Theoretical Discussion And Experimental Study On The Treatment Of Severe Influenza With Fuzheng Jiedu Formula

Objective1.Clarify the cytotoxicity of Fuzheng Jiedu formula on A549 and anti-viral effects on A/WSN/1933 influenza virus in vitro.2.Determine the efficacy of Fuzheng Jiedu formula in preventing and treating lung inflammation caused by influenza infection from the aspects of antiviral,anti-inflammatory and immunomodulation.Methods1.In vitro anti-influenza A（H1N1）virus activity study of Fuzheng Jiedu Formula:（1）In vitro cytotoxicity of the drug was detected by MTT colorimetric assay.（2）The antiviral activity of the drug against influenza virus A/WSN/1933（H1N1）was detected by celigo whole field cell imaging analyzer.2.In vivo anti-H1N1 influenza virus infection efficacy of Fuzheng Jiedu Formula（1）Antiviral effect of the drug on A/PR/8/34 infected miceThe mice were randomly divided into 2 groups of 5 mice in each group,which were solution control group and Fuzheng formula group（concentration of 25 mg/ml）.Two groups of mice were infected with LD₅₀ A/PR/8/34 virus droplets in the state of anesthesia,with a total of 30 μl in each left and right nostril.2 days after infection,200 μl is administered by gavage and 200 μl of water is given to the solution control group,1 dose/day.Continuous administration for 6 days.Mice were anesthetized,sacrificed,dissected,and observed for lung lesions on the afternoon of the 9th day after infection.Lung tissue and oral and snout tissues were stored in a-80℃ refrigerator for viral titer detection.（2）Antiviral effect of the drug on A/Netherlands/602/2009 infected miceExperiment 1:Mice were randomly divided into 6 groups,each group of 5 animals,respectively,solution control-2 days group,Fuzheng formula-2 days group;solution control 0-day group,Fuzheng formula 0-day group;solution control+2 days group and Fuzheng+2 days group.Six groups of mice were infected with LD₅₀ A/Netherlands/602/2009 virus droplets in the state of anesthesia,with a total of 30 μl in each left and right nostrils.The-2-day group started the administration 2 days before infection in the mice,the 0-day group started the dose on the day of infection,and the+2-day group started the administration 2 days after infection.Gastric administration of 200μl,solution control group given 200 μl of water,2 doses/day,the interval between administration is greater than 9 h,continuous administration for 5 days.Mice were anesthetized,sacrificed,dissected,and observed for lung lesions on the afternoon of day 7 after infection.Whole lung tissue was stored in a-80℃ freezer for viral titer detection.Experiment 2:The mice were randomly divided into 8 groups,each group of 5 animals,respectively,solution control group 1,Fuzheng formula 1 group,solution control group 2,Fuzheng formula 2 groups,solution control 3 groups,Fuzheng formula 3 groups,solution control 4 groups and Fuzheng formula 4 groups.Mice were anesthetized under anesthesia to establish a mouse viral pneumonia model with LD₅₀ A/Netherlands/602/2009 influenza virus nasal drops,with a total of 30 μl in each left and right nostrils.From 2 days before infection,200μl of gavage was administered in the Fuzheng group,200μl of water was given to the solution control group,2 doses/day,the interval between administration was greater than 9h,and the maximum intervention was 7 days after infection.Mice were anesthetized,sacrificed,dissected,and observed for lung lesions on days 3,5,7 and 14 after infection according to groups.Whole lung and oral and snout tissues were stored in a-80℃ freezer for viral titer detection.（3）To investigate the effect of drugs on weight loss and mortality in mice infected with A/Netherlands/602/2009Experiment 1:Mice were randomly divided into 6 groups,each group of 5 animals,which were solution control-2-day group,Fuzheng formula-2-day group,solution control 0-day group,Fuzheng formula 0-day group,solution control+2-day group and Fuzheng formula+2-day group.Six groups of mice were infected with LD₅₀ A/Netherlands/602/2009 virus droplets in the state of anesthesia,with a total of 30 μl in each left and right nostrils.-2 days group started gavage administration of 200 μl 2 days before infection of mice,solution control group gave 200 μl of water,0 day group started administration on the day of infection,+2 day group started administration 2 days after infection,2 doses/day,dosing interval is greater than 9h,continuous administration for 5 days.Weigh mice and die daily and plot mice weight change curves and survival curves until day 7 post-infection.Experiment 2:Mice were randomly divided into 4 groups,each group of 5 animals,respectively,solution control Mock group,Fuzheng formula Mock group,solution control group and Fuzheng formula.In the state of anesthesia,the solution control group and the Fuzheng formula group were infected with LD₅₀ A/Netherlands/602/2009 virus droplets for nasal infection,with a total of 30 μl in each left and right nostrils.Solution control Mock group and Fuzheng formula Mock group simulated infection with the same volume of PBS nasal drops.All 4 groups started gastric gavage on the day of infection in mice,2 groups were given 200μl of Fuzheng formula,2 control groups were given 200μl of water,2 doses/day,the interval between administration was greater than 9h,and continuous administration was administered to the experimental endpoint.The weight of mice was recorded every day,the weight change curve of mice was drawn,and the observation end time of the experiment was determined according to the weight change results.Experiment 3:Mice were randomly divided into 4 groups,each group of 5 mice,respectively,solution control Mock group,Fuzheng formula Mock group,solution control group and Fuzheng formula.Four groups of mice were infected with LD₅₀ A/Netherlands/602/2009 virus droplets in the state of anesthesia,with a total of 30μl in each left and right nostril.Solution control Mock group and Fuzheng formula Mock group simulated infection with the same volume of PBS nasal drops.All 4 groups started gastric gavage 2 days before infection in mice,2 groups were administered 200μl of Fuzheng formula,2 control groups were given 200μl of water,2 doses/day,the interval between administration was greater than 9h,and continuous administration was administered to the experimental endpoint.The weight of mice was recorded every day,the weight change curve of mice was drawn,and the observation end time of the experiment was determined according to the weight change results.（4）To investigate the anti-inflammatory effects of drugs on influenza-induced inflammationExperiment 1:Mice were randomly divided into 4 groups,each group of 5,which were solution control 3dpi group,Fuzheng formula 3dpi group,solution control 7dpi group and Fuzheng formula 7dpi group.Four groups of mice were infected with LD₅₀ A/Netherlands/602/2009 virus droplets in the state of anesthesia,with a total of 30 μl in each left and right nostril.All 4 groups of mice started gavage 2 days before infection,2 Fuzheng groups administered 200μl,2 control groups were given 200μl water,2 doses/day,the interval between administration was greater than 9h,and continuous administration was administered to the experimental endpoint.According to the group,on the 3rd day after infection,or on the 7th day after infection,the whole lung was sent to make pathological sections.After scanning,it was observed and described in NDP.view 2 software for lung injury scoring.Experiment 2:Thirty 7-8-week-old C57BL/6J female mice were randomly divided into 6 groups,namely uninfected solution group,uninfected Fuzheng formula,blank control group,blank Fuzheng group,solution control group and Fuzheng formula,with 5 mice in each group.A mouse model of viral pneumonia was established with LD₅₀ A/Netherlands/602/2009 influenza virus nasal drops,with a total of 30 μl in each left and right nostril.From 2 days before infection,200μl of gavage was administered in the Fuzheng group,and 200μl of water was given to the solution control group,2 doses/day,and the interval between administration was greater than 9h.The lungs of the uninfected groups were taken before infection on day 0,and the lungs of the remaining groups were taken on the 7th day after infection and placed at-80℃ for qPCR detection.（5）To investigate the immunomodulatory activity of drugs on pneumonia caused by influenza A/Netherlands/602/2009The mice were randomly divided into 6 groups,each group of 5 mice,which were solution control group 1 group,solution control group 2 group,solution control group 3 group,and Fuzheng formula 1 group,Fuzheng formula 2 group,and Fuzheng formula 3 groups.Solution control 2,solution control 3 groups,Fuzheng formula 2,Fuzheng formula 3 groups.Under anesthesia,LD₅₀ A/Netherlands/602/2009 virus drops were infected with a total of 30 μl in each left and right nostril.Solution control group 1 and Fuzheng formula group 1 nasal drops with volume phosphate buffer were used as negative controls.Each group of mice is gavaged twice a day,starting from day-2 of infection and 6 consecutive days of administration.Fuzheng formula group gavaged 200μl of Fuzheng formula solution once a day in the morning and evening;The solution control group was given 200 μ1 of purified water once in the morning and evening.On the 7th day after infection,the mouse alveolar lavage fluid was taken for flow cytometry.（6）Study on the effect of different preparation methods of Fuzheng formula on weight change of mouse with A/Netherlands/602/2009 influenzaThe mice were randomly divided into 4 groups,each group of 5 animals,which were solution control group,Fuzheng formula 1 group,Fuzheng formula 2 groups and Fuzheng formula 3 groups.Four groups of mice were infected with LD₅₀ concentration A/Netherlands/602/2009 virus droplets in the state of anesthesia,with a total of 30 μl in each left and right nostrils.All 4 groups started gastric gavage on the day of infection in mice,3 groups administered 200μl of Fuzheng formula,20μl of water were given to the solution control group,2 doses/day,the interval between administration was greater than 9h,and continuous administration was administered to the experimental endpoint.14 days after infection as the end point of the experiment,the mice were weighed every day,and a line chart of the mice weight loss was drawn.Results1.Fuzheng Jiedu Formula is not cytotoxic to A549 in vitro,and Fuzheng Jiedu Formula has no anti-A/WSN/1933（H1N1）influenza virus activity in vitro.2.In vivo efficacy of Fuzheng Jiedu Formula（1）The results of plaque assay showed that on the 9th day post infection,compared with the solution control group,Fuzheng Jiedu Formula could significantly reduce the virus titer in the lung tissue of mice with statistical difference（P=0.043）.（2）The results of plaque engulation experiments showed that on the third day after infection,Fuzheng Jiedu Formula could significantly reduce the virus titer of the snouts of mice compared with the solution control group（34000pfu/ml vs 70pfu/ml,P=0.0179）.However,the medication time of Fuzheng Jiedu Formula had no significant effect on the lung virus titer of mice.（3）The weight loss curve showed that administration 2 days before infection significantly slowed weight loss in mice caused by influenza A/Netherlands/602/2009 compared with dosing started on the day of infection and 2 days after infection.It suggests that initiation 2 days before infection is the best initial dosing time for Fuzheng formula.The weight loss curve showed that the Fuzheng Jiedu formula could reduce the weight loss and alleviate the disease in the later stages of the disease course of A/Netherlands/602/2009 infected mice.The survival line chart showed that compared with the solution control group,Fuzheng Jiedu Formula could reduce the mortality rate of mice with severe influenza pneumonia caused by A/Netherlands/602/2009 and prolong the survival days of mice（P<0.05）.（4）From the pathological section,the early stage of infection,that is,the 3rd day after infection,the inflammatory damage caused by the virus was low,most mice in the solution control group had local inflammatory cell infiltration and thickening of the alveolar wall,and the structure of each lobe alveolar in the lungs of the mice in the Fuzheng group was relatively intact.In the middle stage of infection,that is,on the 7th day after infection,the solution control group showed a stronger inflammatory response than in the early stage of infection,manifested as diffuse inflammatory cell infiltration,alveolar damage and hemorrhage in the entire lung lobe,and the degree of lung damage in the Fuzheng group was relatively mild.Compared with the solution control group,mice in the Fuzheng formula group showed less inflammatory cell infiltration,relatively intact alveolar structure,thickened alveolar wall,and less bleeding on the 3rd and 7th days after infection.Semi-quantitative analysis of pathological sections showed that lung damage was lower in the Fuzheng group,and the difference was statistically significant（P=0.048）.The results showed that Fuzheng Jiedu Formula could significantly reduce lung inflammatory infiltrates and lung damage and improve inflammation in influenza mice.（5）On the 7th day after infection,compared with the solution control group,the proportion of dendritic cells and alveolar macrophages in the alveolar lavage solution after Fuzheng Jiedu treatment tended to increase,and there was no difference in the proportion of inflammatory monocytes and neutrophils.Compared with the solution control group,the expression of interferon-related genes IFNβ,ISG15,OAS1,MX1 and IP10 decreased after treatment with Fuzheng Jiedu Formula.The results of qPCR technology showed that there was no difference in the expression of M1 protein in the Fuzheng group compared with the solution control group,indicating that the drug failed to interfere with the replication of the virus in the lungs on the 7th day after infection,and the antiviral effect was not obvious.（6）The weight loss curve showed that there was no difference in the weight change of mice administered on the day of infection by different preparation methods of Fuzheng Jiedu Formula compared with the solution control group.ConclusionFuzheng Jiedu Formula has no cytotoxic effect on A549 in vitro and no antiviral activity against A/WSN/1933.Fuzheng Jiedu Formula has antiviral activity in mice in vivo against A/PR/8/34 in the lung and in the upper respiratory tract in the early stages of infection with A/Netherlands/602/2009.The appropriate initial dosing time of Fuzheng Jiedu Formula is 2 days before infection.In this condition Fuzheng Jiedu Formula could alleviate body weight loss in mice and increases the survival rate.In terms of immunity,Fuzheng Jiedu Formula has anti-inflammatory effect and can significantly reduce the inflammatory infiltration and lung injury in the lungs of influenza mice and improve the inflammation.