The Effect Of Quercetin And Oseltamivir On The TLR7 Signaling Pathway Infected By H1N1 In Vitro

ObjectiveIn this study, we analyzed the effect of quercetin and oseltamivir on TLR7 signaling pathway of influenza virus infected human immune cells. We observed the dynamic changes of NO in influenza virus infected human macrophages and the changes of Toll-like receptor (TLR) 7 signaling pathways of dendritic cells and macrophages. At the same time, we investigated the molecular mechanism that the drug inhibited the influenza virus via the MyD88-dependent pathway mediated the TLR7 and found the antivrial molecular targets of the Chinese medicine.Methods1. To isolate and induce dendritic cells and macrophages from human cord blood, then identify them.2. H1N1 A/E13 virus were increased in chicken embryo allantoic cavity and determinated by TCID50. Using 100TCID50 H1N1 A/E13 to infect human bronchial epithelial cells 16HBE, to establish model of influenza virus infected cells.3. Using MTT to detect inhibition of influenza virus and relationship between effect and times (24 hr,48 hr,72 hr). Aiming at investigate the antiviral efficiency and the characteristics of quercetin and oseltamivir phosphate in vitro.4. To survey the effect of quercetin and oseltamivir phosphate on the kinetic change of nitrous oxide (NO) in influenza virus infected macrophage. Make macrophage co-cultured with model of influenza virus infected cells, and treated with quercetin and oseltamivir phosphate, then collect supernatant at 6 hr,12 hr,24 hr,48 hr respectively. The lever of NO was measured by nitrate reductase assay.5. The dendritic cells and macrophages were co-cultured with the model of influenza virus infected cells, and treated with quercetin and oseltamivir phosphate, then collected immune cells at 24 hr,48 hr for real-time fluorescence quantitative PCR reaction (RT-qPCR). Aiming at observe the changes of genes in TLR7 signaling pathway signaling of dendritic cells and macrophages before and after using drugs. To observe the level of mRNA expressed by the Toll-like receptor 7 (TLR7), myeloid differentiation factor 88 (MyD88), interleukin-1 receptor associated kinase 4 (IRAK4), tumor necrosis factor-related kinase 6 (TRAF6) and nuclear factor-κB p65 (NF-κB p65).6. The dendritic cells and macrophages were co-cultured with the model of influenza virus infected cells, and treated with quercetin and oseltamivir phosphate, then collected immune cells at 24 hr. Using Western-bolt to detect the TLR7, NF-κB p65 protein expression of dendritic cells and macrophages.Results1. Dendritic cells and macrophages were separated and induced successfully.2. Influenza virus was amplified in chick cavity inoculation successfully; hemagglutination titer was 1:640; the TCID50 was 5×10-4.56 according to Reed-Muench.3. MTT test confirmed that quercetin has the the same role as oseltamivir against influenza virus.4. NO levels can be significantly redueced when using quercetin and oseltamivir phosphate on influenza virus infected in macrophages at 6 hr,12 hr,24 hr,48 hr.5. RT=qPCR experiments showed that influenza virus infection could increase mRNA expression ofTLR7 signal transduction pathway of dendritic cells and macrophages; quercetin and phosphate oseltamivir significantly reduced TLR7, MyD88, IRAK4, TRAF6, NF-κB mRNA expression levels of virus-infected dendritic cells and macrophages.6. Western-blot experiments showed that quercetin and oseltamivir phosphate could reduce TLR7,NF-κB p65 protein expression of virus-nfected dendritic cells and macrophages.ConclusionQuercetin played an importent role as oseltamivir, which could regulate TLR7 signaling pathway of H1N1 infection.