Study On The Mechanism Of Mahuang Fuzi Xixin Decoction For Allergic Rhinitis With Yang Deficiency Based On Gut Microbiota And Network Pharmacology

Background:Allergic rhinitis(AR)is a common respiratory allergic disease,which belongs to the category of "Biqiu" in Traditional Chinese Medicine.Gut microbiota are found to influence the development of allergic diseases by regulating Th17/Treg.Previous research proved that Mahuang Fuzi Xixin Decoction(MFXD)would regulate Thl/Th2 balance in treating AR with Yang deficiency,but the mechanism of MFXD on gut microbiota and Th17/Treg balance is not clear.Objective:Providing a basis for clinical application and further development of new drugs though illumination of the action mechanism of MFXD in regulating the gut microbiota structure and Th17/Treg balance of AR rats with Yang deficiency,and clarification of the relationship between the nasal and colonic mucosa,and an analysis the chemical composition of MFXD and its molecular mechanism for the treatment of AR.Methods:(1)AR rat models with Yang deficiency were established to detect indexes of AR and Th17/Treg balances through HE staining,ELISA,flow cytometry,and qRT-PCR.(2)16S rDNA sequencing technique was adopted to detect gut microbiota structure,PICRUSt was used to predict the function of gut microbiota,and GC-MS/MS was conducted to detect the content of short chain fatty acids(SCFAs)in faces(3)HE staining and immunohistochemistry were used to observe the damage of mucosal epithelium and cells,and immunofluorescence was adopted to detect the change of neuropeptide substance P.(4)LC-MS/MS and network pharmacology were used to explore the components of decoction and its molecular mechanism in treating AR.Results:(1)MFXD significantly reduced rhinitis symptom score and IgE and histamine content of AR with Yang deficiency.It decreased the level of serum IL-17,the number of CD4+IL-17A+Th17 cells in peripheral blood mononuclear cells(PBMCs),and the mRNA expression of RORyt in nasal mucosa.MFXD increased the level of IL-10,the number of CD25+Foxp3+Treg cells in PBMCs,and the mRNA expression of Foxp3.(2)MFXD increased the ?-diversity indexes of gut microbiota,upregulated the relative abundance of Firmicutes,Bacteroidetes and Proteobacteria,and downregulate the abundance of Actinobacteria and Cyanobacteria.At genus level,MFXD increased the abundance of Anaerotruncus,Bacteroides,Bifidobacterium,Butyricicoccus,Lachnospiraceae,Ruminococcaceae and Prevotellaceae,and reduced the abundance of Enterococcus and Erysipelotrichaceae.PICRUSt prediction proved that differential bacteria were involved in metabolism,genetic information processing,environmental information processing,cell regulation,human diseases and organic systems.MFXD increased the function of membrane transport,energy metabolism,nucleic acid metabolism,cell movement,enzyme family,signal transduction,environmental adaptation and reduced the function of digestion and excretion.In addition,MFXD increased SCFAs content in feces,especially butyric acid.(3)MFXD repaired epithelial cells,reduced inflammatory cell infiltration,increased protein expression of occludin,claudin-1 and ZO-1,and reduce substance P level in nasal and colonic mucosa.Butyrate can repair mucosal injury and reduce substance P level.(4)Twenty-four components of MFXD were identified,relating to 37 targets of AR.The top 10 contribution components include quercetin,pseudoephedrine,ephedrine,?-asarone,methylephedrine,linolenic acid,cathine,ferulic acid,nardosinone and higenamine.It was found through GO functional annotation that MFXD concentrated in plasma membrane,cell surface,cell membrane and other cell components,participating in drug binding,enzyme binding,receptor activity and other molecular functions,and involve in biological processes such as inflammation and lipid metabolism.KEGG pathway enriched into the immune and inflammatory pathways of Fc epsilon RI/B cell receptor/NF-?B signaling pathway and the energy metabolism pathway such as PI3K/Akt,HIF-1,cAMP and AMPK signaling pathway etc.,which suggested that the target and pathway of components are related to the immunoregulation and energy metabolism.Conclusions:(1)MFXD can adj ust the gut microbiota structure of AR rats with Yang deficiency,increase the content of SCFAs and restore the balance of Th17/Treg.(2)The nasal mucosa and colonic mucosa are related to each other,and the pathological changes are synchronous.Substance P may be their mediators(3)The chemical components in MFXD can act on many signal pathways related to inflammation,immunity and energy metabolism,which has the characteristics of synergistic effect.