Exploration Of The Antiviral Mechanisms And Effective Material Basis Of Ge-Gen Decoction Against Influenza A Virus(H1N1)infection

Objective: Taking the antiviral mechanism and effective material basis of Ge-Gen Decoction against influenza virus(H1N1)as the research focus.The antiviral mechanism of Ge-Gen Decoction against influenza virus was elucidated through in vivo and in vitro antiviral experiments from the aspects of antiviral,inflammatory inhibition and immune reg?Lation.To explore the Spectrum-Effect relationship between the chemical constituents of Ge-Gen Decoction and its antiviral activity,and to explore the effective material basis of Ge-Gen Decoction against influenza virus.To provide scientific basis for Ge-Gen Decoction as an effective anti-influenza drug.Method:1.Anti-influenza virus(H1N1)activity evaluation in vitro: After inoc?Lating monolayer MDCK cells with H1N1 virus(100TCID50),different concentrations of Ge-Gen Decoction extract were added.CCK8 assay was used to detect the inhibitory effect of drugs on the cytopathy induced by influenza virus.At different time points after infection,the progeny viruses released into c?Lture medium were quantified by RT-PCR.The in vitro growth kinetics of influenza virus was analyzed to investigate the ability of Ge-Gen Decoction extract to inhibit the generation of viral progeny.2.Study on the mechanism of action of anti-influenza virus(H1N1)in vitro: The potential antiviral mechanism of Ge-Gen Decoction was preliminarily explored by a time-of-addition assay.The effect of drugs on the attachment phase of viral infection was detected by attachment assay.The effect of drugs on the internalization phase of viral infection was detected by internalization assay.The viral titers in c?Lture medium after 10 hours of infection was detected by RT-PCR to further evaluate the effect of Ge-Gen Decoction on the late biosynthesis of single infection cycle species.3.Evaluation of anti-influenza virus(H1N1)activity in vivo: To detect the effect of Ge-Gen Decoction on the mortality of mice infected with lethal dose(5 LD50)influenza virus and the weight change of mice infected with sub-lethal dose(0.8 LD50)influenza virus.Another group of mice were taken lungs on 2,4 and 6 days after infection(0.8 LD50).In the other mice,lungs were taken on days 2,4,and 6 after infection(0.8 LD50),and the lung index was calc?Lated.The morphological changes of the lungs were observed.The relative expression of virus in lung tissue was detected by RT-PCR on the 2th,4th and 6th day after infection,and the expression of influenza virus in lung tissue was detected by Western-blot method on the 4th day after infection.4.Inflammatory inhibition and immunomod?Latory effects of drugs on virus-infected mice: The effects of Ge-Gen Decoction on the expression of proinflammatory cytokines IL-1?,IL-6 and TNF-? in the lung tissue of mice were detected by RT-PCR and Western-blot.The effects of Ge-Gen Decoction on the expression of IFN-? and IL-4 in lung tissue were detected by RT-PCR and Western-blot assay on the 4th day after infection,and the effects of drugs on Th1/Th2 balance were investigated.The effect of the Ge-Gen Decoction on the proportion of peripheral blood T lymphocyte subsets CD4+IFN-?+ T cells and CD4+IL-4+ T cells in mice on day 4 post infection was further examined by flow cytometry.The effect of Ge-Gen Decoction on CD3+CD4+/CD3+CD8+ ratio in peripheral blood T cell subsets of mice was detected by flow cytometry on the 4th day after infection.5.Effect of study drugs on Toll-like receptor signaling pathway in virus-infected mice: The m RNA expressions of TLR7,My D88,TRAF6,IRF7 and NF-?B in lung tissues of mice infected with influenza virus were detected by RT-PCR.The protein expression of TLR7,My D88 and TRAF6 and the activation of IRF7 and NF-?B in lung tissues of mice infected with influenza virus were detected by Western-blot method.6.Characterization of chemical constituents of Ge-Gen Decoction based on HPLC-Q-TOF-MS: To establish the HPLC chromatogram of the water extract of Ge-Gen Decoction.The chromatographic peaks were qualitatively analyzed by HPLC-Q-TOF-MS.7.To explore the possible pharmacodynamics of Ge-Gen Decoction against H1N1 in vivo: The dichloromethane fraction,the ethyl acetate fraction,the water-saturated n-butanol fraction and the remaining fraction after extraction were respectively obtained by system solvent extraction.The virus titer in the lung tissue of mice on the 4th day after influenza virus infection(0.8 LD50)was detected by RT-PCR,and the anti-influenza activity of the above four sites and Ge-Gen Decoction was investigated.Partial least squares regression analysis was used to study the relationship between peak intensity and anti-influenza virus activity,and to explore the effective material basis of Ge-Gen Decoction against influenza virus in vivo.Res?Lt:1.Ge-Gen Decoction has anti-influenza virus H1N1 activity in vitro: In vitro antiviral experiments showed that Ge-Gen Decoction extract co?Ld significantly inhibit the cytopathic effects caused by influenza virus H1N1 in MDCK cells in a dose-dependent manner.The CC50,IC50 and SI of Ge-Gen Decoction on MDCK cells were 10.88 mg/m L,1.81 mg/m L and 6.01 respectively.The res?Lts of in vitro growth kinetics analysis showed that Ge-Gen Decoction co?Ld significantly delay the growth kinetics of the virus.Compared with the control group,Ge-Gen Decoction significantly reduced the production of offspring virus 4,8,12,18 hours after infection,and delayed the arrival of viral growth platform.2.Anti-influenza virus H1N1 mechanism of Ge-Gen Decoction in vitro:Time-of-addition assay showed that GGD extract was dose-dependent and time-dependent effective against H1N1 in MDCK cells(Figure 2 C).We found that the GGD extract was more effective when given before viral infection.The IC50 was1.42 mg/m L(4 h before),1.58 mg/m L(2 h before),and 4.96 mg/m L(2 h after).No noticeable difference was observed between 4 h before infection and 2 h before infection.Attachment assay showed that Ge-Gen Decoction extract co?Ld inhibit virus adsorption in a dose-dependent manner,IC50 was 2.59 mg/ml,internalization assay showed that Ge-Gen Decoction extract had no effect on the penetration stage of virus infection.After 10 h of infection,the viral-genome copies in GGD treatment group was 5-fold lower than that of the model group.3.Ge-Gen Decoction has anti-influenza virus H1N1 activity in vivo: Ge-Gen Decoction co?Ld significantly reduce the mortality of mice infected with lethal dose of influenza virus(5 LD50),prolong the survival time,and inhibit the weight loss of mice infected with sub-lethal dose of influenza virus(0.8 LD50).RT-PCR res?Lts showed that Ge-Gen Decoction co?Ld significantly reduce the viral titers in lung tissues of mice infected with influenza virus(0.8 LD50)on the 2th,4th and 6th day after infection,and there was no difference between Ge-Gen Decoction and oseltamivir phosphate treatment group on the 4th and 6th day after infection.Compared with the model group,Ge-Gen Decoction can significantly reduce the lung index on the 4th and 6th day after infection,alleviate the pathological changes of lung tissue,and alleviate p?Lmonary inflammation.4.Ge-Gen Decoction has inflammatory inhibition and immunomod?Latory activity: The res?Lts of RT-PCR and Western blot showed that Ge-Gen Decoction co?Ld significantly reduce the expression of pro-inflammatory cytokines IL-1?,IL-6and TNF-? in lung tissues of mice infected with influenza virus(0.8 LD50),decrease the expression of IFN-? and promote the expression of IL-4.The res?Lts of flow cytometry showed that the ratio of CD4+IFN-?+/CD4+IL-4+ in peripheral blood of mice infected with influenza virus increased and the ratio of CD3+CD4+/CD3+CD8+decreased,suggesting that on the fourth day after infection,Th1/Th2 shifted to Th1 direction and cell?Lar immunity was significantly enhanced.Compared with the model group,the ratio of CD4+IFN-?+/CD4+IL-4+ decreased,the ratio of CD3+CD4+/CD3+CD8+ increased and the immune imbalance improved after Ge-Gen Decoction treatment.5.Effect of Ge-Gen Decoction on TLR7 signaling pathway: The res?Lts of RT-PCR showed that the m RNA expressions of TLR7,MYD88,TRAF6 and IRF7 in the Ge-Gen Decoction treatment group were lower than those in the model group.However,there was no difference in the expression of NF-?B m RNA between the groups.Western blot res?Lts showed that compared with the model group,the expression of TLR7,MYD88,TRAF6 and P-IRF7 was decreased,the ratio of P-IRF7/IRF7 was decreased,and the expression of IKB? protein was increased.6.The establishment of HPLC fingerprint of Ge-Gen Decoction: The HPLC fingerprint of Ge-Gen Decoction extract was established,and 31 fingerprint peaks were defined according to the resolution,peak shape and peak symmetry.Another method was used to identify ephedrine and pseudoephedrine,and a total of 33 peaks were defined.The RSD of the precision test,the stability test,and the repeatability test are all less than 3.0%.Nineteen compounds were identified by comparing with the control.Twelve compounds were inferred by comparing mass spectrometry information with mass spectrometry data in relevant literature.Two compounds have not been reported yet,and their structures need further confirmation.The identified 33 components are mainly flavonoids,and include organic acids,mono-stitches,triterpene saponins,alkaloids and other components.7.Study on the spectrum-activity relationship of Ge-Gen Decoction against influenza virus: RT-PCR res?Lts showed that the viral inhibition rates of dichloromethane,ethyl acetate,water-saturated n-butanol and residual parts of Ge-Gen Decoction were 48.87%,70.22%,92.78% and 6.51%,respectively.Among the 33 characteristic peaks,27 peaks were positively correlated with anti-H1N1 activity,and 9 of them had VIP values greater than 1.It was concluded that the 9components contributed significantly to anti-H1N1 activity of Ge-Gen Decoction,namely daidzein,mangiferin,puerarin D,genistein,daidzein,3'-methoxypuerarin,puerarin,paeoniflorin and pseudoephedrine.8.Six components of Ge-Gen Decoction were identified,including eight prototype components(Ephedrine,Pseudoephedrine,Puerarin,3'-Methoxypuerarin,Daidzin,Glycyrrhizic acid,Formononetin and 6-Gingerol)and two metabolites(Paeonimetabolin I and Genistein).Pueroside D or its metabolites were not detected in the blood.Considering that the metabolites of Paeoniflorin,Daidzin,Ononin and Genistin are Paeonimetabolin I,Daidzein,Formononetin and Genistein,we believe that Pseudoephedrine,Puerarin,3'-Methoxypuerarin,Paeoniflorin,Daidzin,Genistin,Ononin and Daidzein are the active components of Ge-Gen Decoction against influenza.Conclusion:This study shows that Ge-Gen Decoction has anti-influenza A virus H1N1 activity in vitro and in vivo,and its targets are manifested in many aspects.In vitro,Ge-Gen Decoction can play a direct antiviral role by inhibiting the stage of adsorption and replication of influenza A virus H1N1.In the study of the potential mechanism of Ge-Gen Decoction in the treatment of influenza infection,this topic pays special attention to the immune reg?Lation and anti-inflammatory activity of Ge-Gen Decoction.Through the comparison of several immune-related indexes between the GGD and OP treated groups,we found that GGD treatment can especially decrease the expression of cytokine TNF-? to avoid the pathological lung injury.GGD treatment also can reg?Late the balance of Th1/Th2 to reduce inflammation.In addition,we also found that TLR7 signaling pathway also participates in the immunomod?Latory effect of Ge-Gen Decoction,and Ge-Gen Decoction treatment can significantly reduce the activation level of IRF7,but the reasons need further study.In addition,31 chemical constituents in Ge-Gen Decoction were qualitatively identified by HPLC-Q-TOF-MS,and the effective materials of Ge-Gen Decoction against influenza A(H1N1)virus in vivo was revealed by the method of spectral pharmacodynamics of traditional Chinese medicine,and the res?Lts were verified by the detection of blood-entry components of Ge-Gen decoction.Finally,we believe that the effective materials of Ge-Gen Decoction against influenza A H1N1 virus are Pseudoephedrine,Puerarin,3'-Methoxypuerarin,Paeoniflorin,Daidzin,Genistin,Ononin and Daidzein.These studies provide a scientific basis for understanding the mechanism of Ge-Gen Decoction in anti-influenza A virus H1N1 infection,and also provide a technical method and data support for improving the quality standard of Ge-Gen Decoction preparation.