Screening Of Effective Components Of Ma Huang Tang Against Influenza A Virus And Mechanism Based On The TLRs Signaling Pathway

Objective1.The influenza virus-infected Madin-Darby canine kidney（MDCK）cells were utilized as the carrier to screen the main effective components in Ma Huang Tang（MHT）,to study the effect and underlying mechanism of MHT and these effective components against influenza A virus in virto.2.An intranasal influenza virus infection in ICR mice were used as the model to investigate the effect of ephedrine（E）and pseudoephedrine（PE）against influenza A virus in vivo,and to study the effect of ephedrine and pseudoephedrine on immunologic function of infected mice.In addition,the possible antiviral mechanism of E and PE in vivo was investigate on the basis of Toll like receptors（TLRs）,which could provided a theoretical basis for the further research.Method1.After infection of influenza A virus,different concentrations of methylephedrine（ME）,E,PE,cinnamic alcohol,cinnamic acid,cinnamic aldehyde,liquiritin,glycyrrhizic acid and amygdalin were given to MDCK cells at the maximum non-toxic concentration（TC₀）.After48 h of intervention,the absorbance（A）value of cells was determined by MTT assay and cell viability was calculated to screen the main active ingredients of MHT against influenza A virus.2.MDCK cells were infected by influenza A virus,blocking influenza virus invading host cells,intervening the adsorption of influenza virus,directly inhibiting influenza virus and inhibiting the biosynthesis of influenza virus were utilized as four different administration methods,MTT assay was used to observe the effect and characteristics of Ma Huang Tang,ME,E and PE against influenza A virus;at 24 h and 48 h after the treatment,the virus load,the level of tumor necrosis factor alpha（TNF-α）,interleukin（IL）-6 and interferon beta（IFN-β）cykotines,TLR3,TLR4,TLR7,Myeloid differentiation factor（My D88）,tumor necrosis factor receptor related factor-3（TRAF3）,tumor necrosis factor receptor related factor-6（TRAF6）,interferon regulatory factor-3（IRF3）,nuclear factor-κB p65（NF-κB p65）and IFN-βm RNA expression levels of MDCK cells infected with influenza A virus were detected.3.ICR mice were randomly divided into the normal group,the virus group,the oseltamivir group,the low,moderate and high dose of E and PE treated group.At the 3th day and 7th day after intranasal influenza virus infection,the lung index,the spleen index,thymus index,the virus load and pathologic changes of lung tissues of mice were obversed,ELISA method was used to detecte the level of IL-1β,6,10 in serum,RT-PCR assay was employed to measure the m RNA expression level of interferon gamma（TFN-γ）and TNF-αcykotines as well as TLR3,TLR4,TLR7,My D88,NF-κB p65 and retinoic acid inducible gene-1（RIG-1）,in addition,Western blot assay was used to detecte the protein expression level of TLR4,TLR7,My D88 and NF-κB p65.Results1.In the nine major bioactive components of MHT,ME,E and PE all significantly increased the A values in the treatment groups with the drug concentration dependence（P<0.01）,the cell survival rate could reach more than 70%at each TC₀;the A values in most concentrations groups of cinnamic aldehyde and glycyrrhizic acid were also significantly increased（P<0.05,P<0.01）,in which the survival rate of the cells increases were not obvious,and the survival rates of the cells were only about 47%at each TC₀;while cinnamic alcohol,cinnamic acid,liquiritin and amygdalin had little effect on the A value and cell survival rate of each group after influenza A virus infection.2.TC₀ of MHT,ME,E and PE for MDCK cells were 5.00 mg·m L^-1,31.25μg·m L^-1,15.63μg·m L^-1,15.63μg·m L^-1,respectively.In the TC₀ range,MHT,ME,E and PE could inhibit the proliferation of influenza A virus by blocking influenza virus invading host cells,intervening the adsorption of influenza virus,directly inhibiting influenza virus and inhibiting the biosynthesis of influenza virus.At 24 h and 48 h after the treatment,MHT,ME,E and PE significantly inhibited the virus load in infected MDCK cells（P<0.05,P<0.01）and the inhibitory effect at 24 h after the treatment was more obvious than that at 48 hours.Furthermore,MHT,ME,E and PE significantly decreased the level of TNF-αin infected MDCK cells（P<0.05,P<0.01）,the high and middle dosages of MHT and the high dosage of ME,E and PE significantly increased the level of IFN-β（P<0.05,P<0.01）,and the high dosage of MHT,ME,E and PE had a certain promoting effect for the secretion of IL-6（P<0.05,P<0.01）.Additionally,the high and middle dosages of MHT could significantly inhibit the m RNA expression level of TLR3,TLR4,TLR7,My D88 and TRAF6（P<0.05,P<0.01）,and induce the expression of IFN-βm RNA（P<0.05,P<0.01）;the high and medium dosages of ME,E and PE all inhibited the expression of TLR3,TLR4,TLR7,My D88,TRAF3,IRF3and NF-κB p65 m RNA in infected MDCK cells（P<0.05,P<0.01）,and significantly induced the expression of IFN-βm RNA（P<0.05,P<0.01）.3.At the 3th day and 7th day after infection,the high and middle dosages of E and PE could significantly decrease the virus load in the lung（P<0.05,P<0.01）,the lung index and thymus index of influenza virus-infected mice（P<0.05,P<0.01）,significantly attenuate lung histopathological changes,markedly down-regulate the level of IL-1β（P<0.05,P<0.01）and up-regulate the level of IL-10（P<0.05,P<0.01）in serum,significantly reduce the m RNA expression of TNF-α,TLR3,TLR4,TLR7,My D88,NF-κB p65 and RIG-1（P<0.05,P<0.01）,increase the m RNA expression of IFN-γ（P<0.05,P<0.01）,and significantly decrease the protein expression of TLR4,TLR7,My D88 and NF-κB p65（P<0.05,P<0.01）.Conclusion1.ME,E and PE in MHT have obvious protective effects on MDCK cells infected with influenza A virus,indicating that they have a certain inhibitory effect on influenza A virus.2.In the non-toxic concentration range,MHT,ME,E and PE can inhibit influenza A virus in various ways,have a certain influence on the process of influenza virus invading cells,adsorbing on cells and the biosynthesis of influenza virus in cells,even can directly kill the influenza virus,which may be closely associated with the down-regulation of m RNA expression of related genes in TLR3,TLR4 and TLR7 signaling pathways and subsequent regulation of TNF-α,IL-6 and IFN-βcytokine.3.E and PE have a certain protect effects on the influenza virus-infected mice in vivo,which may be related to their abilities of effectively alleviating lung histopathological changes,improving the immunologic function of infected mice by regulating the imbalance of inflammatory cytokines secretion and adjusting the m RNA expression level of TLR3 and RIG-1 as well as the m RNA and protein expression level of TLR4,TLR7,My D88 and NF-κB p65.