Antiviral Activities Of Ginsenoside Rk1 And Rg5 Against Influenza A Viruses In Vitro And In Vivo

Influenza is an acute infectious disease of the respiratory tract both in human and animals caused by the influenza virus.With characters of wide host range,multiple subtypes,rapidly spread,high mortality and the high genetic variability.influenza viruses continually pose serious threats to human and animals.Vaccination and medication are two most commonly employed effective measures to control influenza infections.However,vaccination is ineligible to respond quickly and effectively against outbreaks of newly influenza pandemics because its long production period and no cross-immunization between subtypes.Nowadays,there are two clinically approved anti-influenza drugs:M2ion channel inhibitors including amantadine and rimantadine and neuraminidase（NA）inhibitors including zanamivir,paramivir and oseltamivir.The resistance of two kinds of drugs are increasing.Thus,the need for developing novel antivirals with newly mechanism is very urgent.Drug therapy is one of the important pathways to deal with influenza virus infections.The development of antivirals with characters of multi-targets and multi-mechanisms should be preferentially considered.Containing abundant of compounds with rich chemical structures and various pharmacological activities,Traditional Chinese Medicines（TCM）and natural products are regarded as a treasure-house for searching effective antiviral compounds and provide a shortcut for the development of antivirals.Ginsenosides are the main active ingredients of medicinal plant Ginseng with various functions,including immunity-enhancement,anti-cancer,anti-inflammation and anti-virus and et al.Ginsenoside rg5（G-rg5）is an isomer to ginsenoside rk1,which belong to rare ginsenosides and are deglycosylated products from original ginsenosides.Both of them have been reported to have anti-inflammatory and anti-tumor effects.However,the anti-influenza virus activity of G-rk1 and Grg5 has yet to be determined.In this study,an in vitro model of influenza viruses infecting A549 cells and an in vivo model of PR8 infecting BALB/c mice were established to evaluate anti-influenza activity of G-rk1 and G-rg5.The in vitro activities of G-rk1 and G-rg5 against influenza virus infections were determined by a series of experiments including cytopathic inhibition assay,viral titration and etc.The in vivo effects were evaluated by the protection of G-rk1 on PR8infected mice.The antiviral mechansims of G-rk1 were investigated by a series of experiments including time of addition assay,hemagglutinin inhibition assay and etc.Our results showed that the 50%cytotoxic concentration(CC₅₀)of G-rk1 and G-rg5 in A549cells was 34.78μM and 41.06μM,respectively.G-rk1 and G-rg5 inhibited A549 cells pathogenesis induced by PR8 infections in a dose-dependent manner at concentrations of5μM,10μM and 15μM.Virus titration showed that G-rk1 and G-rg5 inhibited the replication of PR8 on A549 cells in a dose-dependent manner.Indirect immunofluorescence assay（IFA）showed that G-rk1 and G-rg5 significantly inhibited the NP protein expression of PR8 on A549 cells in a dose-dependent manner.Futher more,G-rk1 also inhibited the NP protein expression of H5N1 and H9N2 IAVs on A549 cells,and the inhibitory effect of G-rk1 was better than that of G-rg5.In the experiments of different drug-administration modes,G-rk1 exhibited inhibition on the replication of influenza virus in the pre-treatment mode,co-treatment mode and post-treatment mode.Among three modes,G-rk1 showed the highest inhibition against viral replication in the post-treatment mode.Time of course assay showed the virus replications were significantly inhibited when cells were treated with G-rk1 during 0～2 h,2～4 h and 0～8 h post virus infection.However,hemagglutination inhibition assay showed that G-rk1 could not inhibit the adsorption of influenza virus to cells.The nuclear export observation assay showed that G-rk1 could not block the export of viral ribonucleoprotein complexes and the ultrafiltration centrifugal assay showed that G-rk1 cannot interact with virus directly and inhibits its cell-to-cell spreading.Subcutaneous injection of G-rk1（50 mg/kg/day）could not only significantly increase the survival rate and mean survival time of mice infected with PR8,but also reduced the weight loss of infected mice.In conclusion,our study proved that G-rk1 and G-rg5 has anti-influenza virus activity in vitro and in vivo G-rk1 can significantly protect mice from influenza virus infection.Our results suggest that G-rk1 and G-rg5 may potentially be used as a novel strategy to treat severe or pandemic influenza infections.