Studies On The Effect And Mechanism Of Myricetin And Dihydromyricetin Against Influenza Virus In Vitro

BackgroundInfluenza virus are classified into four groups,A,B,C,and D,and its infection can causec highly contagious respiratory diseases and widely spread between humans and animals.Influenza A virus(IAV)is the major cause of seasonal and pandemic influenza with characteristic of high morbidity and mortality,seriously threatened public health and social economic development.In present,the clinical use of anti-inflenza drugs mainly included M2 ion channel inhibitors and neuraminidase inhibitors.However,constantly emerging drug-resistance and side-effects limit usage of these anti-influenza drugs.In order to improve the statue,enormous efforts have been undertaken to develop anti-influenza drugs targeting different stages of IAV life cycle for treatment and prevention of influenza infection.The past of antiviral research more focused on the replication process of the viruses,which resulted in drug resistance or cross resistance.It is very vital significance to discover antiviral drugs with dual function which not only inhibit the virus replication,but protect the host cells.Therefore,novel drugs with dual-actions mechanism are superior to drugs with the single target.The natural products have received extensive attention owing to multiple pharmacological activities,especially low-toxicity and uneasily induced drug-resistance.Therefore,the natural products are regarded as excellent screening library for screening antiviral drugs.It's worth noting that most of natural products have dual pharmacological activity against influenza viruses from the host and virus.In previous work,we screened small molecules from TagerMol natural product library,which could bind to the cap-binding domain of the PB2 subunit(PB2cap)of IAV.In the project,fluorescence polarization assay were established to screen the natural molecular on basis of PB2cap activity of influenza A virus.Furthermore,the results of fluorescence polarization assay demonstrated that myricetin and dihydromyricetin could competitively bind to PB2cap in does-dependent manner.ObjectionsIn present study,we explored the anti-influenza effects and action mechanism of myricetin and dihydroyricetin.Their immune adjustment mechanisms were also investigated.The study aimed to explore and develop new anti-influenza drugs from natural products,meanwhile provided new idea for solving problems with single pharmaceutical effect,drug-resistance and side effects.Methods1.Cap-binding assay and fluorescence polarization assay were served as evaluation methods to screen active agents from natural compounds library.2.MTT-based cell activity assay was applied to determine evaluate the efficacy and cytoxicity of myricetin and dihydromyricetin on MDCK and A549 cells.Plaque assay and qRT-PCR were used to determine anti-influenza viral activity.3.Immunofluorescence microscopy,western blot and quantitative RT-PCR were adopted to measure the effect of myricetin and dihydromyricetin on replication of influenza virus.4.H5N1 pseudo virus neutralization assay,time-of-addition were used to explore which periods of time myricetin and dihydromyricetin exerted inhibitory activity.5.The influence of myricetin and dihydromyricetin on influenza viral vRNP activity were detected by mini-replicon,FP,SPR,and molecular docking were performed to analyze the binding between compounds and PB2cap.6.Quantitative RT-PCR was applied to evaluate influenza A virus induced the release of inflammatory cytokines,and western blot was ultilized to detect the expression level of NF-?B,P3 8 MAPK and TLR3 protein.Results1.Based on screening system targeted PB2cap activity in vitro,myricetin and dihydromyricetin were selected as the objects of study.2.Myricetin and dihydromyricetin could suppress different influenza A virus.The IC50 of myricetin for A/Puerto Rico/8/34(H1N1),A/FM-1/1/47(H1N1),and A/Aichi/2/68(H3N2)were 9.82±1.81,17.44±4.44,10.74±3.33 ?M,respectively.While the IC50 of dihydromyricetin were 15.18±0.92,23.33±4.83,11.67±2.28?M.3.Deverse infectious manners and H5N1 pseudovirus neutralization experiment showed myricetin and dihydrohydromyricetin exerted inhibitory activity of influenza virus at post-infection of influenza virus.4.During the influenza viral single life-cycle,the specific action periods of myricetin and dihydromyristin were 1-3,3-5 h respectively.5.Myricetin and dihydromyricetin affected the activity of influenza vRNP complex in dose-dependent manner.6.TLR3 signaling pathway could be activated during influenza viral infection,but the expression of TLR3 and cell cytokines could reduce significantly with treated with myricetin and dihydromyricetin.Conclusions1.Myricetin and dihydromyricetin effectively inhibited influenza A virus in the early stage of viral replication.2.Myricetin and dihydromyricetin supressed vRNP activity of influenza virus by competitively binding to PB2cap.3.Myricetin and dihydromyricetin not only inhibited the replication of IAV,but reduced excessive inflammatory immune response by down-regulating TLR3 signaling pathway.