mito-TEMPO干预24 h)、Mito-TEMPO对照组(500μmol/L Mito-TEMPO干预24 h)、3-MA干预组、3-MA对照组和PR8+3-MA+XDY组,流式细胞术检测mtROS水平。结果:流感病毒感染后,J774A.1细胞内LC3-II和P62蛋白水平显著升高,线粒体膜电位下降,mtROS生成增多,IL-1β分泌显著增多;与模型组相比,XDY可降低P62的表达,促进线粒体自噬,升高线粒体膜电位,减少mtROS产生及IL-1β分泌;以3-MA抑制细胞自噬可加重mtROS累积,XDY降低mtROS的作用也受到抑制。结论:XDY可通过促进流感病毒感染的小鼠巨噬细胞自噬而降低mtROS水平,从而抑制炎症反应。
AIM:To investigate the effects of Xijiao-Dihuang decoction combined with Yinqiao powder(XDY)on the autophagy of mouse macrophages infected with influenza virus.METHODS:Mouse macrophage J774A.1 infected with influenza virus PR8 was used as the model.The J774A.1 cells were divided into normal group,model group(infected with influenza virus PR8 for 24 h),XDY intervention group(infected with PR8 and treated with XDY for 24 h),3-methyladenine(3-MA)intervention group(3-MA pretreatment for 3 h and infected with PR8 for another 24 h),3-MA control group(3-MA preteatment for 3 h and cultured for another 24 h)and PR8+3-MA+XDY group(3-MA preteatment for 3 h and PR8+XDY for another 24 h).The protein levels of P62 and microtubule-associated protein 1 light chain 3-II(LC3-II)were detected by Western blot,and the release of interleukin-1β(IL-1β)in the cell culture supernatant was measured by ELISA.Co-localization of autophagosomes and mitochondria was detected by immunofluorescence.The level of mitochondrial reactive oxygen species(mtROS)was detected by flow cytometry.RESULTS:Influenza virus infection increased the protein levels of LC3-II and P62,the production of mtROS,and the secretion of IL-1βin the J774A.1 cells.Compared with model group,XDY decreased P62 expression,promoted mitophagy,elevated mitochondrial membrane po⁃tential,and reduced mtROS production and IL-1βrelease.The inhibition of autophagy by 3-MA increased the accumula⁃tion of mtROS,and the effect of XDY on mtROS was also inhibited.CONCLUSION:The XDY promotes the autophagy of influenza virus-infected mouse macrophages to regulate the mtROS levels and inhibit the inflammatory response.
作者
毛钦
马璐瑶
刘天怡
周娜
刘梦华
吴珺
郝钰
MAO Qin;MA Lu-yao;LIU Tian-yi;ZHOU Na;LIU Meng-hua;WU Jun;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
2022年第4期704-711,共8页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81573723)。
关键词
流感病毒
巨噬细胞
细胞自噬
线粒体活性氧
犀角地黄汤合银翘散
Influenza virus
Macrophage
Autophagy
Mitochondrial reactive oxygen species
Xijiao-Di⁃huang decoction combined with Yinqiao powder
