Expression Of Influenza A Virus Nucleoprotein Interfere With Forsythia Extracts In Vitro

Background：Influenza seasonal epidemic or pandemic influenza severely impaired humanityhealth. Now the major side effects and increasing drug resistance limit the antiviralwestern medicine to be used. More and more people paid widespread attention totraditional Chinese medicine on prevention and treatment of influenza in clinical andbasic theory research in domestic and overseas, to search for new antiviral medicineand explore its action mechanism, will help pushing traditional medicine to the worldand will be beneficial to mankind.Objective：To study the intervention effects of forsythia extracts (phillyrin, ethanol extractof fructus forsythia, decoction of forsythia) on influenza virus A nucleoproteingene in vitro, to provide scientific theoretical foundation for the forsythia Anti-influenza virus.Methods：1. The research were divided into hela cell group (hela cells), empty plasmidgroup (empty plasmid and hela cells), liposome group (liposome and hela cells),recombinant plasmid group (recombinant plasmid gene transfect hela cells), phillyringroup(phillyrin was added into recombinant plasmid group), ethanol extract offructus forsythia group (ethanol extract of forsythia was added into recombinantplasmid group), decoction of forsythia group (decoction of forsythia was added intorecombinant plasmid group).2. Used cytopathic effect method to determine the fructus forsythia extracts thebiggest non-toxic boundary and experimental concentrations.3. Used liposome transient transfection method transfected hela cells asexperimental target cells.4. Used colloidal gold immunoassay chromatography to detect the experimentalgroup supernatant and intracellular nucleoprotein expression. 5. Extracted the total RNA of colloidal gold tested negative group andrecombinant plasmid group, synthesized cDNA.6. Used fluorescent quantitative reverse transcription PCR to detectrecombinant plasmid and colloidal gold tested negative group nucleoprotein genecopies number, used statistical software for analysis.Results：1. The cell culture,s supernatant and intracellular of recombinant plasmid groupwere positive tested by colloidal gold method; the cell culture,s supernatant ofethanol extract of forsythia and decoction of forsythia two groups were weaklypositive, ethanol extract of forsythia was less positive; the intracellular of phillyringroup, ethanol extract of forsythia group, decoction of forsythia group were weaklypositive, phillyrin was the weakest, decoction group was the secondly. The cellculture,s supernatant of phillyrin group、The cell culture,s supernatant and theintracellular of hela cells group, empty plasmid group, liposome group were allnegative.2. The nucleoprotein gene copies number of phillyrin group intracellular was(2.07±0.2)×105, the recombinant plasmid group intracellular was (61.2±13)×105,the phillyrin group intracellular was lower than the recombinant plasmid groupintracellular，the difference was statistically significant (t=9.836, p <0.05).Conclusion：1. Phillyrin not only could reduce the nucleoprotein gene dosage in be transienttransfected hela cells, but also strongly inhibited the expression of the nucleoprotein.2. The ethanol extract of forsythia and decoction of forsythia could also inhibitthe expression of the nucleoprotein in be transient transfected hela cells, but wasweaker than phillyrin; the decoction of forsythia was weakest.