The Study Of Antiviral Effect And Mechanism Of Jinyin Qingre Oral Liquid On Influenza Virus In Vitro

ObjectiveInfluenza is the cute respiratory infectious diseases which was caused by influenza virus invade the body.Influenza virus has a unique single-stranded RNA structure,an effective vaccine for the influenza virus is difficult because of its mutable feature.The chemical antiviral drugs has strong side-effect and easy induced drug resistance.Traditional Chinese medicine has low toxicity,multiple targets,and has both control and prevention.So to find effective anti-influenza virus traditional Chinese medicines has a very important theoretical,economic and social significance.Jinyin qingre Oral Liquidmain components include honeysuckle,capillaris,Gardenia,Forsythia,rhubarb,red sage root,astragalus,licorice.The multi-flavor drugs in the prescriptions have the effect of anti-influenza virus alone or in combination.To clarify the anti-influenza virus effect and mechanism of Jinyin qingre Oral Liquid,experiments are conducted in vitro.To investigate the effects of Jinyin qingre Oral Liquid on cell pathogenic inhibition on influenza A/H1N1 influenza virus infection and on regulation the immune functions of Hep-2 cell,Carrying out three different treament on the Flu/MDCK and Hep-2 cell model,including virucidal assay,drug daaed before or after infection assay.Methods1.The research on method of Jinyin qingre Oral Liquid against the H1N1influenza virus in vitroThe cytopathic effect（CPE）and MTT assays were used to examine the effect of Jinyin qingre Oral Liquid on the cytopathogenic effects of the H1N1 influenza virus.Recovery the MDCK cell and Hep-2 cell and adjusted the concentration of 1.5×10⁵per milliliter and 2.0×10⁵ per milliliter.According to the virus infection process,three experimental groups were designed:prevention group（A）,replication inhibition group（B）,and direct killing group（C）.MDCK cell and Hep-2 cell infected with influenza A/H1N1 influenza virus were detected by the MTT assay and the cell survival rate was calculated.2.The influence of Jinyin qingre Oral Liquid to theexpression of levels of the cellular cytokines in Hep-2 cell which infected with influenza A/H1N1 influenza virus or not.Recovery the Hep-2 cell and adjusted the concentration of 2.0×10⁵ per milliliter.And two experimental groups were designed:normal cell control group（A）and Jinyin qingre Oral Liquid optimal dose group（B）.Jinyin qingre Oral Liquid was diluted with virus growth solution and used for group B cells.Group A was treated with the same amount of virus growth solution.After 48 hours,the supernatant was discarded.The surface of cells was washed three times with PBS,and Trizol lysate was added to collect the cells.The lysates were extracted into RNase-free EP tubes.Total RNA was extracted from each group of cells.The m RNA expression levels of the cellular cytokines（TNF-α,IFN-1α,IL-1α,IL-6 and IFN-1β）in Hep-2 cells treated with the Jinyin qingre Oral liquid were determined by quantitative real time PCR（qRT-PCR）.The study of the influence of Jinyin qingre Oral Liquid to the expression of levels of the cellular cytokines in Hep-2 cell which infectedwith influenza A/H1N1 influenza virus.Jinyin qingre Oral Liquid treated with the Hep-2 cell were infected with influenza A/H1N1 influenza virus.The methods of cells collection,total RNA extraction was performed and detection are the same as above.Results1.The research on method of Jinyin qingre Oral Liquid against the H1N1influenza virus in vitroThe TC₀f Jinyin qingre Oral Liquid forMDCK cell and Hep-2 cell were 3.11mg/mL and 4.095 mg/mL,respectively.The TCID₅₀0 for MDCK cell and Hep-2 cell of influenza A H1N1 virus was 10^-4.94.9 and 10^-3.4,respectively.The TC_O of Jinyin qingre Oral Liquid forMDCK cell and Hep-2 cell were 3.11 mg/mL and 4.095 mg/mL,respectively.The TCID₅₀0 for MDCK cell and Hep-2 cell of influenza A H1N1 virus was 10^-4.94.9 and 10^-3.44,respectively.In the three modes of action of this experiment,the survival rate of the cells in each concentration group in the replication inhibition group was higher,Jinyin qingre Oral liquid(3 mg·mL^-11 and 4 mg·mL^-1)significantly increased the survival rate of MDCK and Hep-2 cells（p<0.05）by 61.60%and40.60%,respectively.2.The influence of Jinyin qingre Oral Liquid to theexpression of levels of the cellular cytokines in Hep-2 cell which infected with H1N1 or not.The mRNA expression levels of TNF-α,IFN-1αof Hep-2 cells were significantly up-regulated by 2.39 and 1.98 folds（p<0.05）,respectively,while the mRNA expression levels of IL-1α,IL-6 were down-regulated by 90%and 64%（p<0.05）,respectively.After Hep-2 cells treated with the Jinyin qingre Oral liquid.Compared with the virus control group,the mRNA expression levels of IFN-1αof Hep-2 cells were significantly up-regulated by 24.92 folds（p<0.05）,respectively,while the m RNA expression levels of IL-1α,IL-6 and TNF-αwere down-regulated by 98.60%、88.84%and 98.34%（p<0.05）,respectively.After Jinyin qingre Oral Liquid treated with the Hep-2 cell were infected with influenza A/H1N1influenza virus.ConclusionJinyin Qingre Oral Liquid can significantly increase the survival rate of cells infected were infected with influenza A/H1N1 influenza virus.And Jinyin qingre Oral Liquid can significantly inhibits inflammatory responses,and may have the effect of regulating cell innate immunity.At the same time,Jinyin qingre Oral Liquid can reduce the inflammatory damage of Hep-2 cell induced by influenza A/H1N1influenza virus and enhance the immune function of Hep-2 cell,which its stimulatory effect on cellular innate immunity.