去氢二异丁香酚通过TFEB/自噬溶酶体途径抵抗H1N1病毒感染  

Dehydrodiisoeugenol resists H1N1 virus infection via TFEB/autophagy-lysosome pathway

作　　者：刘哲 李俊良 周义翔 刘霞 禹艳丽 罗政 王遥 贾鑫 LIU Zhe;LI Jun-liang;ZHOU Yi-xiang;LIU Xia;YU Yan-li;LUO Zheng;WANG Yao;JIA Xin(School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 100029,China;School of Life Science,Beijing University of Chinese Medicine,Beijing 100029,China;Beijing Institute of Traditional Chinese Medicine,Beijing University of Chinese Medicine,Beijing 100029,China)

机构地区：[1]北京中医药大学中药学院,北京100029 [2]北京中医药大学生命科学学院,北京100029 [3]北京中医药大学北京中医药研究院,北京100029

出　　处：《中国中药杂志》2025年第6期1650-1658,共9页China Journal of Chinese Materia Medica

基　　金：2023年度北京中医药大学-优莎纳联合研究中心(BURC)基金重点项目(BUCM-2023-JS-KF-032);中华中医药学会青年人才托举工程项目(CACM-2023-QNRC2-A02);北京市科技新星计划课题(20230484342);北京市自然科学基金项目(7242239)。

摘　　要：基于转录因子EB(transcription factor EB,TFEB)/自噬溶酶体生物发生探究去氢二异丁香酚(dehydrodiisoeugenol,DEH)抗病毒感染的细胞学机制。采用细胞计数试剂盒(cell counting kit-8,CCK-8)检测DEH对人非小细胞肺癌细胞系(A549)细胞活力的影响;实时荧光定量聚合酶链式反应(real-time quantitative polymerase chain reaction,RT-qPCR)检测其对甲型流感病毒(influenza A virus,H1N1)复制的影响;蛋白免疫印迹(Western blot)法检测DEH对H1N1核蛋白(H1N1 nucleoprotein,H1N1-NP)表达的影响;免疫荧光检测DEH对H1N1-NP荧光强度的影响;构建H1N1滴鼻感染小鼠模型,评价30 mg·kg^(-1)去氢二异丁香酚对H1N1感染的治疗作用;在胚胎成纤维细胞(mouse embryonic fibroblasts,MEFs)中通过转录组测序技术(RNA sequencing,RNA-seq)分析药物的转录表型组;利用微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)荧光质粒检测DEH对细胞自噬的影响;Western blot检测病毒感染下DEH对LC3Ⅱ/LC3Ⅰ蛋白自噬流的影响;最后,在野生型及TFEB敲除的永生化骨髓源性巨噬细胞(iBMDM)中评价TFEB表达缺失对DEH抗H1N1感染的影响。结果显示,DEH对A549细胞的半数抑制浓度(half-maximal inhibitory concentrations,IC_(50))为(87.17±0.247)μmol·L^(-1);体外以剂量依赖方式抑制H1N1复制;与H1N1滴鼻感染小鼠模型组相比,DEH治疗显著提升了小鼠的体质量和生存时间;药物处理后引起LC3聚集;在iBMDM中TFEB表达缺失显著限制了DEH抵抗H1N1复制的作用。综上所述,DEH通过激活TFEB/自噬溶酶体途径抵抗H1N1感染。The present study delves into the cellular mechanisms underlying the antiviral effects of dehydrodiisoeugenol(DEH)by focusing on the transcription factor EB(TFEB)/autophagy-lysosome pathway.The cell counting kit-8(CCK-8)was utilized to assess the impact of DEH on the viability of human non-small cell lung cancer cells(A549).The inhibitory effect of DEH on the replication of influenza A virus(H1N1)was determined by real-time quantitative polymerase chain reaction(RT-qPCR).Western blot was employed to evaluate the influence of DEH on the expression level of the H1N1 virus nucleoprotein(NP).The effect of DEH on the fluorescence intensity of NP was examined by the immunofluorescence assay.A mouse model of H1N1 virus infection was established via nasal inhalation to evaluate the therapeutic efficacy of 30 mg·kg^(-1)DEH on H1N1 virus infection.RNA sequencing(RNA-seq)was performed for the transcriptional profiling of mouse embryonic fibroblasts(MEFs)in response to DEH.The fluorescent protein-tagged microtubule-associated protein 1 light chain 3(LC3)was used to assess the autophagy induced by DEH.Western blot was employed to determine the effect of DEH on the autophagy flux of LC3Ⅱ/LC3Ⅰ under viral infection conditions.Lastly,the role of TFEB expression in the inhibition of DEH against H1N1 infection was evaluated in immortalized bone marrow-derived macrophage(iBMDM),both wild-type and TFEB knockout.The results revealed that the half-maximal inhibitory concentration(IC_(50))of DEH for A549 cells was(87.17±0.247)μmol·L^(-1),and DEH inhibited H1N1 virus replication in a dose-dependent manner in vitro.Compared with the H1N1 virus-infected mouse model,the treatment with DEH significantly improved the body weights and survival time of mice.DEH induced LC3 aggregation,and the absence of TFEB expression in iBMDM markedly limited the ability of DEH to counteract H1N1 virus replication.In conclusion,DEH exerts its inhibitory activity against H1N1 infection by activating the TFEB/autophagy-lysosome pathway.

关 键 词：去氢二异丁香酚 TFEB 自噬 甲型流感病毒(H1N1) 

分 类 号：R285[医药卫生—中药学]