Study On The Intervention Of Active Component Of Principal And Ministerial Drug Of Lonicerae And Forsythiae Powder On Multiplication Of Ⅳ And Apoptosis Related Gene Expression Of Influenza Viral Pneumonia In Mice

Objective1. To identify and assay chlorogenic acid, phillyrin and arctiin in FlosLonicerae extract, Forsythia suspense extract, Arctium Lappa extract and theircompatibility extract with the methods of thin-layer chromatography and highefficiency liquid chromatography in order to elucidate the substance basis ofLonicerae and Forsythiae Powder inhibiting influenza virus.2. To study the effect that Flos Lonicerae extract, Forsythia suspenseextract, Arctium Lappa extract and their compatibility extract inhibitingproliferation of influenza A virus FM1 strain in vitro, to observe theirprecautionary effect and therapeutic effect towards embryonated egg infectedbyâ…£, and to approach the dependability of time- effect and dose- effect.3. To observe the therapeutic effects of the Flos Lonicerae extract,Forsythia suspense extract, Arctium Lappa extract and their compatibilityextract on the mice pneumonia infected byâ…£, and to explore the mechanism ofthe effect through regulating the expression of the apoptotic singnaltransduction related gene Fas/FasL and TNF-α/TNFRâ… .Methods1. Prepare Flos Lonicerae extract, Forsythia suspense extract, Arctium Methods1. Prepare Flos Lonicerae extract, Forsythia suspense extract, ArctiumLappa extract and their compatibility extract with the preparing process byalcohol solution.2. Use the method of hemagglutination (HI) test, observe the effect thatFlos Lonicerae extract, Forsythia suspense extract, Arctium Lappa extract andtheir compatibility extract inhibitingâ…£in vitro and in embryonated egg.3. young mice were inoculated influenza A virus FM1 strain in cavitas nasi,and batch sacrificed late. On the base of the models, following experimentwere carried.(1) Theâ…£-RNA were detected by reverse transcription-polymerasechain reaction (RT-PCR), the lung histopathologic were observed by the lightmicroscope.(2) 70 young mice were divided into 7 groups randomly, normal group,model group, ribavirin control group, Flos Lonicerae extract group, Forsythiasuspense extract group, Arctium Lappa extract group and the compatibilityextract group. We cured the pneumonia of mice by intragastric administrationfor 6 days, and observed the symptom of mice. We calculated the rate betweenthe lung mass and weigh of the body, observe macro-histopathologic andultramicro changes of lung, detected theâ…£-RNA by RT-PCR.(3) We detected the apoptosis related gene expression of Fas, FasL,TNF-α, TNFRâ… by immunohistochemistry method and RT-PCR method.Result Experiments in vitro:1. At the density of their TD₀, 1/2TD₀ and 1/4TD₀, Flos Lonicerae extract,Forsythia suspense extract, Arctium Lappa extract and their compatibilityextract had precautionary effect and therapeutic effect towards embryonatedegg infected byâ…£, and the precautionary effect and therapeutic effectdecremented by the stepping down of their density, the precautionary effect ofall the Chinese crude drug group was better than the ribavirin control group, thetherapeutic effect of Forsythia suspense extract group was better than theribavirin control group. The difference of the four Chinese crude drug groups isnot significant.2. Flos Lonicerae extract, Forsythia suspense extract, Arctium Lappaextract and their compatibility extract can inhibit proliferation of influenza Avirus FM1 strain in vitro directly, the action time was keeping on from 1 hour to24 hour. At their max density witch cannot make blood coagulated, all thegroups can killâ…£completely. The effect was decremented by the steppingdown of their density. The difference of the four Chinese crude drugs group isnot significant.Experiments on animal:1. We can detected IV RNA expression in a great quantity in the lung ofmouse 5 days afer they were infected by influenza A virus FM1 strain. Thelungs histopathologic change was alveolar septum dropsy, inflammatory cellinfiltration, and exfiltration in alveolar space.2. The drugs' therapeutical effect towards pneumonia mice: pneumonia mice. The rate between the lung mass and weigh of the body werelower than the model group after the treatment, and the histopathologic changesof mice lungs of the five drugs were better than the model group too. And theeffect of the compatibility extract group was better than the Arctium Lappaextract group.(2) The content ofâ…£-RNA in the lungs of the five drugs groups werelower than the model group. There was no significant difference between thecompatibility extract group and the ribavirin control group, and the effect of thecompatibility extract group was better than the Forsythia suspense extract groupand the Arctium Lappa extract group, the difference was significant.(3) the protein expression of Fas and FasL in the lungs of five drugsgroup were lower than the model group. There was no significant differencebetween the compatibility extract group and the ribavirin control group. Andthe expression of Fas of the compatibility extract group was lower than theArctium Lappa group, the difference was significant. The TNF-αand TNFRâ… gene expression of the five drugs group were lower than the model group, therewas no significant difference between the compatibility extract group and theribavirin control group. And the expression of TNF-αof the compatibilityextract group was lower than the Arctium Lappa group, the difference wassignificant.Conclusion1. Flos Lonicerae extract, Forsythia suspense extract, Arctium Lappaextract and their compatibility extract had precautionary effect and therapeutic effect towards embryonated egg infected byâ…£, the effect had positivecorrelation with the drug's density. At the density of their TD₀, the effect of thefour Chinese crude drugs group was better than the ribavirin control group.2. Flos Lonicerae extract, Forsythia suspense extract, Arctium Lappaextract and their compatibility extract can inhibit proliferation of influenza Avirus FM1 strain in vitro directly, there was dependability between effect andtime, and was dependability between effect and density.3. we can make the model of viral pneumonia by inoculating influenza Avirus FM1 strain in the cavitas nasi of young mice. Flos Lonicerae extract,Forsythia suspense extract, Arctium Lappa extract and their compatibilityextract can had therapeutical effect towards pneumonia mice.4. Flos Lonicerae extract, Forsythia suspense extract, Arctium Lappaextract and their compatibility extract can inhibit apoptosis by down-regulatingFas and FasL protein expression, and by down-regulating TNF-αand TNFRâ… mRNA expression.5. chlorogenic acid, phillyrin and arctiin are the fundamental substancebasis of Lonicerae and Forsythiae Powder inhibiting influenza virus.