| Objective The research investigate the Ephedra decocted first Maxing Shigan Decoction(hereafter this text will be abbreviated as MXSGD)the effect mechanism of anti type A influenza virus from the TLR7/8-MyD88 signaling pathway regulating the IFNalpha/beta generation.It is Expected that this will provide theoretical support for its clinical application.Methods 1.Effects of influenza A virus infection and the Ephedra decocted first MXSGD rat containing serum on the secretion and expression of IFN-alpha/beta in macrophage culture media mediated by TLR7/8.(1)The maximum non-toxic concentration of different rat drug-containing serum——MXSGD four kinds of herbals decocted at the same time rat serum,MXSGD Ephedra decocted ahead for 20 min rat serum,MXSGD Ephedra decocted ahead for 25 min rat serum,MXSGD Ephedra decocted ahead for 30 min rat serum,oseltamivir rat serum and antiviral granule rat serum on macrophage(RAW264.7 cell)was measured by four methyl tetrazolium blue(MTT)colorimetric assay after 12 h,24h and 48 h of the interventions.(2)Using different concentrations(high,middle and low)of TLR7/8 activator(R848)and TLR7/8 inhibitor(antibody TLR7 and antibody TLR8)as a reference during the experiment to intervene mouse macrophages,the levels of IFN-alpha/beta in the macrophage culture media of intervened 24 h and 48 h by different concentrations(high,middle and low)of type A influenza virus dilution were detected by enzyme-linked immunosorbent assay(ELISA for short in this text).This will screen the best concentration of influenza virus fluid for use in the experiment.(3)ELISA method was used to detect IFN-alpha/beta level in macrophage culture media,of which the mouse macrophage were the intervened by the type A influenza virus infection dilution and then used the different rat serum cultured for 24 h and 48 h.And it will clear the effects of influenza A virus infection and the Ephedra decocted first MXSGD on the secretion and expression of IFN-alpha/beta in macrophage culture media mediated by TLR7/8.2.Effects of influenza A virus infection and the Ephedra decocted first MXSGD rat containing serum on the expression of major target genes and proteins in macrophage cells mediated by TLR7/8.Quantitative Real-Time PCR(q RT-PCR for short in this text)and Western Blot(WB for short in this text)method was used to detect the the expression of genes(TLR7,TLR8,MyD88,IFN-? and IFN-? m RNA)and proteins(TLR7,TLR8,MyD88,IFN-? and IFN-? protein)in macrophage cells which were the intervened by the type A influenza virus infection dilution and then used the different rat serum cultured for 24 h and 48 h.After the contrastive analysis,it will clear the effects and mechanism.3.Study on the mechanism of Maxing Shigan Decoction Ephedrae being decoctered first against influenza virus in vivo.The animal model was established that several SPF grade KM mice after intranasal inoculation of influenza virus——a mouse lung adapted strains(A/PR/8/34).Then the expression of TLR7,TLR8 and MyD88 proteins in lung tissues was detected by WB and analyzed the proteins correlation with body weight and lung index after intragastric administration(oseltamivir,antiviral particles,MXSGD four kinds of herbals decocted at the same time,MXSGD ephedrae being decoctered first or saline)for 7 days.Results 1.Effects of influenza A virus infection and the Ephedra decocted first MXSGD rat containing serum on the secretion and expression of IFN-alpha/beta in macrophage culture media mediated by TLR7/8.(1)The result of 12 h,24h and 48 h MTT shows that the blank rat serum of concentration of 50%,25%,12.5%,and 6.25% have no toxic effect on RAW264.7 cells compared with that of the 12.5% concentration‘s fetal bovine serum.It also shows that rat serum drug containing of concentrations of 50%,25%,12.5% and 6.25% have no toxic effect on RAW264.7 cells compared with the corresponding concentrations of fetal bovine serum and blank rat serum.(2)ELISA result show that the levels of IFN-alpha/beta in the macrophage culture media of activator of R848 group and virus intervention group were significantly increased(P< 0.05),compared with that of fetal bovine serum group and blank rat serum group;The levels of IFN-alpha/beta in the macrophage culture media of MXSGD serum group were significantly decreased(P< 0.05),compared with that of virus intervention group;At 48 h time point,the levels of IFN-alpha/beta in the macrophage culture media of MXSGD ephedra decocted ahead for 25 min rat serum group were significantly decreased(P< 0.05),compared with that of MXSGD four kinds of herbals decocted at the same time rat serum group.2.Effects of influenza A virus infection and the Ephedra decocted first MXSGD rat containing serum on the expression of major target genes and proteins in macrophage cells mediated by TLR7/8.(1)q RT-PCR test result shows that the expression of TLR7,TLR8,MyD88,IFN-? and IFN-? m RNA in macrophage cells of activator of R848 group and virus intervention group were significantly increased(P< 0.05),compared with that of fetal bovine serum group and blank rat serum group;The expression of TLR7,TLR8,MyD88,IFN-? and IFN-? m RNA in macrophage cells of MXSGD serum group were significantly decreased(P< 0.05),compared with that of virus intervention group;The expression of TLR7,TLR8,MyD88,IFN-? and IFN-? m RNA in macrophage cells of the ephedra decocted first MXSGD group were significantly decreased(P< 0.05),compared with that of MXSGD four kinds of herbals decocted at the same time rat serum group;The expression of TLR7,TLR8 and IFN-? m RNA in macrophage cells of MXSGD ephedra decocted ahead for 25 min rat serum were significantly decreased(P< 0.05),compared with that of MXSGD four kinds of herbals decocted at the same time rat serum group;At 48 h time point,the expression of MyD88 m RNA in macrophage cells of MXSGD ephedra decocted ahead for 20 min rat serum and MXSGD ephedra decocted ahead for 25 min rat serum were significantly decreased(P< 0.05),compared with that of MXSGD four kinds of herbals decocted at the same time rat serum group.(2)The results of Western Blot show that the expression of TLR7,TLR8,MyD88,IFN-? and IFN-? protein in macrophage cells of activator of R848 group and virus intervention group were significantly increased(P< 0.05),compared with that of fetal bovine serum group and blank rat serum group;The expression of TLR7,TLR8,MyD88,IFN-? and IFN-? protein in macrophage cells of MXSGD serum group were significantly decreased(P< 0.05),compared with that of virus intervention group;The expression of TLR7,TLR8,MyD88,IFN-? and IFN-? protein in macrophage cells of the ephedra decocted first MXSGD rat containing serum group were no significant difference(P > 0.05),compared with that of MXSGD four kinds of herbals decocted at the same time rat serum group.3.Study on the mechanism of Maxing Shigan Decoction ephedrae being decoctered first against influenza virus in vivo.(1)The effect of Maxing Shigan Decoction ephedrae being decoctered first on body weight of mice infected with A-type influenza virus.The results show that the mice infected with A-type influenza virus led to body weight decreased in model group,and there was a significant difference compared with that of the normal control group(P<0.05);The mice body weight increased in Maxing Shigan Decoction four kinds of herbals decocted at the same time group and Maxing Shigan Decoction ephedrae being decoctered first group,there were significant difference compared with that of the model group(P<0.05).The mice body weight increased in Maxing Shigan Decoction ephedrae being decoctered first group,there was significant difference compared with that of the Maxing Shigan Decoction four kinds of herbals decocted at the same time group(P<0.05).The body weight decreased inhibition rates in Maxing Shigan Decoction four kinds of herbals decocted at the same time group and Maxing Shigan Decoction ephedrae being decoctered first group were 29.57%,37.68%.(2)The effect of Maxing Shigan Decoction ephedrae being decoctered first on lung index of mice infected with A-type influenza virus.The results show that the mice infected with A-type influenza virus led to lung index increased in model group,and there was a significant difference compared with that of the normal control group(P<0.05);Lung index decreased in Maxing Shigan Decoction four kinds of herbals decocted at the same time group and Maxing Shigan Decoction ephedrae being decoctered first group,there were significant difference compared with that of the model group(P<0.05).Lung index decreased in Maxing Shigan Decoction ephedrae being decoctered first group,there were significant difference compared with that of the Maxing Shigan Decoction four kinds of herbals decocted at the same time group(P<0.05).The lung index increased inhibition rates in Maxing Shigan Decoction four kinds of herbals decocted at the same time group and Maxing Shigan Decoction ephedrae being decoctered first group were 39.79%,45.60%. (3)The results of Western Blot show that the mice infected with A-type influenza virus led to the expression level of TLR7,TLR8 and MyD88 protein of the lung tissue increased in model group,and there was a significant difference compared with that of the normal control group(P<0.05);The 7 days' drug intervention after mice infected with A-type influenza virus led to the expression level of TLR7,TLR8 and MyD88 protein of the lung tissue decreased in Maxing Shigan Decoction four kinds of herbals decocted at the same time group and and Maxing Shigan Decoction Ephedrae being decoctered first group,there were significant difference compared with that of the model group(P<0.05).;The expression level of TLR7,TLR8 and MyD88 protein of the lung tissue decreased in Maxing Shigan Decoction Ephedrae being decoctered first group,there were significant difference compared with that of the Maxing Shigan Decoction four kinds of herbals decocted at the same time group(P<0.05).(4)The Pearson correlation analysis of the antiviral effect index(the lung index and mice body weight)and(TLR7,TLR8 and MyD88 protein)protein expression level after modeling which made mice infected with A-type influenza virus and intervening for 7 days showed that the mice body weight was inversely correlated with TLR7 protein expression level(r=-0.841,P< 0.05);the mice body weight was inversely correlated with TLR8 protein expression level(r=-0.898,P< 0.05);the mice body weight was inversely correlated with MyD88 protein expression level(r=-0.823,P< 0.05);the lung index was positively correlated with TLR7 protein expression level(r=0.899,P< 0.05);the lung index was positively correlated with TLR8 protein expression level(r=0.917,P< 0.05);the lung index was positively correlated with MyD88 protein expression level(r=0.763,P< 0.05).Conclusions 1.Mouse macrophages can identify the molecular pattern of disease related to influenza A virus,which can be used as the carrier of the antiviral mechanism;2.MXSGD has an obvious regulation effect on IFN-alpha and IFN-beta expression level in the macrophage culture media,of which the mouse macrophage were the intervened by the type A influenza virus infection dilution.At 48 h time point,the MXSGD Ephedra decocted ahead for 25 min rat containing serum is better than that of the Maxing Shigan Decoction four kinds of herbals decocted at the same time rat containing serum on regulating IFN-alpha expression level.3.MXSGD has an obvious regulation effect on the expression of major target genes(TLR7,TLR8,MyD88,IFN-? and IFN-? m RNA)and proteins(TLR7,TLR8,MyD88,IFN-? and IFN-? protein)in macrophage cells mediated by TLR7/8.At some time points,Maxing Shigan Decoction Ephedrae being decoctered first rat containing serum is better than that of the Maxing Shigan Decoction four kinds of herbals decocted at the same time rat containing serum on regulating certain genes expression level.4.MXSGD has a protective effect on influenza A virus infection mice.The MXSGD can also inhibit the loss of weight and decreased lung index led by the influenza virus infection;ephedra decocted MXSGD control effect is better than that of siyao with decocted MXSGD.Also the regulation function of Maxing Shigan Decoction Ephedrae being decoctered first is better than that of the Maxing Shigan Decoction four kinds of herbals decocted at the same time.5.MXSGD has an obvious regulation effect on TLR7,TLR8 and MyD88 protein expression level in the lung tissue which was infected by influenza A virus.Also the regulation function of Maxing Shigan Decoction Ephedrae being decoctered first is better than that of the Maxing Shigan Decoction four kinds of herbals decocted at the same time.6.There was a correlation between the effects of influenza virus(the lung index and the mice body weight)and the immune effect index(TLR7,TLR8 and MyD88 protein expression level).In conclusion,MXSGD can act on the TLR7/8-MyD88 signaling pathway regulating the IFN-alpha/beta generation to exert antiviral effect of influenza virus A infection model.It indicated that MXSGD prevent and treat influenza by "Internal regulating" and "external fighting" equate to strengthening healthy qi and eliminating pathogens in Traditional Chinese Medicine(TCM)to some extent.At some time points,the regulation function of Maxing Shigan Decoction Ephedrae being decoctered first is better than that of the Maxing Shigan Decoction four kinds of herbals decocted at the same time. |
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