摘要
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/Hoechst33342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.
Abstract
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 EGFP-mCherry-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 Al,blocking the autophagy pathway,causing viral particles to remain in the autophagy,and destroying the life cycle of the virus.
基金项目
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(82001663)
NETL
NSTL
万方数据