Effects Of Qingrexuanfei And Jianpiyiqi Prescriptions On Intestinal Flora, DC And T Cell Subsets In Influenza Mic

Influenza is a kind of acute respiratory tract infection caused by influenza virus.It has epidemic and infectious characteristics.Its clinical manifestations include severe exogenous symptoms,body ache,fatigue and other systemic symptoms.A large number of intestinal microorganisms constitute the most complex micro-ecosystem in the human body.Intestinal flora and host are mutually beneficial and interdependent.Intestinal flora plays an important role in regulating metabolic pathways,maintaining the development and function of immune system and maintaining the body’s stability.The host intestine provides a nutritious and suitable living environment for the intestinal flora.However,once the stable balance between host and intestinal flora is broken,it may cause a series of disorders and pathophysiological reactions,thus promoting the occurrence of diseases.Studies have shown that intestinal flora not only participates in intestinal immune damage after influenza virus infection,but also regulates lung immune response to anti-influenza virus.Normal intestinal flora plays a vital role in maintaining lung anti-viral immunity.Immune response of mice with intestinal flora imbalance is impaired.In addition,the intestinal flora of mice infected with influenza virus changed compared with normal mice.Influenza is caused by the evil of six prostitutions and the epidemic virus invading the human body,which belongs to the category of exogenous febrile diseases in traditional Chinese medicine.Weakness of body,insufficient of healthy qi and unsound external defense are also important reasons for influenza occurrence.Maxingshigan Decoction is a representative prescription of Xinliang Jiebiao.It has the functions of clearing heat,promoting lung and relieving asthma.It is widely used in the treatment of influenza,viral pneumonia and other lung diseases.In the treatment of exogenous diseases,traditional Chinese medicine pays attention to both dispelling pathogens and strengthening health.Spleen and stomach are the acquired basis and source of qi.When the spleen and stomach are normalized,the defensive force is strong against disease.Therefore,the care of spleen and stomach is particularly important.Some clinical influenza patients can see the symptoms of lungs and spleen qi deficiency,such as weakness,laziness,weakness and shortness of breath.When mice are infected with influenza,their asthma,constipation or thin feces accord with the symptoms of this syndrome.It is appropriate to Treat Spleen and qi,so as to nourish the healthy qi.Sijunzi Decoction is a basic prescription for invigorating qi and invigorating.spleen.It is used clinically to treat various diseases with deficiency of stomach and spleen.In previous studies,we confirmed that Sijunzi Decoction had significant promoting effect on mice infected with influenza:Sijunzi Decoction alone had no obvious effect on Anti-influenza and improving symptoms of mice,but combined with Maxingshigan Decoction had significant effect on influenza mice;Maxingshigan Decoction had better effect on influenza mice than Sijunzi Decoction alone.Modern studies have confirmed that Sijunzi Decoction can improve intestinal flora imbalance in mice with spleen deficiency and has immunomodulatory effect.Therefore,whether the synergistic effect of Sijunzi Decoction on the treatment of influenza mice with Maxingshigan Decoction is related to its regulation of intestinal flora needs to be further explored.ObjectiveBased on the important role of intestinal flora in influenza infection and anti-influenza immune response,and on the basis of our previous research,this study further explored the effects of Sijunzi Decoction,Maxingshigan Decoction and their combination on intestinal flora in normal mice,influenza-infected mice,and T cell and DC cell subsets in lung tissue of influenza-infected mice.In this study,Illumina Miseq high-throughput sequencing technology was used to detect the effects of Maxingshigan Decoction,Sijunzi Decoction,Maxingshigan Decoction and Sijunzi Decoction on intestinal flora of normal mice,to determine whether drugs can directly regulate the structure and diversity of intestinal flora;The influenza virus mice model was established,and to clarify the effects of Maxingshigan Decoction,Sijunzi Decoction,Maxingshigan Decoction and Sijunzi Decoction on the structure and diversity of intestinal flora in mice infected with influenza.The influenza virus mice model was established.The number of CD4+T,CD8+T cells,CD 11 c+,CD103+DC in lung tissue of influenza-infected mice was detected by flow cytometry to determine the effect of each drug on T cells and DC cell subsets in lung tissue of influenza-infected mice.Methods1.Effects of Maxingshigan Decoction,Sijunzi Decoction,Maxingshigan Decoction and Sijunzi Decoction on Intestinal Microflora Structure and Diversity in Normal MiceNormal group（group Z）,Maxingshigan Decoction group（group M,group M）,Sijunzi Decoction group（group S）,Maxingshigan Decoction and Sijunzi Decoction group（group MS）were set up.The feces of mice in each group were collected in sterile environment on the 4th and 7th day after administration,Illumina Miseq high-throughput sequencing was used to detect the effect of drugs on intestinal flora in normal mice.2.Effects of Maxingshigan Decoction,Sijunzi Decoction,Maxingshigan Decoction and Sijunzi Decoction on Intestinal Microflora Structure and Diversity of Influenza Virus Infected MiceThe sub-lethal influenza virus infection model of mice was established.Normal group,virus group（shown in group B）,Maxing group,Sijun group and Masi group were set up.The feces of mice in each group were collected in sterile environment on the 4th and 7th day after administration.Illumina Miseq high-throughput sequencing was used to detect the effect of drugs on intestinal flora of influenza-infected mice.3.Effects of Maxingshigan Decoction,Sijunzi Decoction,Maxingshigan Decoction and Sijunzi Decoction on T cells and DC cell subsets in lung tissue of influenza miceThe sublethal influenza virus infection model of mice was established.Normal group,virus group,Maxing group,Sijun group and Masi group were set up.The number of CD4+T cells,CD8+T cells and DC in lung tissues of influenza-infected mice were detected and evaluated by Maxingshigan decoction,Sijunzi decoction,Maxingshigan Decoction and Sij unzi decoction.Results1.Effects of Maxingshigan Decoction,Sijunzi Decoction,Maxingshigan Decoction and Sijunzi Decoction on intestinal flora structure and diversity in normal miceThe structure and proportion of intestinal flora in mice were changed by the use of drugs in each group.At the taxonomic level,there were differences in different genera between each group and the normal group.1.1 On the 4th day after taking the Sijunzi Decoction,the proportions of lactobacillus,Alistipes,Candidatus_Arthromitus,Roseburia,Tyzzerella₃ were significantly decreased,while the Eubacterium_ventriosum_group,Blautia,Anaerofustis,Erysipelatoclostridium and others were significantly increased.Day 7:the lactobacillus fraction increases,Alistipes fraction decreases,Eubacterium_ventriosum_group increases significantly,Ruminococcaceae_UCG₀09 and Lachnospiraceae_UCG₀01 decrease significantly.1.2 After taking Maxingshigan Decoction,day 4:the proportion of Alistipes increased,Peptococcaceae_guncultures and Coprococcus₁ increased significantly,lactobacillus and Candidatus_Arthromitus decreased significantly.Day 7:the proportion of Alistipes increased,and there was a significant increase in the genus Coprococcus₁,Oscillibacter and Roseburia.1.3 After taking Maxingshigan and Sijunzi Decoction,the fourth day:the proportion of Alistipes decreased,the Lactobacillus and segmented Candidatus_Arthromitus decreased significantly,Ruminococcaceae_gunclassified significantly increased;Day 7:the proportion of Alistipes decreased,Bifidobacterium,Eubacterium ventriosum_group,Ruminococcaceae_gunclassified Significantly increased.2.Effects of Maxingshigan Decoction,Sijunzi Decoction,Maxingshigan Decoction and Sijunzi Decoction on Intestinal Microflora Structure and Diversity of Influenza Virus Infected MiceInfluenza infection changes the structure and proportion of intestinal flora in mice and causes the imbalance of intestinal flora.The use of drugs in each group has an effect on the imbalance of intestinal flora in influenza mice.At the genus level,there are different genera of bacteria between the viral group and the normal group,the administration group and the viral group.2.1 On day 4 after influenza infection:Tyzzerella increased significantly,Eubacterium_Ventriosum_group decreased significantly;Day 7:Alistipes increased,Candidatus_Arthromitus,Lactobacillus,Anaerofustis,Eubacterium_Ventriosum_group,Ruminococcaceae_UCG₀14 significantly reduced,Tyzzerella,Anaerrotruncus,Alloprevotella have increased significantly.2.2 On day 4 after influenza infection:Candidatus_Arthromitus,Tyzzerella,norank_fLachnospiraceae,norank_fRuminococcaceae decreased significantly in the Sijun group,Eubacterium_Ventriosum_group,Ruminococcaceae_UCG₀02,Ruminococcaceae_UCG₀14 increased significantly;Day 7:Akkermansia,Desulfovibrio,Enterorhabdus,Eubacterium_Ventriosum_group,Ruminococcaceae_UCG₀05,Ruminococcaceae_UCG₀14 significant Increased,Oscillibacter,Tyzzerella,norank_fRuminococcaceae decreased significantly,and the proportion of Alistipes decreased.2.3 On the 4th day after infection,Candidatus_Arthromitus,Desulfovibrio,Oscillibacter,Tyzzerella,Blautia,norank_fLachnospiraceae,Anaerotruncus,Ruminococcaceae_UCG₀09 have significantly decreased in Maxing group,Eubacterium_Ventriosum_group has significantly increased and Alistipes has increased.Day 7:Anaerofustis,Ruminococcaceae_UCG₀14,Ruminococcaceae NK4A214_group and Subdoligranulum increased significantly,while norank_fLachnospiraceae,unclassified_fLachnospiraceae and Blautia decreased significantly.2.4 On the 4th day after infection,Tyzerella,Ruminococcaceae_UCG₀09,Blautia,and Butyricicoccus were significantly decreased in Masi group,and Eubacterium_Ventriosum_group was significantly increased;Day 7:Oscillibacter,Tyzzerella,Anaerrotruncus,Butyricicoccus,Blautia,norank_fRuminococcaceae,Lachnospiraceae_FCS020_group significantly decreased,Ruminococcaceae_UCG₀14,Anaerofustis significant Increased,the proportion of Alistipes increased.3.Effects of Maxingshigan Decoction,Sijunzi Decoction,Maxingshigan Decoction and Sijunzi Decoction on T cells and DC cell subsets in lung tissue of influenza mice3.1 On the 7th day of infection,the percentage of CD4+T lymphocyte in the lung of mice in virus group was significantly lower than that of normal group（P<0.01）,and the percentage of CD4+T lymphocyte in each administration group was higher than that of virus group,but there was no statistical significance;the percentage of CD8+T lymphocyte in the lung of mice in virus group was significantly lower than that in normal group（P<0.05）,and the percentage of CD8+T lymphocyte in Sijun group was higher than that in virus group.Maxing group and Masi group were slightly lower than virus group,but there was no statistical significance.3.2 On the 7th day of infection,the number of CD11c+ cells in the lungs of mice in virus group was significantly higher than that in normal group（P<0.05）.The number of CDllc+cells in the lungs of mice in virus group was lower than that in virus group,but there was no statistical significance.The number of CD 103+DC cells in the lungs of mice in virus group was higher than that in normal group,the number of CD 103+DC cells in Maxing group was higher than that in virus group,and the number of CD 103+DC cells in Sijun group and Masi group was lower than that in virus.There was no statistical significance.3.3 On the 10th day of infection,the number of CD11c+ cells in the lungs of mice in virus group was significantly higher than that in normal group（P<0.05）,and the number of CD11c+cells in each drug group was higher than that in virus group（P<0.01）;the number of CD 103+DC cells in the lungs of mice in virus group was significantly higher than that in normal group（P<0.001）;the number of CD103+DC cells in the lungs of mice in Maxing group and Masi group was significantly higher than that in virus group（P<0.001,P<0.001）.Conclusions1.The composition and proportion of intestinal microflora can be changed by these traditional Chinese medicine prescription.The use of Sijunzi Decoction,Maxingshigan Decoction and Sijunzi Decoction will reduce the proportion of pathogenic bacteria Alistipes,and the combination of Sijunzi Decoction and Maxingshigan Decoction will significantly increase the abundance of bifidobacteria as a probiotic.Maxingshigan Decoction decreased the content of probiotics such as Lactobacillus and increased the content of pathogenic bacteria such as Coprococcus₁ and Alistipes,which had a significant impact on the structure and diversity of intestinal flora in normal mice.In addition,the use of Sijunzi Decoction,Maxingshigan Decoction and Sijunzi Decoction significantly increased the abundance of bacteria associated with SCFA production.2.Infection with influenza virus can increase the pathogenic bacteria Alistipes and Tyzzerella in mice,decrease the number of probiotic Candidatus_Arthromitus and lactobacillus,decrease the number of bacteria related to SCFAs production,and cause serious disorders.The use of Sijunzi Decoction,Maxingshigan Decoction,Maxingshigan Decoction and Sijunzi Decoction can improve the disorder caused by influenza in varying degrees:the use of Sijunzi Decoction can reduce the content of pathogenic bacteria Alistipes and Tyzzerella,and increase the probiotic Akkermansia;the use of Maxingshigan Decoction can reduce the number of pathogenic bacteria such as Desulfovibrio,Oscillibacter and Tyzzerella.At the same time,the increase of pathogenic bacteria Alistipes caused a significant impact on the intestinal flora of influenza mice.The use of Maxingshigan Decoction and Sijunzi Decoction significantly reduced the pathogenic bacteria Tyzzerella and Oscillibacter.In addition,the number of probiotic Candidatus_Arthromitus in intestinal tract of viral mice decreased on the 4th day when Sijunzi Decoction and Maxingshigan Decoction were used alone,while the combination of Sijunzi Decoction and Maxingshigan Decoction inhibited the decrease of Candidatus_Arthromitus and the increase of Alistipes.Whether in normal mice or in influenza mice model,the use of Maxingshigan Decoction will increase the proportion of Alistipes,indicating that Qingre traditional Chinese medicine may have some side effects on bacteria,killing probiotics will also increase the proportion of pathogenic bacteria,and Sijunzi Decoction can neutralize such side effects.In addition,the use of each group of drugs has complicated changes in the genera associated with SCFAs production.3.During the adaptive immune response period of influenza infection,Sijunzi Decoction can promote the effect of Maxingshigan Decoction on increasing the number of DC cells with the prolongation of medication time,thus mediating further antiviral cellular immune response in mice.