Regulating Intestinal Flora: An Potential Mechanism Of Qingfei Oral Liquid Improves Airway Inflammation In Asthmatic Mice

Objective: Through the intervention of Qingfei Oral Liquid in the mouse model of asthma and the intervention of fecal bacteria transplantation,the microbial characteristics of the intestinal flora of asthmatic mice and the effect of the intervention of Qingfei Oral Liquid on the intestinal microecology effects in improving asthma airway inflammation.Method: There were a total of 30 female BALB / c mice of 5-6 weeks of age.In order to study the characteristics of intestinal flora in asthmatic mice,a blank control group(CON group)and asthma group(OVA group)were set up,with 6 mice in each group.The asthma group was sensitized by intraperitoneal injection of OVA solution(OVA 10 ug + AL(OH)3 2 mg / 100ul)0.2 ml on days 0,7,and 14 of the experiment.From day 21,20 mg / ml OVA solution was nebulized Inhalation stimulates asthma,30 minutes each time,a week in a row,rest for 3 days,continue to stimulate a week.The blank group was given equal amount of PBS to simulate the same stimulation.Record the growth status and asthma attack status of the mice,and retain the lung tissue sections for HE and PAS staining to observe the infiltration of inflammatory cells and mucus secretion in the lung tissue;use flow cytometry to detect IL-4 and IL-17 a in spleen cell samples 1.The ratio of IFN-γ cytokines;highthroughput sequencing to detect the distribution of bacteria in the intestinal contents.In order to confirm the success of the asthma model,the mice showed typical symptoms of wheezing attacks,laboratory examination of eosinophilia in the peripheral blood of the mice,and typical pathological changes in the lung tissue.In order to observe the relationship between asthma airway inflammation and intestinal flora after intervention of Qingfei Oral Liquid,set Qingfei Oral Liquid to intervene in asthma group(QF group),and take budesonide inhalation as a control(BUD group),6 animals in each group.Asthma model preparation method is the same as above.After the success of the asthma model,from the39 th day,the treatment group was given 0.2ml / day of Qingfei Oral Liquid for 5 days.In the BUD group,the budesonide suspension was nebulized at 30 min / time.D for one week.Twenty-four hours after the last dose,leave the specimen as before and wait for testing.In order to study the relationship between intestinal microflora changes and airway inflammation,fecal bacteria transplantation intervention asthma group(FMT group),each group 6 animals.The preparation method of the asthma model is the same as above.From the 39 th day onwards,0.3ml of normal mouse fecal liquid was infused rectal per mouse for5 days.The mice were sacrificed within 48 hours after the last enema,and specimens were also taken for testing.Results: Part Ⅰ: Asthma mice in the OVA group showed typical symptoms of asthma attacks such as scratching of the face,deep breathing or shortness of breath,and elevation of the back of the arch.Compared with the CON group,the lung histopathology showed that the OVA group had thickened tracheal walls,increased inflammatory cell infiltration,and increased bronchial mucus secretion.Flow cytometry results showed that compared with the CON group,the proportion of IL-4 and IL-17 a positive cells in the splenocytes of the OVA group decreased(P <0.05),while the proportion of IFN-γ positive cells did not change.High-throughput sequencing of intestinal contents showed that the intestinal flora of the OVA group was significantly different from the CON group.Among them,the dominant flora of the CON group are:Adlercreutzia,Coriobacteriaceae,Coriobacteriaceae,Coriobacteriia,Coriobacteriales.The predominant flora of the OVA group are: Y-S2,Mogibacteriaceae,4C0d＿2.Part Ⅱ: The mice in QF group and BUD group had reduced asthma attacks after intervention.Lung histopathology showed that compared with the OVA group,the interstitial eosinophil infiltration of mice in the QF group and the BUD group was reduced,the murine trachea wall was thin,and the secretion of bronchial mucus was reduced.The results of flow cytometry showed that compared with OVA group,the percentage of cells with positive expression of IL-4 and IL-17 a in splenocytes of QF group and BUD group decreased(P <0.05),and there was no significant positive expression of IFN-γ.High-throughput sequencing of intestinal contents showed that the intestinal flora of the two groups was quite different.Among them,the predominant flora of the BUD group were: Parabacteriaceae,Adlercreutzia,Prevotella,Porphyromonadaceae;the predominant flora of the QF group were: Actinomycetals,Sediminibacterium,Chitinophagacea,Saprospirales,Rhizobiales,Anaeroplasma,Firmicutes,Clostridia,Clostridiales,Sediminibacterium,Ruminococcaceae,Ochrobactrum,Brucellaceae,Anaeroplasma,Tenericutes,Roseburia,Mollicutes,Anaeroplasmatales.Part Ⅲ:In the FMT group,the symptoms of asthma attack were alleviated after the intervention of fecal bacteria transplantation.Lung histopathology showed that compared with the OVA group,the infiltration of eosinophils in the lung tissue of the FMT group was reduced,the bronchial wall of the mouse was thinner,and the secretion of bronchial mucus was reduced.Flow cytometry showed that compared with the OVA group,the proportion of cells with positive expression of IL-4 and IL-17 a in the splenocytes of the FMT group decreased(P <0.05),and there was no significant positive expression of IFN-γ.Sequencing of intestinal contents showed that the predominant flora of the FMT group was S24＿7.The intestinal contents of the CON group,OVA group,QF group,BUD group and FMT group were compared and analyzed.The results showed that in the intestinal contents of mice,the microbial flora of each group was quite different.At the phylum level,compared with the OVA group,the abundance of Bacteroidetes and Proteobacteria was higher in the CON and FMT groups,and the abundance of Firmicutes was lower.The abundance of Proteobacteria and Firmicutes in the QF group was higher,and Bacteroidetes and TM7 in the BUD group were higher.At the genus level,compared with the OVA group,the abundances of S24-7,Lachnospiraceae and Odoribacter in the FMT group were lower,and the abundance of Clostridiales,Prevotella and Ruminococcus were higher.The abundances of S24-7,Lachnospiraceae,Clostridiales and Odoribacter in the QF group,BUD group and CON group were significantly reduced,while the abundance of Prevotella,Rikenellaceae,Helicobacter and Ruminococcus increased compared with the OVA group.Conclusion: Both Qingfei Oral Liquid and budesonide nebulization intervention can significantly improve the airway inflammation in asthmatic mice and affect the digestive tract flora in asthmatic mice.Changes in the intestinal microflora of asthmatic mice are related to airway inflammation,and transplantation of normal fecal bacteria can improve airway inflammation in asthmatic mice.Although the changes in the intestinal flora caused by different interventions are not exactly the same,the dominant bacteria in each group are still significantly different from the normal mouse flora,but the existing experimental results can confirm: intestinal flora disorder and airway There is an interaction between inflammation,and this conclusion is consistent with the theory of "the surface of the lung and the large intestine" of traditional Chinese medicine.It may be one of the mechanisms of Qingfei oral liquid to improve asthma airway inflammation by affecting the intestinal flora to regulate immune inflammation.