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.
Hantaan virusHemorrhagic fever with renal syndromeLuteolinAntiviral infection
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