The Effect And Mechanism Of Fufang-Yinhua-Jiedu Decoction Against Influenza A Virus

Aim:To investigate the effect and mechanism of Fufang-Yinhua-Jiedu Decoction(FFYH,one Chinese patent medicine with clearing heat and detoxicating)treating influenza A virus infection,the antiviral effect of FFYH was firstly evaluated with in vitro and in vivo models and the underlying mechanisms of FFYH against influenza A virus infection were investigated with network pharmacology.Furthermore,based on the results of network pharmacology,the potential mechanism of FFYH regulating the innate immunity or autophagy to control the replication of influenza A virus and excessive inflammatory responses were discoveried,which will lay a theoretical foundation for interpreting the action mechanism of representative prescriptions of heat-clearing and detoxifying-like Chinese traditional medicine(TCM)in the treatment of respiratory tract virus infection.Methods:(1)MDCK and A549 cells infected with H3N2 virus as in vitro models were used to investigate the inhibitory effect of FFYH on virus replication with CPE inhibition assay and HA assay.Secondly,qRT-PCR,Western blot,and immunofluorescence were used to further confirm the antiviral effect of FFYH on H3N2 virus replication.Moreover,ICR mice infected with H3N2 virus as an in vivo model was used to evaluate the protective effect of FFYH on influenza virus infection confirmed by diet loss,body weight loss,survival time,lung index,mortality,and lung pathological injury.Finally,a multi-cytokine microarray were used to examine the inhibitory effect of FFYH on excessive cytokine storm.(2)Using the active components of single Chinese medicine of FFYH and key active compounds derived from FFYH as research objects,the pharmacological components and corresponding targets were searched and screened from TCMSP and Pubchem database,and then network pharmacology maps of TCM,Compound-Target-Disease,Protein-Protein Interaction(PPI),Gene Ontology(GO),and Kyoto Encyclopedia of Genes and Genomes(KEGG)were constructed using network analysis softwares to investigate the potential drug targets and related signal pathways of FFYH treating influenza.Molecular docking was performed to futher identify the potential targets and underlying mechanism of FFYH against influenza virus infection.(3)Based on the significant increase of monocyte-associated inflammatory cytokines and the results of network pharmacology analysis,human THP-1 monocytes were used to investigate the inhibitory effect of FFYH on inhibiting the replication of influenza A virus and excessive inflammatory responses by regulating host immunity with qRT-PCR and Western blot methods.Moreover,an in vivo model of mice infected with H3N2 virus was used to further validate the underlying mechanism of FFYH regulating the host innate immunity and excessive inflammatory responses.(4)Based on the results of network pharmacology indicating the effect of FFYH against influenza virus infection may be highly associated with the autophagy-related pathways,A549 and THP-1 cells were used as in vitro models to investigate the effect of FFYH regulating autophagy to inhibit virus replication with qRT-PCR and Western blot methods.And then,blocking autophagy with pharmacological methods were used to confirm the effect of FFYH regulating autophagy to inhibit virus replication.Finally,A549 cells with stable expressing mCherry-GFP-LC3B cells were used to investigate the blockage effect of FFYH on autophagic flux induced by influenza A(H3N2)virus with laser confocal microscopy.Results:(1)Our results indicated that FFYH not only dose-dependently inhibited influenza A(H3N2)virus replication,RNA transcription,and protein expression in vitro,but also effectively prolonged the survival time,significantly reduced mice mortality,inhibited the levels of inflammatory factors,and improved lung tissue pathological damage in vivo,suggesting that FFYH had a good protective effect on influenza H3N2 virus infection.(2)Network pharmacology results showed that the core targets shared by the active ingredients of single traditional Chinese herbs and key active components derived from FFYH mainly included MAPK1,SRC,HSP90AA1,and EGFR.Key signaling pathways associated with the mechanism of FFYH against IAV infection mainly included PI3K-AKT signaling pathway,MAPK signaling pathway,TNF signaling pathway,NF-κB signaling pathway,autophagy,mTOR signaling pathway,Toll-like receptor signaling pathway and RIG-Ⅰ-like receptor signaling pathway.(3)Our results indicated that FFYH dose-dependently inhibited influenza A(H3N2)virus RNA transcription and protein expression,and effectively inhibited the expression of proinflammatory factors such as IL-6,CXCL-10,IFN-α,and NLPR3 in THP-1 monocytes.Mechanistic studies showed that FFYH not only could effectively inhibit excessive inflammatory responses through regulating TLR7/MyD88/IRF7 signaling pathway,but-also could inhibit influenza virus replication by upregulating the RIG-I/MDA5-IRF3 antiviral immune signaling pathway to promote the secretion of ISGs such as PRK,Mx1,ISG15,TRIM25.Therefore,we speculated that FFYH may exert protective effect against influenza virus infection by the dual mechanism of inhibiting the TLR7/MyD88/IRF7 inflammatory signaling pathway and enhancing the RIG-I/MDA5-IRF3 antiviral immune signaling pathway.(4)Our results indicated that H3N2 induced autophagy in THP-1 and A549 cells through increasing the aggregation of autophagy marker LC3B-Ⅱ,promoting the degradation of P62,and inducing autophagic flux.However,blocking autophagy with pharmacological methods could enhance the expression accumulation of LC3B-Ⅱ and P62,while inhibit the viral replication,suggesting that H3N2 may promote its replication by enhancing autophagy.Similar to autophagy blockers such as CQ and BafA1,FFYH could inhibit the replication of H3N2 virus by blocking H3N2-mediated autophagy,which was confirmed by enhancing the aggregation of LC3B-II and P62,blocking the formation of autophagic flux,and inhibiting the expression of viral NP.Furthermore,blocking autophagy with pharmacological methods could synergistically enhance the inhibitory effect of FFYH on viral replication,suggesting that blocking H3N2-induced autophagy may be one key mechanism of FFYH against influenza A virus infection.Conclusion:In summary,our results demonstrated that FFYH exhibited a good therapeutic effect on influenza A(H3N2)virus infection in vitro and in vivo.Mechanistic studies indicated that FFYH not only directly inhibits the replication of influenza A H3N2 virus by inhibiting viral RNA transcription and protein expression,but also indirectly inhibits influenza virus replication by regulating the host innate antiviral immune through RIG-I/MDA5-MAVS/IPS1-IRF3 signaling pathway and autophagy.In addition,FFYH exhibited a good inhibitory effect on the excessive inflammatory responses,of which may be associated with inhibition of the TLR7/MyD88/IRF7 inflammatory signaling pathway.Collectively,our results clearly indicated that FFYH may exert comprehensive efficacy against influenza through multiple targets and signaling pathways.