| ObjectiveBronchial asthma(asthma)is a complex chronic allergic inflammatory disease characterized by Airway Hyper Reactivity(AHR),airway remodeling,and airway inflammation,and it is one of the most prevalent and fastest growing diseases today.In addition,the intestinal flora plays an important role in improving the immune function of the body.Based on this,we used the classical Chinese medicine formula Si Jun Zi Decoction to improve the immune balance and intestinal flora homeostasis by benefiting Qi and strengthening the spleen and cultivating earth and gold.By comparing the Th17/Treg balance and flora balance indexes of asthmatic mice and asthmaABX(Antibiotic mixture)mice,we investigated the underlying mechanism and the inner link between Si Jun Zi Decoction and the regulation of Th17/Treg balance and intestinal flora homeostasis to alleviate asthma.Methods1.6-8 weeks Balb/c females were selected for the replication and excitation of Balb/c female mouse asthma model using OVA and aluminum hydroxide.Mice in the clear bacteria group were selected for gavage with antibiotic mixture on the basis of asthma model,and the intervention components of SiJunZi Decoction were high,medium and low dose groups,and budesonide was selected as the positive drug.2.The general condition of mice was observed during nebulization excitation,airway resistance values were measured by mouse ventilator,and lung histopathology was observed by H&E staining.3.ELISA was used to detect IL-17,TGF-β,IL-6,IL-10 in lung tissues of asthmatic mice,OVA-specific IgE(OVA-IgE)in serum,and SIgA in small intestine lavage fluid.Q-PCR was used to detect the expression of RORyt,Foxp3,and TLR2,TLR4,TLR7 mRNA in lung intestinal tissues.Western Blot assay was performed to detect the expression of RORyt,Foxp3 proteins in lung tissues.Flow cytometry was used to detect the number of Th17/Treg cells in peripheral blood.16SrDNA high-throughput sequencing was used to detect the diversity and structure of intestinal flora in colonic feces.Results1.Therapeutic effects of SiJunZi Decoction on asthmatic mice(1)Compared with the model group,mice in the Si Jun Zi Decoction administration group showed a reduction in the main signs of asthma onset,such as ear scratching,coughing and arching of the back,a decrease in airway Pennh values,a decrease in bronchial inflammatory cell infiltration in lung tissues,a reduction in tissue edema,epithelial detachment and vacuolation,and a decrease in OVE-IgE expression in serum.(2)The main secretory cytokines of Th17,IL-17,IL-6,and TGF-,were significantly less expressed.The percentage of Th17 cells in peripheral blood was decreased,and there was a trend toward decreased expression of the specific transcription factor ROR mRNA and protein in lung tissue.(3)The percentage of Treg cells in peripheral blood increased along with the expression of the cytokine IL-10 and the specific transcription factor Foxp3 mRNA and protein.(4)In addition,TLR2 and TLR4 mRNA expression was decreased and TLR7 expression was increased in pulmonary intestinal tissues.SIgA expression was increased in small intestinal lavage fluid.(5)The differences between groups shrank as intestinal flora diversity increased.At the phylum level,the abundance of the Patescibacteria-dominated phylum decreased while the abundance of the phyla Bacteroidota,Desulfobacterota,and Campilobacterota increased.Alistipes,Odoribacter,and other significant 8 genera increased while the 5 genera based on Lactobacillus decreased.2.The therapeutic effect of SiJunZi Decoction on asthma-ABX mice.(1)Compared with the asthma group,mice in the asthma-ABX group had a higher frequency of scratching and coughing and appeared to have loose stools.The lung tissue was heavily infiltrated with inflammatory cells around the bronchus,and the tissue oedema,epithelial detachment and vacuolation were aggravated,with higher Penh values and OVAIgE expression levels.Compared with the asthma administration group,the asthma-ABX group of Sijunzi Decoction still had an effect,but the effect was diminished.(2)Compared with the asthma group,IL-17,IL-6,TGF-β,as well as RORy mRNA and protein expression and Th17 cell percentage tended to increase more significantly in the asthma-ABX group.Compared with the asthma-administered group,Si Jun Zi Decoction still had an effect in the asthma-ABX group,but the effect was diminished.(3)Compared with the asthma group,IL-10,Foxp3 mRNA,protein and Treg percentage expression were significantly decreased in the asthma-clearing group.Compared with the asthma-administered group,Sijunzi Decoction was still effective in the asthma-ABX group,but the effect was diminished.(4)Compared with the asthma group,the expression of TLR2 and TLR4 in the asthmaABX group was significantly higher and accompanied by a significant decrease in the expression of TLR7 and SIgA.Compared with the asthma-administered group,the asthmaABX group still had an effect of Sijunzi Decoction,but the effect was diminished.(5)Compared with the Control group,the intestinal flora diversity Shannon index and Chao index of asthma-ABX group decreased significantly,the abundance of Bacteroidota phylum decreased,the abundance of Staphylococcus,prevotella and other genera increased,Alistipes,Helicobacter,Bacteroides The abundance of Staphylococcus and Prevotella decreased,and the abundance of Alistipes,Helicobacter and Bacteroides decreased,and Sijunzi Decoction could regulate the structure and diversity of the above enterobacteria;in addition,correlation analysis showed that TLR2,TLR4 and Th 17 were positively correlated with Staphylococcus,negatively correlated with Alistipes and Bacteroides,TLR7 and Treg were positively correlated with Alistipes and Bacteroides,and were negatively correlated with Staphylococcus.Conclusions1.Th17/Treg immune imbalance exists in asthmatic and asthma-ABX mice,and Sijunzi Decoction can regulate the levels of cytokines and specific transcription factors secreted by Th17 and Treg,as well as the percentage of Th17 and Treg,and alleviate the Th17/Treg immune imbalance in asthmatic and asthma-ABX mice2.Si Jun Zi Decoction can benefit Qi,strengthen spleen,cultivate earth and generate gold to regulate asthma,asthma-ABX mice intestinal flora homeostasis,lung and intestinal tissue cell surface pattern recognition receptors and SIgA expression;intestinal flora plays an active role in promoting the regulation of immune homeostasis by Si Jun Zi Decoction. |
