The Effect And Mechanism Of Representative Prescriptions With Heat-clearing And Detoxification Activity Against Influenza A Virus

Aim:To clarify the effect and potential common mechanism of Chinese patent medicines with activities of heat-clearing and detoxifying against influenza A virus,the relationship between their compositions and efficacy of anti-influenza A virus in vivo were firstly investigated in ICR mice infected with influenza A(H1N1)virus.To further investigate the potential common mechanism of these Chinese patent medicines mainly derived from Yin-Qiao-San for clinical treatment of influenza,the antiviral activities of one representative Chinese patent medicines,Fufang-Yinhua-Jiedu Granules(FFYH)against influenza A virus in vivo and in vitro,was further evaluated and its potential mechanism against influenza A virus were in-depth explored.To achieve a more ideal therapeutic effect on influenza,the synergistic effect and potential mechanism of combined administration of FFYH and oseltamivir against influenza A virus were further investigated.Our work is expected to provide a theoretical basis for the clinical treatment of influenza with traditional Chinese medicines and the development of innovative Chinese Patent Medicines.Methods:In this study,the antiviral effect of 40 kinds of Chinese Patent Medicine with heat-clearing and detoxifying activity against influenza A virus were firstly evaluated in ICR mice infected with influenza A(H1N1)virus by comparing their difference in mortality and survival period,and then one MDCK cell model was used to evaluate the in vitro broad-spectrum antiviral effects of FFYH against influenza A virus such as H1N1,H3N2,H5N1,H7N9 and H9N2 with CPE inhibition test and HA assay.The therapeutic effect of FFYH on influenza A virus in vivo was comprehensively investigated by measuring the changes in diet,weight loss,survival time,death protective rate,lung injury and blood parameters.The expression level of inflammatory factors in sera caused by influenza A virus was examined by ELISA.qRT-PCR and Western blot methods were used to investigate the underlying mechanism of FFYH against influenza A virus in vitro and in vivo.Furthermore,the synergistic effect and potential mechanism of combined administration of FFYH and oseltamivir against influenza A virus in vivo and in vitro were explored using the above-mentioned similar methods.Results:(1)The results of in vivo efficacy evaluation showed that 19 of 40 kinds of Chinese Patent Medicine with heat-clearing and detoxifying activity,which are derived from Yin-Qiao-San showed siginficant therapeutic effect on diseases caused by influenza A virus,while others don’t have any obvious effect on this diseases.(2)One representative prescription(FFYH)showed a good therapeutic effect on influenza A virus,which not only had a good inhibitory effect on the replication of H1N1,H3N2,H5N1,H7N9 and H9N2 subtypes of influenza virus in vitro,but also can markedly increase the survival rate of mice by prolonging the survival time of mice,improving blood parameters and reducing lung injury.Mechanistic studies have demonstrated that the therapeutic effect of FFYH in vivo on influenza A virus is directly related to its inhibition of transcription of TLR7,MyD88,IRAK4,IRF7,TRAF3 and TRAF6 and the expression of inflammatory factors(TNF-α,IL-6,IFN-γ,IL-1β and IP 10).Moreover,this conclusion was further confirmed using human lung adenocarcinoma A549 cell,suggesting that targeting TLR7/MyD88 signaling pathway might be the critical mechanism of FFYH against influenza A virus.(3)The in vivo results showed that the combined administration of FFYH and oseltamivir phosphate(Ose)can more markedly prolong the survival time of mice than that of FFYH or Ose alone(FFYH 10.6±3.1 day,Ose 12.6±2.6 day,combination 13.7±2.1 day,respectively),and more significantly increase the survival rate of mice(FFYH 30%,Ose 50%,and combination 70%,repectively).Furthermore,the combination of FFYH and Ose also significantly improved hematological parameters and lung injury caused by influenza A virus.Mechanistic study in vivo indicated that the combination of FFYH and Ose not only synergisticly enhanced the inhibitory effect on influenz A virus,but also synergisticly inhibited TLR7/MyD88 signaling pathway to reduce acute lung injury caused by overstress inflamatory response.Conclusion:Our results indicated that most of heat-clearing and detoxifying prescriptions with anti-influenza viral activity are mainly derived from the classic prescription Yin-Qiao-San.One representative prescription,FFYH,not only can effectively inhibit the replication of multiple subtypes of influenza virus in vitro,but also in vivo significantly reduce the mortality caused by lethal influenza virus infection.Furthermore,the combined administration of FFYH and Ose showed a good synergistic effect on influenza A virus.FFYH or combination with Ose not only can markedly inhibit the replication of influenza A H1N1 virus,but also significantly block the TLR7/MyD88 signaling pathway to reduce the excessive inflammatory responses caused by influenza A virus,suggesting that targeting inhibition of the TLR7/MyD88 signaling pathway may be a common mechanism of these heat-clearing and detoxifying Chinese patent medicines for clinical treatment of influenza.