| ObjectiveInfluenza viruses are pathogens of high risk,which often results in catastrophic pandemics worldwide.Among all viruses,influenza A virus is evolved by mutation and recombination,so it is more susceptible to mutations.Flu is called "exogenous fever" in traditional Chinese medicine.The motherland medicine has accumulated a lot of experience in the prevention and treatment of influenza.The unique advantages of traditional Chinese medicine will contribute to the prevention and treatment of influenza,which has important and far-reaching significance.In this study,we used the influenza A virus as the infection source and took advantage of A549 cell line infected with H1N1 as model to study the immune mechanism of luteolin monomer.To further study it's possible molecular mechanisms,we checked out differentially expressed genes by gene microarrays and found the relevant factors.In this way,we can provide the theory for the application of luteolin in the treatment of influenza at the stage of biology molecular.Methods1.Luteolin's intervention effect induced by H1N1 in vitroThe maximum of non-toxic concentration?TCO?and the median toxic concentration?TC50?were detected by the cytopathogenic effect?CPE?of cells.Furthermore,The median inhibitory concentration?IC50?and treatment indexes?TI?in the best mode were calculated by Probit regression.2.Effect of luteolin on the expression of inflammatory cytokines induced by influenza virus H1N1 in vitroThe chips were used to screen the differentially expressed of IL-1,IL-10,IFN-r and TNF-a.The mRNA and protein expressions of IL-1,IL-10,IFN-r and TNF-a were verified by Real-Time PCR and Western-blot.3.The regulatory effect of Luteolin on TLR Signaling Pathway in Influenza Virus Infected CellsThe chips were used to screen the differentially expressed genes which selected from TLR signaling pathway.The mRNA and protein expressions of TLR3/7and Myd88 were verified by Real-Time PCR and Western-blot.4.Luteolin's intervention effect and mechanism of apoptosis induced by H1N1 in vitroThe chips were used to screen the differentially expressed genes which selected from the apoptotic signaling pathway.The mRNA and protein expressions of Caspase3?Caspase8 and Myd88 were verified by Real-Time PCR.Results1.Luteolin's intervention effect induced by H1N1 in vitroA significant inhibitory effect was shown when added luteolin from 12.5 to 25.0?g·mL-1 after 48-60 h compared with the model without the drug.In addition,the best method against viral infection was adding luteolin at 2 hours after cells infected and the best survival rate was?87.0±1.71?%with 25.0 ?g·mL-1 of luteolin.Under this condition,IC50 was 11.282 ?g·mL-1 and TI was3.528.2.Effect of luteolin on the expression of inflammatory cytokines induced by influenza virus H1N1 in vitroThe gene chip tips:oseltamivir and luteolin could down-regulated the expressions of IL-10?IL-1?IFN-r?TNF-a compared with the virus-infected group.Real-Time PCR and western-blot experiments showed that luteolin could significantly decrease the mRNA and protein expressions of IL-10?IL-1?IFN-r?TNF-a.3.The regulatory effect of Luteolin on Toll Receptor Signaling Pathway in Influenza Virus Infected CellsThe gene chip tips:oseltamivir and luteolin could down-regulated the expressions of Myd88 8?TLR3?TLR7?IRAK4?IRF7?TRAF6 compared with the virus-infected group.Real-Time PCR and western-blot experiments showed that luteolin could significantly decrease the mRNA and protein expressions of Myd88?TLR3?TLR7 compared with the virus-infected group.4.Luteolin's intervention effect and mechanism of apoptosis induced by H1N1 in vitroThe gene chip tips:oseltamivir and luteolin could down-regulated the expressions of Caspase3?Caspase6?Caspase7?Caspase8?Caspase9 and Myd88 compared with the virus-infected group.Real-Time PCR experiments showed that luteolin could significantly decrease the mRNA expressions of Caspase3?Caspase8?Myd88 compared with the virus-infected group.ConclusionsIn this study,we used gene chip?RT-PCR and western blot to analyze the effect and mechanism of luteolin on influenza virus H1N1 infected A549 cells.The results showed that luteolin had a strong inhibitory effect on influenza virus.It can affected the expression of inflammatory cytokines?TLR signaling pathway and the expression of apoptosis-related factors,in which way reduce the immune damage to the host. |
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