Activities Of Astragaloside IV Against Influenza A Viruses In Vitro And In Vivo

Influenza is a highly contagious respiratory disease caused by influenza virus infection, which is characterised with strong infectivity, rapid transmission and highly morbidity. Pandemics caused by influenza A viruses(IAV) usually cause higher mortality rates than that of seasonal influenza epidemics, and result in seriously threatens on human welfare and animal health worldwide. Although vaccination remains the best strategy of preventing influenza virus infections currently, it’s use has been restricted by the shortcomings of highly variability of influenza viruses, delay development of viral vaccine and inability of cross protection to another subtype influenza virus strain. To date, two classes of antiviral drugs: M2 ion channel inhibitors(amantadine and rimantadine) and neuraminidase inhibitors(NAIs, zanamivir and oseltamivir) have bee approved for the treatment and prevention of influenza. However,almost 100% IAV strains are resistant to M2 ion-channel blockers, as they have been used for decades. As for NAIs, increasing incidences of resistance development against NAIs have been reported. Consequently, available strategies are far from enough for dealing with influenza pandemics because of shortcomings of available vaccinations and drugs. Therefore, the urgency of the development of novel anti-influenza drugs has been highlighted.Astragalus membranaceus is a famouse Traditional Chinese Medicine plant and has being used for thousands of years in China and East Asia. Astragaloside IV(AST), one of lanolin alcohol tetracyclic triterpenoid saponins, is a major bioactive ingredient extracted from Astragalus membranaceus roots. A lot of documented studies show that AST possesses a wide spectrum of activities, including immunoregulation,organ protection, anti-inflammation and antivirus, anti-hypoglycemic, anti-aging, improving hemorheology and et al. However, as far as we know, the antiviral activity of AST against influenza viruses has not been reported. In this study, in vitro cell infection models were established by infecting lung adenocarcinoma epithelial cells(A549) with three different influenza viruses strains including A/PR/8/34(H1N1), A/Guangzhou/03/2009(pan H1N1) and A/Duck/Guangdong/99(H5N1) respectively. The inhibition of AST on proliferation of progeny virus in A549 cells was assessed by titrating virus titer with the endpoint dilution assay. A BALB/c mouse model infected with PR8 was used to evaluate protective effects of AST on infected mice. The parameters used for efficacy evaluation included body weigh change and survival. In addition, a multi-passing experiments was used to address whether AST has a tendency to induce resistant variants. To elucidate the underlying mechanisms, a series of experiments were conducted using cellular model infected with PR8. In the time-of-addition experiment, the expression of viral nucleoprotein(NP) m RNA were analyzedby real-time PCR to determine which stages were inhibited by AST in the single replication circle of influenza virus. Hemagglutinin-inhibition(HI) test, hemolytic inhibiton assay and NA inhibition assay were used to investigate the effect of AST on hemagglutinin(HA) and neuraminidase(NA) of influenza virus. Results indicated that AST ranging from 6.25 to 25 μg/m L showed significant inhibitory activity against PR8、pan H1N1 and H5N1 progeny virus in A549 cell cultures with an advantage of being not easy to induce viral resistance. AST at 400 μg/m L and lower concentrations have no cytotoxicity on A549 cells. Subcutaneous injection of AST at 30 mg/kg/d showed significantly protective activities for BALB/c mice infected PR8, including significant increase of the survival rate and the mean survival days, and decrease of body weight lose of infected mice. In the time-of-addition assay, AST was found to exhibit a significant inhibition on multiplication of IAV when it was added during 0~2 h post infection. However, results of hemagglutinin-inhibition test and hemolytic inhibiton assay showed that AST had no effect on the absorption of PR8 towards A549 cells and the fusion between the virus and endsomal membranes. Results of NA inhibition assay showed that AST had no inhibitory effects on NA. Thinking together, it can be speculated that AST exhibitedantiviral activities by inhibiting replications of IAV in the early phase.To our best knowledge, it is the first time for this study to reveal the antiviralactivities of AST against influenza A virus infection in vitro and in vivo with the advantage of being not easy to induce viral resistance. This finding has provided theprimary basis for the clinical use of Astragalus membranaceus against influenzas, aswell as for the development of AST as a novel antiviral drug or a lead compound against influenza viruses.