Effects And Mechanism Of Yu-Ping-Feng-San On Relieving Allergic Asthma Based On The Regulation Of Respiratory Microbiomes

Allergic diseases,induced by foreign antigens,includes urticaria,atopic dermatitis,asthma,allergic enteritis and allergic rhinitis,etc.[1].Asthma is one the most common allergic diseases with growing incidence.Although genetic factors and autoimmunity play an important role in the etiology of allergic diseases such as asthma[2],changes in lifestyles and living environment also contribute to the rise in asthma incidence.The irrational use of antibiotics and some other antibacterial drugs,especially,are considered by many researchers as one of the culprits causing the increasing incidence and prevalence of asthma[3].Considering that the human respiratory tract connects with the outside world as a channel,the upper and lower respiratory tract should be the place where the microorganisms are colonized.It is virtually found that whether in the lungs or in the bronchi,all kinds of flora are colonized.A large number of studies have shown that intestinal flora is closely related to intestinal diseases[4],which also proves that intestinal flora is related to asthma diseases[5]Meanwhile,more and more researchers believe that the respiratory flora also plays an important role in the regulation of respiratory diseases[6].Yu-ping-feng-san(YPFS)is from the Yuan dynasty work named "Danxi’s Mastery of Medicine",which has the effect of replenishing qi and strengthening the muscles.Consisting of Astragalus membranaceus,Saposhnikovia divaricate(Turcz.)Schischk and Atractylodes macrocephala Koidz,YPFS has been widely used in allergic diseases.The effect of YPFS on asthma might also be closely related to the regulation of respiratory microbiomes.However,it is almost impossible to study the relationship between respiratory flora and asthma based on clinical and published data.The lack of suitable asthmatic animal models with respiratory flora disturbance is the key obstacle which makes in-depth mechanism research unpractical.This study aimed to explore the relationship between allergic asthma and respiratory flora.An experimental asthmatic mice model with respiratory flora disorder was established to evaluate the efficacy of the Chinese prescription YPFS from the perspective of balancing respiratory bacterial.Method:1.To investigate the effect of airway microbiomes disorder on the animal model of asthma,and to analyze the respiratory tract flora,(1)We established an antibiotic treated(intranasal administration of cefoperazone sulbactam sodium-vancomycin hydrochloride)asthma mice model with respiratory flora disorder,and colonized bronchoalveolar lavage fluid(BALF)from healthy mice to model group mice to study the role of the microbiomes in asthma.(2)16S rRNA(16S ribosomal RNA)sequencing was performed using BALF samples collected from the control group,asthma model group and airway dysbacteriosis asthma model group tostudy the difference in bacterial composition and abundance between these groups.According to the literature,the known sequencing results of the dominant and harmful bacteria in the respiratory tract were statistically screened,as the basis for further research.2.To investigate the efficacy of YPFS in athmatic mice with airxway dysbacteriosis,and to confirm the effect of YPFS on regulating respiratory microbiomes of mice,we(1)Administered YPFS in asthmatic mice.And by analyzing various representative asthmatic changes,xwe confirmed the efficacy of YPFS;(2)Results of 16SrRNA sequencing was analyzed to evaluate whether the abundance of the airway dysbacteriosis in asthmatic mice was restored after YPFS administration,and the beneficial bacteria and harmful bacteria were screened based on the sequencing results.3.To explore the mechanism of action of related disordered bacteria and the effect of YPFS,we(1)Evaluated the actual effects of the colonization of Pseudomonas aeruginosa(PAE)and Flavobacterium(FLA)selected based on sequencing in asthmatic diseases by analyzing the representative asthmatic responses;(2)Co-cultured PAE or FLA with human bronchial epithelial cells 16HBE to study the mechanism of FLA or PAE on epithelial cell junction proteins such as E-cadhernn,occludin and Toll-like receptor-2(TLR2)receptor-related pathway,through which we attempted to study the effect of YPFS on the action of PAE on 16HBE,and to explain the mechanism of YPFS in the treatment of asthma by regulating the microbiomesResearch results:1.Airway dysbacteriosis remarkably aggravated asthma responses in mice,and the colonization from the BALF of healthy mice could significantly alleviated the asthmatic symptoms of model mice.16SrRNA sequencing revealed observable changes in respiratory microbiomes of in model mice.Based on the asthma model established previously by our laboratory,a mouse model of intranasal administration antibiotic-HDM induced asthmatic mice model with respiratory dysbacteriosis was established.We confirmed that respiratory microbiomes(PAE and FLA)played a pivotal role in asthma pathogenesis by analyzing TH2 cytokine levels in mouse lung and BALF,Eosinophils(EOS)in blood,Immunoglobulin E(IgE)levels in serum,lung histopathology and 16SrRNA sequencing results in mouse BALF.2.YPFS could rebalance the disorder of airway dysbacteriosis in asthmatic mice model.YPFS was administered to asthmatic model mice with airway dysbacteriosis,and results showed that YPFS could significantly alleviate the asthmatic responses.16SrRNA sequencing suggested that YPFS could rebalance the disordered flora of model mice,confirming that the anti-asthma efficacy of YPFS might be correlated with regulating respiratory microbiomes in asthmatic mice.3.PAE could aggravate asthmatic responses whereas FLA could reverse its action;The underlying mechanism might be related to regulating TLR2 pathway and restoring epithelial integrity.PAE was found to aggravate the occurrence and severity of asthma in model mice.In vitro studies suggested that PAE could activate TLR2 pathway,disrupt epithelial junction proteins such as E-cadherin and occludin,and thereby lead to the increased production of thymic stromal lymphopoietin(TSLP).In contrast,FLA alone had negligible effect on epithelial junctions,while it could significantly inhibit the indicating responses caused by PAE.4.YPFS could restore epithelial junction dysfunction and inhibit the activation of TLR2 pathway caused by PAE.YPFS was administered in the co-culture system of 16HBE and PAE,and results indicated that YPFS could restore the destruction of epithelial junction proteins,and inhibit the activation of TLR2 and its downstream proteins induced by PAE.Collectively,results suggested that YPFS could alleviate asthma by inhibiting the effect of PAE on epithelial cells and regulating the composition of microbiomes.Research conclusion:1.The respiratory tract flora plays an important role in asthma.By activating the TLR2 pathway,PAE could destroy the bronchial epithelial junction proteins and up-regulate TSLP expression,aggravating the responses of asthma.Meanwhile,FLA could obviously inhibit the effect of PAE.2.The anti-asthma efficacy of YPFS was highly correlated with regulating of respiratory tract flora in asthmatic mice.Inhibition of PAE induced TLR2 activation and epithelial junction protein dysfunction might be its mechanisms.