摘要
目的 探索木犀草素对汉滩病毒(HTNV)的抑制作用.方法 用0、3.125、6.25、12.5、25、50、100、150、200 μmol/L 的木犀草素处理 Huh-7 细胞,用 0、10、20、25、50、75、100、150、200 µmol/L 的木犀草素处理 A549 细胞,培养48 h后,细胞计数试剂盒8(CCK-8)法检测细胞活力并计算木犀草素半数毒性浓度(CC50).在HTNV感染同时Huh-7细胞分别加入0、8、10、14、16、22μmol/L木犀草素,A549细胞分别加入0、4、6、8、10、12 μmol/L木犀草素,2 h后换液为含对应浓度药物的培养基,培养48 h后,实时荧光定量PCR检测细胞内HTNV RNA水平并计算半数抑制浓度(IC50).在Huh-7细胞感染HTNV的不同时间阶段(感染前1～48h、感染前1 h、感染0～2h、感染后2～48 h、感染后6～48 h、感染后12～48 h、感染后24～48 h),给予25 μmol/L木犀草素处理,感染48 h后,通过蛋白质印迹、免疫荧光、实时荧光定量PCR检测细胞内HTNV蛋白和RNA水平,通过酶联斑块形成实验检测子代HTNV滴度.分别在HTNV入胞阶段(感染0～2 h)及复制阶段(感染后2～48h),用高、中、低浓度(25、12.5、6.25 μmol/L)木犀草素处理感染HTNV的Huh-7细胞,感染48 h后检测细胞内HTNV蛋白和RNA水平.最后通过分子对接模拟木犀草素与HTNV的核衣壳蛋白(NP)和RNA依赖的RNA聚合酶(RdRp)的相互作用效果.结果 与0 μmol/L木犀草素处理比较,25 μmol/L木犀草素处理的Huh-7和A549细胞的细胞存活率差异无统计学意义(F=79.94、14.64,P均＞0.05);后续实验选择25 µmol/L为木犀草素的最大安全浓度;木犀草素对于Huh-7、A549细胞的CC50分别为141、83μmol/L.Huh-7、A549 细胞的木犀草素的 IC50 分别为 16.18、11.64 μmol/L.在 HTNV 感染后 2～48 h、6～48 h、12～48 h、24～48 h给予木犀草素处理,HTNV蛋白、RNA及滴度水平降低,差异均有统计学意义(F=3.48、6.29、18.69,P均＜0.05).木犀草素抑制复制组的HTNV蛋白、RNA水平随着药物浓度的增加而降低,差异均有统计学意义(F=122.90、49.49,P均＜0.05).分子对接结果显示,木犀草素可与HTNV NP和RdRp相互作用,结合能分别为-8.0、-8.1kcal/mol.结论 木犀草素有效抑制HTNV感染,该抑制效果主要作用于HTNV复制周期的转录翻译阶段.
Abstract
Objective To confirm the anti-Hantaan virus(HTNV)activity of luteolin.Methods Huh-7 cells were treated with luteolin at the concentrations of 0,3.125,6.25,12.5,25,50,100,150 and 200 μmol/L;A549 cells were treated with luteolin at the concentrations of 0,10,20,25,50,75,100,150,200 μmol/L.After 48 h of culture,the viability of Huh-7 and A549 cells was detected by cell counting kit-8(CCK-8).Then,the 50％cytotoxic concentration(CC50)of luteolin was calculated.Huh-7 cells infected with HTNV were treated with luteolin at 0,8,10,14,16 and 22 μmol/L;A549 cells infected with HTNV were treated with luteolin at 0,4,6,8,10 and 12 μmol/L.After 48 h of culture,the HTNV RNA level in the cells was detected by qRT-PCR and the half maximal inhibitory concentration(IC50)was calculated.At different stages of HTNV infection(1-48 h before infection,1 h before infection,0-2 h after infection,2-48 h after infection,6-48 h after infection,12-48 h after infection,24-48 h after infection),Huh-7 cells were treated with 25 μmol/L luteolin.After 48 h infection,the levels of HTNV protein and RNA in cells were detected by Western blot,immunofluorescence and qRT-PCR,and the HTNV titer was detected by enzyme-linked focus formation assay test.Huh-7 cells infected with HTNV were treated with high,medium and low concentrations(25,12.5,6.25 μmol/L)of luteolin at the cell entry stage(0-2 h after infection)and replication stage(2～48 h after infection),and the HTNV protein and RNA levels in the cells were detected after 48 h of infection.To detect the interaction between luteolin and HTNV,luteolin is docked with Nucleocapsid protein(NP)and RNA dependent RNA polymerase(RdRp)of HTNV.Results Compared with 0 µmol/L luteolin-treated,there was no statistical significance in cell viability between 25 µmol/L luteolin-treated Huh-7 and A549 cells(F=79.94,14.64;both P＞0.05).In the following experiments,25 μmol/L was selected as the maximum safe concentration of luteolin.The CC50 of luteolin to Huh-7 and A549 cells were 141 and 83 μmol/L respectively.The IC50 of luteolin on Huh-7 and A549 cells were 16.18 and 11.64 μmol/L,respectively.After 2-48 h,6-48 h,12-48 h,24-48 h of HTNV infection,luteolin treatment reduced the HTNV protein,RNA and titer level,with statistical significance(F=3.48,6.29,18.69;all P＜0.05).The HTNV protein and RNA level of replication-inhibited group decreased with the increase of drug concentration,and the differences were statistically significant(F=122.90,49.49;both P＜0.05).Molecular docking results show that luteolin can interact with HTNV NP and RdRp,and the binding energies were-8.0 and-8.1 kcal/mol,respectively.Conclusion Luteolin can effectively inhibit HTNV infection,and this inhibitory effect mainly acts on the transcription and translation stage or the assembly stage of the virus replication cycle.
基金项目
陕西省重点研发计划(2022ZDLSF01-08)
NETL
NSTL
万方数据