目的 探索防己诺林碱在细胞水平抗H1N1病毒的作用，阐明防己诺林碱调控细胞自噬抑制H1N1病毒复制的分子机制。方法 CCK-8法检测0.312 5、0.625 0、1.250 0、2.500 0、5.000 0、10.000 0、20.000 0、40.000 0、60.000 0 μmol·L^-1的防己诺林碱对MDCK细胞活力的影响；MDCK细胞设置对照组、模型组和防己诺林碱（2.5、5.0、10.0 μmol·L^-1）组，除对照组外，以感染复数（MOI）为0.1的H1N1病毒感染MDCK细胞，加入防己诺林碱共同孵育12 h，同时设置防己诺林碱（10.0 μmol·L^-1）与病毒共同孵育4、8 h组，通过实时荧光定量PCR （qRT-PCR，检测HIN1 mRNA表达）和Western blotting ［H1N1病毒核蛋白（NP）］检测防己诺林碱对病毒复制的抑制作用；PI/Hoechst 33342染色检测防己诺林碱（10.0 μmol·L^-1）对MDCK细胞死亡的影响；qRT-PCR法检测防己诺林碱检测防己诺林碱预处理、病毒感染早期药物处理、病毒感染晚期药物处理以及吸附和进入阶段给药对病毒复制的影响；MDCK细胞转染EGFP-LC3或EGFP-mCherry-LC3双荧光质粒，观察防己诺林碱对EGFP-LC3的荧光强度、EGFP-mCherry-LC3共定位的影响，设置自噬诱导剂雷帕霉素（0.5 μmol·L^-1）、自噬晚期抑制剂氯喹（10 μmol·L^-1）、巴佛洛霉素A1 （100 nmol·L^-1）对照。转染过EGFP-LC3质粒的MDCK细胞感染H1N1病毒，免疫荧光检测防己诺林碱对LC3与胞内的病毒NP蛋白共定位的影响；体外培养A549细胞，以MOI为0.1的H1N1病毒感染，Western blotting检测防己诺林碱对LC3II/LC3I蛋白表达的影响。结果 防己诺林碱对MDCK细胞的半数抑制浓度（IC₅₀）为40.19 μmol·L^-1 ；与模型组比较，防己诺林碱以浓度相关性的方式抑制HIN1 mRNA表达，以浓度和时间相关性的方式抑制NP蛋白表达（P＜0.01、0.001）；显著减少H1N1诱导的细胞死亡（P＜0.001）；抑制H1N1病毒进入过程。与对照组比较，防己诺林碱处理诱导LC3荧光聚集，诱导EGFP-LC3和mCherry-LC3双荧光共定位显著增加（P＜0.001）。与模型组比较，防己诺林碱处理明显减少NP的荧光数量（P＜0.001），同时造成LC3荧光积累并与胞内的病毒NP蛋白共定位；引起LC3II积累（P＜0.01、0.001）。结论 防己诺林碱同氯喹和巴佛洛霉素A1一致，阻断自噬途径，使病毒粒子在自噬体内滞留，破坏病毒生命周期。

Objective To investigate the antiviral effect of fangchinoline against the H1N1 virus and elucidate its molecular mechanism in regulating cellular autophagy. Methods CCK-8 method was used to detect the effect of tetrandrine of 0.312 5, 0.625 0, 1.250 0, 2.500 0, 5.000 0 0, 10.000 0 0, 20.000 0, 40.000 0, and 60.000 0 0 μmol·L^?1 on viability of MDCK cells. MDCK cells were divided into control group, model group, and tetrandrine (2.5, 5.0, and 10.0 μmol·L^?1) groups. Except for control group, MDCK cells were infected with H1N1 virus with a multiple infection index (MOI) of 0.1, and co-incubated with tetrandrine for 12 h. At the same time, tetrandrine (10.0 μmol·L^?1) was set up incubated with the virus for 4 and 8 h, and detected the inhibitory effect of tetrandrine on virus replication through real-time fluorescence quantitative PCR (qRT-PCR, detecting HIN1 mRNA expression) and Western blotting [H1N1 virus nucleoprotein (NP)]. PI/Hoechst 33342 staining was used for detection of tetrandrine on MDCK cell death. QRT-PCR method was used to detect the effects of pre-treatment with tetrandrine, early drug treatment for viral infection, late drug treatment for viral infection, as well as adsorption and entry stage administration on virus replication. MDCK cells were transfected with EGFP-LC3 or EGFP-mCherry-LC3 dual fluorescent plasmids to observe the fluorescence intensity of EGFP-LC3 and EGFPmCherry-LC3 co-localization. MDCK cells transfected with EGFP-LC3 plasmid were infected with H1N1 virus, and immunofluorescence was used to detect the effect of tetrandrine on LC3 and co-localization with intracellular viral NP protein. A549 cells were cultured in vitro and infected with H1N1 virus with a MOI of 0.1. Western blotting was performed to detect the effect of tetrandrine on expression of LC3II/LC3I protein. Results The half inhibitory concentration (IC₅₀) of tetrandrine on MDCK cells is 40.19 μmol·L^?1. Compared with model group, tetrandrine inhibited HIN1 mRNA expression in a concentration dependent manner and NP protein expression in a concentration and time dependent manner (P < 0.01, 0.001), significantly reduced H1N1 induced cell death (P < 0.001), and inhibited the entry process of H1N1 virus. Compared with control group, treatment with tetrandrine induced LC3 fluorescence aggregation and significantly increased co-localization of EGFP-LC3 and mCherry-LC3 dual fluorescence (P < 0.001). Compared with model group, treatment with tetrandrine significantly reduced the fluorescence quantity of NP (P < 0.001), while causing the accumulation of LC3 fluorescence and co-localization with intracellular viral NP proteins, causing accumulation of LC3II (P < 0.01, 0.001). Conclusion Fangchinoline is consistent with bafilomycin A1, blocking the autophagy pathway, causing viral particles to remain in the autophagy, and destroying the life cycle of the virus.

R285.5

北京市科技新星计划（2023107）；中华中医药学会青年人才托举工程项目（CACM-2023-QNRC2-A02）；国家自然科学基金资助项目（82001663）