| Objective To investigate the effects of Yinhuapinggan granule(YHPG)against influenza A/PR8/8/34(H1N1)virus in vitro,the model was established by murine macrophage cells(RAW264.7)infected with influenza A/H1N1 virus.At the same time,an influenza A/H1N1 virus(IFV)infection in RAW264.7 cells was utilized as the model,based on the pattern recognition receptors in innate immune TLR7(toll-like receptor 7),the research investigated the immunomodulatory effects of YHPG and its related mechanisms in vitro,and the regulation effects of YHPG on inflammatory cytokines in macrophages infected with influenza virus.Moreover,a model of influenza virus pneumonia was established to study the activity of YHPG against influenza A/H1N1 virus in vivo.Based on the key targets of apoptosis signal pathway,the research investigated the effects of YHPG on apoptosis related factors in mice infected with influenza virus,which could provide experimental basis and molecular biological basis for its clinical application.Method(1)The effects of YHPG against influenza A/H1N1 virus in vitro: The MTT colorimetric method was used to observe the antiviral efficiency of YHPG.According to the replication cycle of influenza virus,three antiviral drug modes of administration were designed in RAW264.7 cells: drug prevention group,antiviral adsorption group and drug treatment group.Furthermore,to observe the antiviral effect of YHPG and identify the best method of administration,and to explore the anti-influenza virus effects of YHPG in vitro in various aspects.(2)The effects of YHPG on the TLR7 signaling pathway of IFV-infected RAW264.7 cells in vitro: TLR7,My D88,IRF7 and NF-κB p65 m RNA expression levels in the key target of TLR7 signaling pathway of IFV-infected RAW264.7 cells were detected by real-time fluorescence quantitative PCR(RT-PCR).(3)The effects of YHPG on the expression of NF-κB and the downstream inflammatory cytokines of IFV-infected RAW264.7 cells in vitro: IFN-β,IL-6 and TNF-α m RNA and protein expression levels in IFV-infected RAW264.7 cells were detected by RT-PCR and ELISA,respectively.The phosphorylation of NF-κB p65 protein expression in IFV-infected RAW264.7 cells was detected by western blot.(4)The effects of YHPG on IFV-infected mice with pneumonia and immune organs index: The model of pneumonia by nasal dropping influenza virus was established in ICR mouse,and then divided randomly into normal control group(Normal-C),IFV-infected control group(IFV-C),Oseltamivir group(positive control,21.63 mg/kg),high dose(18 mg/kg)of YHPG group,moderate dose(12 mg/kg)of YHPG group,low dose(6 mg/kg)of YHPG group.The mice were sacrificed on the3 rd day and the 5th day after infection,respectively.The lung index,the spleen index,the thymus index,the histopathological changes of lung tissue and the changes of lung viral load in each group lung tissues of mice were observed,and to the protective effects of YHPG in IFV-infection mice.(5)The effects of YHPG on the apoptosis signaling pathway of IFV-infected mice: At the 3rd day after infection,Bax,Bcl-2 and Caspase-3 m RNA and protein expression levels in lung tissues of IFV-infected mice were detected by RT-PCR and immunohistochemical analysis,respectively.Results(1)YHPG has a certain antiviral effect in vitro.Within a certain concentration range,three antiviral drug methods of administration all have certain antiviral effect,and the antiviral efficiency of the drug-treated group was higher.(2)YHPG could dose-dependently reduce the gene expression level of TLR7,My D88,IRF7 and NF-κB p65 in TLR7 signal pathway in IFV-infected RAW264.7cells.(3)YHPG could dose-dependently increase the gene and protein expression level of IFN-β in IFV-infected RAW264.7 cells,and decrease the gene and protein expression levels of IL-6 and TNF-α;it could down-regulate the expression of NF-κB p65 phosphorylation protein.(4)YHPG could dose-dependently decrease the lung index and the lung tissue viral load of IFV-infected mice,attenuate the pathological consolidation of lung tissue caused by influenza virus,and increase the spleen index and thymus index of IFV-infected mice,which indicated that YHPG could play a protective role in influenza viral pneumonia mice.(5)YHPG could dose-dependently decrease the gene and protein expression of Bax,Caspase-3,and increase the gene and protein expression of Bcl-2 in the lung tissue of IFV-infected mice,which are the key targets of apoptotic signaling pathway in the lung tissue of IFV-infected mice.Conclusion(1)The first part: YHPG could inhibit influenza virus replication in vitro.YHPG has inhibitive effects on influenza A/H1N1 virus through a variety of methods,such as drug prevention,antiviral adsorption and drug treatment,and the main impact of YHPG in IFV-infected cells was the host late biosynthesis activity.Antiviral mechanisms of YHPG may be related to regulate the gene expression in the key targets of TLR7 signaling pathway and affect the release of inflammatory cytokines.(2)The second part: YHPG could inhibit lung tissue inflammation caused by influenza virus infection in vivo,and its antiviral mechanism may be related to the improvement of immune organ index induced by IFV-infection mice,reduce the replication of influenza virus,attenuate the pathological consolidation of lung tissue caused by influenza virus and regulate the gene and protein expression of Bax,Bcl-2,and Caspase-3 in the key targets of apoptosis signaling pathway. |
PDF Full Text Request