Mechanism Of Yinhuapinggan Granule Against Influenza A/H1N1 Virus Based On The TLRs/MyD88 Signaling Pathway In Vitro

Objective The present study aimed to investigate the therapeutical effect of Yinhuapinggan granule(YHPG)against influenza A/PR8/34(H1N1)virus in vitro,and to explore the mechanism of toll-like receptor 7(TLR7)/myeloid differentiation factor 88(MyD88)signaling pathway in anti-influenza.In this study,pathological changes in cell,inflammatory factors and viral mRNA expression level were being examining index after the H1N1 influenza virus infection in the Madin-Darby canine kidney(MDCK)to observe the proliferation inhibition effect of drugs to the H1N1 virus,and to explore the mode of action of the influenza virus.Method 1.Effect of Yinhuapinggan Granule on Anti-Influenza Virus in Vitro:H1N1 influenza virus through the chicken embryo proliferation reserve.Using MDCK cells as virus host to establish virus infection model,according to Reed-Muench method to calculate tissue infection(TCID50).Normal,model,YHPG with high,medium and low dose(0.625,0.313,0.156 mg·mL-1)and ribavirin groups(0.780 mg·mL-1)were set up.Median toxic concentration(TC50)and maximum non-toxic concentration(TC0)of MDCK cells were measureed by MTT colorimetry and cytopathic(CPE)method.Inhibitory concentration 50(IC50)and therapeutic index(TI)were analyzed to inhibit the proliferation of influenza virus in different modes of action.2.Mechanism of Yinhuapinggan Granule on Anti-Influenza Virus in Vitro:The levels of interferon-β(IFN-β)and interleukin 6(IL-6)in MDCKs were detected by enzyme-linked immunosorbent assay(ELISA).The levels of TLR7,MyD88,TRAF6,JNK,p38 MAPK,and p65 NF-κB gene expression in MDCK were detected by real-time quantitative fluorescence PCR.Results 1.The results show that the TCID50 of influenza H1N1 virus was 10-3.5/0.1 mL.The TC50 and TC0 for YHPG and ribavirin granules were 0.970 mg·mL-1,0.625 mg·mL-1 and 1.316 mg·mL-1,0.780 mg·mL-1.Compared with the virus control group,the high and medium dose(0.625,0.313 mg·mL-1)can significant decrease the cytopathic effect,and had a marked therapeutic effectt,direct inactivation and anti-adsorption effect on influenza virus.This indicated that YHPG can significant inhibit the influenza virus proliferation and can significant decrease the cytopathic effect.2.The levels of IFN-β and IL-6 in the cell supernatant and expression levels of cellular TLR7,MyD88,TRAF6,JNK,p38 MAPK,and p65 NF-κB genes significantly decreased after influenza virus-infected MDCK cells were treated with high and medium doses(0.625,0.313 mg·mL-1)of YHPG(P<0.01,P<0.05).These results indicated that the high and medium dose of Yinhuapinggan Granule had a moderating effect on the gene expression of TLRs signaling pathway.Conclusion This indicated that YHPG can significant inhibit the influenza virus proliferation,which is mainly manifested in the direct inactivation,anti-adsorption effect and therapeutic effect on influenza virus.The mechanism of action may be associated with inhibition of the activation of TLR7/MyD88 signaling,thus inhibiting secretion of IFN-β and IL-6.