| Purpose:By constructing an influenza model of H1N1 in vitro infection of A549 cells,the effect of Xuanbai Chengqi Decoction on the viral load of H1N1 infected A549 cells at different time points and the expression of RIG-I,MAVS and IFN-β were explored to clarify the Xuanbai Chengqi Decoction Anti-influenza virus mechanism against H1N1 infection of A549 cells.Material and method:First,perform H1N1 in vitro chicken embryo hypertoxicity and detect H1N1 half tissue infection(TCID50),then determine the test drug Xuanbai Chengqi Decoction and the optimal drug concentration(TC50)of oseltamivir phosphate granules,and use drugs to intervene in the cells After the model,the virus inhibition rate was calculated,and the median effective concentration(IC50)of the drug was obtained through Probit regression analysis,and then the drug efficacy evaluation index TI was calculated.Finally,A549 was inoculated in a 4-well culture chamber and randomly divided into normal group,model group,traditional Chinese medicine group,and western medicine group.In addition to the normal group,the other three groups were infected with H1N1 A549 cells.After the virus was adsorbed for 1 hour,the traditional Chinese medicine group was replaced with Xuan Baicheng Qi Decoction with the best concentration of liquid,the western medicine group replaced the best concentration of oseltamivir phosphate,the normal group and the model group replaced the maintenance liquid,at 8h,24 h,48h,72 h after drug intervention H1N1 in vitro infection of A549 cells Take out the culture chamber of the corresponding group at each time point and use immunofluorescence technique to determine the cellular viral load and the fluorescence expression of RIG-I and MAVS protein.Take pictures with a fluorescence microscope and analyze the fluorescence intensity data to explore the changes and differences.ELISA method is used to determine the differences.The expression level of IFN-β in the cell supernatant at the time point.Results:1.H1N1 optimal hemagglutination titer and half of the tissue infection: the optimal hemagglutination titer(1:1024)determined by the virus after chicken embryo amplification,H1N1 virus TCID50 is 10-3.2.Drug therapeutic index: Xuanbai Chengqi Decoction Modified TI=6.82,Oseltamivir Phosphate Granules TI=9.10.3.The viral load of H1N1: The 24 h model group has the highest expression intensity,the traditional Chinese medicine group is less than the model group,there is a difference(P<0.05),the western medicine group is significantly different from the model group(P<0.01),and the traditional Chinese medicine group is less than There are differences in the model group(P<0.05).At 48 h and 72 h,there were differences between the traditional Chinese medicine group and the western medicine group compared with the model group(P<0.01).The 48 h effective rate of the Chinese medicine group was significantly higher than that of 24 h.4.Fluorescence intensity of RIG-I protein expression: The protein intensity of the model histone was significantly increased at 8h(P<0.001),and the drug group was less than the model group;at 24 h,the expression of RIG-I in the western medicine group was significantly reduced(P<0.0001);48h,There were differences between the drug group and the model group at 72h(P<0.05);the model group had the highest protein expression intensity at 24 h at the three time points.5.MAVS protein expression fluorescence intensity: at 8h,the MAVS normal group expressed a small amount,and the expressions of the model group and the drug group were higher than the normal group.There was a difference between the drug group and the model group(P<0.01);the model group continued at 24 h Highly expressed,the expression intensity of the drug group is lower than that of the model group(P<0.01);the MAVS expression intensity of the drug group is lower than that of the model group at 48 h and 72 h,and there is a significant difference(P<0.01);the MAVS fluorescence intensity of the model group reaches the peak at24 h.6.The content of IFN-β in cell supernatant: Compared with the normal group,the expression content of IFN-β in the model group,traditional Chinese medicine group,and western medicine group increased(P<0.05),and the IFN-β expression in the drug group was at 24 h,48h,and 72 h.The expression of β is greater than that of the model group.Conclusion:1.Xuanbai Chengqi Decoction can reduce H1N1 viral load at different time points after infection of A549 cells,and may have the effect of inhibiting influenza virus replication.2.Xuanbai Chengqi Decoction may inhibit the excessive activation of RIG-I protein,down-regulate the expression of MAVS,and release IFN-β to play an anti-influenza virus effect. |
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