Effect Of Baicalin Combined With Phillyrin On Gene Expression Of Influenza A Virus Nucleoprotein (NP) In Vitro

Objective: To investigate preliminarily the effect of Baicalin combined with Phillyrin on gene expression of influenza A virus nucleoprotein（NP） in Vitro, to provide theoretical and experimental basis for the search of effective chinese herbal compound which can anti-influenza virus.Methods: 1. Used cytopathic effect（CPE） method to determine drugs（Baicalin, Phillyrin） the biggest non-toxic boundary and experimental concentrations on Hela cells;2. Used liposome transfection method transfected eukaryotic expression plasmid of NP of Influenza A Virus pc DNA3.1（+）/NP into hela cells as experimental target cells and used RT-qPCR to determine successfully transfected into HeLa cells;3. Used RT-qPCR to evaluate the effect of Baicalin combined with Phillyrin on gene expression of influenza A virus nucleoprotein（NP） in target cells and used statistical software for analysis;4. Used RT-qPCR to evaluate the inhibitory effect of different concentration of Baicalin, Phillyrin on gene expression of influenza A virus nucleoprotein（NP） in target cells and calculate IC50 of Baicalin, Phillyrin;5. Used RT-qPCR to evaluate the combined effect（synergism or additive or antagonism） of IC25、IC50 concentration of Baicalin combined with IC25、IC50、IC75 concentration of Phillyrin on gene expression of influenza A virus nucleoprotein（NP） in target cells;The research were divided into hela cell group, liposome group, recombinant plasmid group（recombinant plasmid gene transfect hela cells）, Baicalin group, Phillyrin group, Baicalin combined with Phillyrin group.Results:1. Used cytopathic effect method to get the biggest non-toxic boundary concentrations of various drugs on HeLa cells, baicalin: 31.25μg·ml^-1, phillyrin: 25μg·ml^-1, the experimental concentrations of various drugs: baicalin: 15.625μg·ml^-1, phillyrin: 12.5μg·ml^-1, and Baicalin(15.625μg·ml^-1) combined with phillyrin(12.5μg·ml^-1) demonstrated non-toxic.2. NP gene expression of recombinant plasmid group was significantly higher than that of HeLa cell group, and the difference was statistically significant（t=7.742,P<0.01）; NP gene expression of recombinant plasmid group was significantly higher than that of liposome group, and the difference was statistically significant（t=7.802,P<0.01）; demonstrated eukaryotic expression plasmid of NP of Influenza A Virus pc DNA3.1（+）/NP successfully transfected into HeLa cells.3. NP gene expression of Baicalin(15.625μg·ml^-1) combined with phillyrin(12.5μg·ml^-1) group was significantly lower than that of recombinant plasmid group, and the difference was statistically significant（t=6.966,P<0.01）.4. Within a certain concentration range, Baicalin, Phillyrin significantly inhibited gene expression of influenza A virus nucleoprotein（NP）, this effect was in concentration-dependent manner. The target cell IC50 values were different between Baicalin and Phillyrin: Baicalin: 10.4627μg·ml^-1, Phillyrin: 4.16832μg·ml^-1.5. CI of IC25 concentration of Baicalin + IC25 concentration of Phillyrin is 1.24169; CI of IC25 concentration of Baicalin + IC50 concentration of Phillyrin is 0.95414; CI of IC25 concentration of Baicalin + IC75 concentration of Phillyrin is 0.94745; CI of IC50 concentration of Baicalin + IC25 concentration of Phillyrin is 0.89460; CI of IC50 concentration of Baicalin + IC50 concentration of Phillyrin is 0.85482; CI of IC50 concentration of Baicalin + IC75 concentration of Phillyrin is 0.89201.Conclutions: Baicalin combined with Phillyrin could decrease gene expression of influenza A virus nucleoprotein（NP）, and had synergism within a certain concentration range.