Study On The Treatment Of Allergic Rhinitis In Rats With Yang Deficiency And The Functional Components Of Regulating Th1/Th2 Balance Of Mahuang Fuzi Xixin Decoction

BackgroundAllergic rhinitis(AR),called the "Biqiu" in Chinese medicine,has a high incidence and seriously affects people's normal life,and Western medicine has limitations in the treatment of AR currently.Studies have shown that Th1/Th2 imbalance may be the key to the pathogenesis of AR.Mahuang Fuzi Xixin decoction(MFXD)is widely used in the treatment of AR caused by Yang deficiency exogenous cold.In our previous studies,it proved that the therapeutic effect of MFXD on AR in guinea pigs is stronger than that of Xinqin granule,and the inhibitory effect of IgE mediated release of inflammatory mediators from mast cells of MFXD and the mechanism of some functional ingredients were also studied.The Regulation of MFXD in Thl/Th2 balance remains to be explored.Aim of the studyTo clarify the therapeutic effect of MFXD on AR with Yang deficiency in rats and to search for the functional components of MFXD to regulate Thl/Th2 balance,which we hope to provide the basis for the clinical application and drug discovery of this prescription.Methods1?Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry(HPLC-MS/MS)was used to establish the fingerprint of MFXD,and the similarities of 10 batches of MFXD were evaluated by Similarity Evaluation System for Chromatographic Fingerprint of TCM(Version 2004 A).2?Ovalbumin and hydrocortisone injection were used to prepare AR rat model with Yang deficiency;after oral administration of MFXD,the AR symptom scores were recorded,nasal mucosa sections were made and stained with hematoxylin and eosin(HE)and toluidine blue(TB,for mast cells),and the serum levels of cyclic adenosine monophosphate(cAMP),cyclic guanosine monophosphate(cGMP),total immunoglobulin E(IgE),histamine(His),interferon-y(IFN-y)and interleukin-4(IL-4)were measured via enzyme-linked immunosorbent assay(ELISA);the percentages of CD3?CD4+IFN-?+Th1 and CD3+CD4+IL-4+Th2 cells in peripheral blood mononuclear cells(PBMCs)were detected through Flow Cytometry;the mRNA levels of T-bet?GATA-3?Signal transducer and activator of transcription(STAT)-1 and STAT-6 in nasal mucosa were detected via quantitative real-time(qRT)-PCR.3?A small molecule ligand database of MFXD was built through consulting Traditional Chinese Medicine Systems Pharmacology Database(TCMSP)and literature reviews;target proteins related to AR and Th1/Th2 balance were collected through consulting the related protein databases and literature reviews;molecule docking was carried out by software SYBYL,and the Total Score values were recorded;the drug-target network built by software Cytoscape.4?PBMCs in normal rats were separated to examine the effects of potential functional components on the survival rates;PBMCs in AR rat model with Yang deficiency were separated to examine the effects of potential functional components on the regulation of Th1/Th2 imbalance.Results1?HPLC-MS/MS fingerprint of MFXD was successful established,25 peaks in the total ion chromatograms were assigned as common peaks and 9 compounds were detected based on the reference standards,and all similarities among the 10 batches of MFXD were more than 0.987.2?AR rat model with Yang deficiency can also exhibit symptoms of AR and Yang deficiency,and the cAMP/cGMP value significantly decreased compared with that in the control group(P<0.05);when compared with those in the AR rat model with Yang deficiency,the AR symptom scores,total IgE and His levels in serum in MFXD groups significantly decreased,and the histopathological injuries in the nasal mucosa such as nasal respiratory epithelium disruption,leucocyte and mast cell infiltration,and ciliated cell reduction were improved effectively,the cAMP/cGMP and IFN-y/IL-4 values in high concentration group of MFXD increased significantly(P<0.05,P<0.01);when compared with those in the AR rat model with Yang deficiency,the ratio of CD3+CD4+IFN-?+ Th1 and CD3+CD4+IL-4+ Th2 cells increased significantly in high and middle concentration groups of MFXD(P<0.01,P<0.05),the T-bet/GATA-3 value in high concentration group of MFXD increased significantly(P<0.05),the ratio of STAT-1 and STAT-6 mRNA expression increased significantly in high and middle concentration groups of MFXD(P<0.05,P<0.05).3?A ligand database containing 83 compounds of MFXD was built and 7 target proteins related to AR and Th1/Th2 balance were collected;there were 56 small molecule ligands which have at least one Total Score value greater than 5 in molecular docking experiment;the degree values of 19 compounds were greater than the average degree valuein drug-target network,and 10 of them were chosen to be tested in vitro.4?The concentrations of compounds were determined according to the cell viability experiments;naringenin,higenamine,?-asarone,kaempferol,pseudoephedrine,sesamin,squalene and ?-linolenic acid can regulate the Th1/Th2 balance at different levels,apart from ephedrine and elemicin.Conclusion1?MFXD samples have stable sources and controllable quality.2?MFXD can effectively treat AR in rats with Yang deficiency and regulate the Th1/Th2 balance,which might be related to regulating the mRNA expression of T-bet,GATA-3,SATA-1,and STAT6.3?8 out of 10 compounds can regulate Th1/Th2 balance in vitro,which demonstrated that they might be the main functional ingredients of MFXD to treat AR and regulate the Thl/Th2 balance.It suggests that molecular docking technology can be used to explore the functional ingredients of traditional Chinese medicines.