Study On The Mechanism Of Dehydroandrographolide Against Influenza Virus

Zoonotic influenza(abbreviated to influenza)is a highly contagious acute respiratory disease caused by influenza virus(IV).Since influenza has caused a great damage to public health,the prevention and treatment of influenza are becoming increasingly urgent.Vaccine currently used has a low protection rate and no effect on new influenza viruses due to antigenic drift or transformation.Meanwhile,there are two classes of clinically approved antivirals against influenza:M2 channel inhibitors(rimantadine and amantadine)and neuraminidase(NA)inhibitors,such as oseltamivir.These drugs are limited in clinical practice due to their toxicity,big side effects,rapid emergence of drug resistance strains,incomplete role and so on.This situation underlines the urgent need for inventing novel antivirals.To find natural drugs from plants is an important strategy to develop novel antivirals.Traditional medicine and ethnic folk medicine in China is rich with treasures and has a bright future in the discovery of novel anti-IV drugs and natural medicine.Andrographis is dry ground part of a traditional Chinese medicine,Andrographis paniculata(Burm.f)Nees.The major and active components of A.paniculata are diterpenoid lactones,such as andrographolide,14-deoxy-11,12-didehydroandrographolide.A.paniculata and its diterpenoid lactones have multiple pharmacological activities,including anti-inflammatory,antipyretic,analgesic,anticancer,antiviral and anti-bacterial effects.To date,only few studies have shown that andrographolide and dehydroandrographolide can inhibit IV.The mechanism of its anti-IV,the actions of link with multiple targets of active ingredients of anti-IV is still unknown.And the influence on the innate immunity of host,the influence on the apoptosis induced by IV and the related signal transduction pathways as well,remain unclear.In this paper,dehydroandrographolide has been studied on its effect and mechanisms against IV.In vitro experiments,we found that dehydroandrographolide can significantly inhibit the proliferation of influenza A virus(IAV),such as A/chicken/Hubei/327/2004(H5N1),A/duck/Hubei/XN/2007(H5N1),A/PR/8/34(H1N1),A/NanChang/08/2010(H1N1)and A/HuNan/01/2014(H3N2)in MDCK and A549 cells.In vivo experiments,we found that DAP can decline the body weight of mice,improve the survival rate,reduce virus titers and lesions in lung tissue caused by A/chicken/Hubei/327/2004(H5N1),A/duck/Hubei/XN/2007(H5N1)and A/PR/8/34(H1N1)within non-toxic concentrations.We also found that DAP can reduce the over-expression of cytokines(TNF-a,IL-6 and IL-1β)and chemokines(CCL-2)in the lung tissue,compared with the control group.Furthermore,we have set A/chicken/Hubei/327/2004(H5N1)as an example to elucidate the underlying mechanisms.Our studies demonstrated that DAP robustly inhibited H5N1 replication by reducing the production of NP mRNA,NP and NS1 protein,whereas DAP had no effect on the absorption and release of H5N1 towards/from A549 cells.DAP also effectively restrained the nuclear export of viral ribonucleoprotein(vRNP)complexes.Meanwhile,DAP significantly reduced the upregulated expression of all of the tested proinflammatory cytokines(TNF-α,IL-6,IL-8,IFN-α,IL-1β and IFN-β)and chemokines(CXCL-10 and CCL-2)stimulated by H5N1.Moreover,we found that DAP can inhibit the NF-kB signalling pathway activated by IAV.So we conclude that DAP can modulate the host innate immune system,decrease the host damage caused by cytokine storm to exert an antiviral activity.In addition,DAP can reduce the number of apoptotic cells caused by H5N1.DAP can inhibit the expression of Cleaved-PARP p89,Caspase-3(active)and activation of Caspase-8,Caspase-9.DAP can inhibit the decrease of mitochondrial membrane potential,hinder the release of Cytochrome C from mitochondria into cytoplasm and reduce of the ratios of pro-apoptotic protein Bax/anti-apoptotic protein Bcl-2 of mitochondria.Our further experiments has shown that DAP can inhibit apoptosis induced by IV by activating PI3K/Akt pathway,improve the survival rate of host cells and play a critical role against IV.