黄连素通过诱导线粒体自噬干预呼吸道合胞病毒感染HEp-2细胞的作用机制  

Berberine interferes with RSV infection of HEp-2 cells by inducing mitophagy autophagy

作　　者：崔玉娟 赵辉 苏东霞 胡丹东 CUI Yu-juan;ZHAO Hui;SU Dong-xia;HU Dan-dong(School of Life Science,Northwest Normal University,Lanzhou 730070,China;Beijing Yanqing Center for Disease Prevention and Control,Beijing 102100,China;Beijing Yanqing Market Supervision Inspection and Testing Monitoring Center,Beijing 102100,China)

机构地区：[1]西北师范大学生命科学学院,甘肃兰州730070 [2]北京市延庆区疾病预防控制中心,北京102100 [3]北京市延庆区市场监管检验检测监控中心,北京102100

出　　处：《中国药理学通报》2024年第2期308-316,共9页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 51873175)。

摘　　要：目的探讨黄连素(berberine,BE)对呼吸道合胞病毒(respiratory syncytial virus,RSV)感染HEp-2细胞的影响及相关机制。方法将HEp-2细胞感染RSV,并用BE处理。CCK-8实验检测HEp-2细胞存活率;Western blot检测HEp-2细胞NLRP3、ASC、caspase-1、PINK1、Parkin、Beclin1、p62、LC3Ⅰ、LC3Ⅱ、BNIP3蛋白表达;ELISA检测HEp-2细胞IL-1β分泌水平;流式细胞术检测HEp-2细胞的凋亡率和线粒体膜电位;MitoSOX染色检测HEp-2细胞线粒体ROS(mtROS);免疫荧光染色检测HEp-2细胞线粒体-自噬小体共定位;环孢素A进行验证实验。结果BE能提高RSV感染HEp-2细胞的活性、降低凋亡率(P<0.05),并降低NLRP3炎性小体活化水平和IL-1β水平(P<0.05);BE通过提高线粒体膜电位和ATP水平、降低mtROS改善线粒体功能;同时,BE促进RSV感染细胞中线粒体-自噬小体共定位,诱导PINK1/Parkin和BNIP3介导线粒体自噬;环孢素A加重了RSV的感染。结论BE对RSV感染的HEp-2细胞具有保护作用,其机制可能与BE通过诱导PINK1/Parkin和BNIP3介导的线粒体自噬,进而抑制mtROS产生改善线粒体功能,并抑制NLRP3炎性小体活化有关。Aim To explore the effect of berberine(BE)on RSV infected HEp-2 cells and the related mechanism.Methods HEp-2 cells were infected with RSV and treated with BE.Cell viability was assessed using the CCK-8 assay.Protein expression levels of NLRP3,ASC,caspase-1,PINK1,Parkin,Beclin1,p62,LC3Ⅰ,LC3Ⅱ,and BNIP3 in HEp-2 cells were detected by Western blot.The secretion level of IL-1βin HEp-2 cells was measured using ELISA.Apoptosis rate and mitochondrial membrane potential of HEp-2 cells were examined by flow cytometry.Mitochondrial ROS(mtROS)in HEp-2 cells was detected through MitoSOX staining.Colocalization of mitochondria and autophagosomes in HEp-2 cells was investigated using immunofluorescence staining.Cyclosporin A was used for validation experiments.Results BE could significantly improve the activity of RSV-infected HEp-2 cells,reduce the apoptosis rate(P<0.05),and decrease the activation level of NLRP3 inflammasomes and IL-1βlevel(P<0.05);BE improved mitochondrial function by increasing mitochondrial membrane potential and ATP levels,and reduced mtROS.BE significantly promoted the colocalization of mitochondria-autophagosome in RSV infected cells,inducing PINK1/Parkin and BNIP3 to mediate mitochondrial autophagy;cyclosporine A aggravated RSV infection.Conclusions BE has protective effects on HEp-2 cells infected by RSV.The mechanism may be related to the inhibitory effect of BE on the production of mtROS and the activation of NLRP3 inflammasomes by inducing PINK1/Parkin and BNIP3-mediated mitochondrial autophagy.

关 键 词：黄连素 线粒体自噬 RSV NLRP3炎性小体 线粒体膜电位 ROS 线粒体-自噬小体共定位 

分 类 号：R282.71[医药卫生—中药学] R329.24[医药卫生—中医学] R364.5R725.6R978.7