Screening The Active Components For Regulating The Key Factors Derived From Epithelial Cells Which Promote Allergic Diseases In Yu-ping-feng-san

In the clinical Yu-ping-feng-san is widely used to treat asthma,allergic rhinitis,urticarial and other allergic diseases,mainly to reduce the recurrence of the allergic diseases.Most studies focused on the mechanism of regulating the balance of Thl and Th2.But the mechanism of preventing and treating the allergic diseases is still unclear.In addition,the components in is also relatively scare,most studies have focused on the polysaccharides in immune regulation while other components is few.So the components research and the mechanism is worth to explore.Firstly,we established fingerprint to provide the material basis for further screening effect components.More and more studies show that epithelial cells(EC)play a key role in stimulating and regulating the local immune response and some cytokines from EC play a key role in starting the allergic diseases.So we established the human bronchial epithelial cell extraction to screen the components specifically binding with the cells.Most Chinese medicine were absorbed into the blood to play the appropriate efficacy after administration.Therefore,we observed whether these components binding to the cells were aslo detected in serum containing.Finally,we explored the possible effect components for immune systerm by pharmacodynamics experiments in vitro and in vivo.Methods and Results:Ⅰ、Establish YPFS fingerprintEstablished YPFS ethanol extrac fingerprint by HPLC-MS to provide the appropriate analytical conditions and material basis for human bronchial epithelial cell extraction.Use Chrom-matrix GP C18 ODS column,acetonitrile-0.05%formic acid as the mobile phase system,the liquid detected wavelength was 200-400 nm,flow rate was 1ml/min,total analysis time was 100min.MS was in positive ion mode,dry gas flow rate:240L/hr;capillary voltage:2800V;cone voltage:40V;drying temperature:310 ℃;ion source tempetrature:120℃;scanning range:200-1000 m/z.There were 32 main peaks in the fingerprint of YPFS ethanol extrac at 254nm,all of the main peaks were well separated.Among these,eight components were determined after comparing with the standard products.They were Prim-O-glucosylcimifugin,Calyeosin-7-O-β-D-glucoside,Cimifugin,4’-O-glucopyranosyl-5-O-methylvisamminol,Ononin,Claycosin,Sec-o-glucosylhamaudol and Formononetin.Ⅱ、Identification of chemicals binding to 16HBE by HPLCIt was reported that epithelial cell played a key role in allergic diseases,so we selected human bronchial epithelial cell(16HBE)to screen potential active components by HPLC-MS based on YPFS fingerprint.Five components(I to V)were found out by 16HBE-HPLC-MS,which indicated that they might bind to 16HBE.Retention time and UV spectrum of the five peaks was corresponding with YPFS fingerprint.Identification by MS,in negative ion mode,the product ion[M+H]+at m/z 447 was Calyeosin-7-O-β-D-glucoside and the transition m/z 447→285 was chosen.The subtraction of the two ions was m/z 162(neutral loss glucose).The product ion[M+H]+at m/z 431 was Ononin,the transition m/z 431→269 was chosen.The product ion[M+H]+at m/z 285 was Claycosin.The product ion[M+H]+at m/z 439 was Sec-o-glucosylhamaudol,the transition m/z 439→277 was chosen.The product ion[M+H]+at m/z 269 was Formononetin.Ⅲ、The efficacy of the components binding to 16HBE in vitroCell extraction and serum containing YPFS showed that Calycosin,Ononis,Formononetin,Sec-o-glucosylhamaudol and Calyeosin-7-O-β-D-glucoside may be the active components regulating allergic factors.TSLP derived from EC plays a key role in allergic diseases,and NF-κB can active TSLP.Then we observed the effect of these components on TSLP in vitro.1.Effect of components on TSLP in vitro:16HBE cell was induced by TNF-α for 12 h togenerate a large number of TSLP,then we observe the effect of these components binding to 16HBE on TSLP.The results showed that Calycosin,Ononis,Formononetin and Calyeosin-7-O-β-D-glucoside could inhibit TSLP in 16HBE significantly,but Sec-o-glucosylhamaudol had no obvious effect.2.Effect of components on the proliferation for 16HBE cells:Cells were seeded in 96-well plate and adding MTT for 4 h,then adding the triple medium overnight,detecting OD value at 570nm.Results showed these components had no effect on the proliferation of 16HBE cells.Ⅳ、Analyse of Components in serum containing YPFSThere were five components binding to 16HBE,so it is worth to know whether these components screened by HB-HPLC-MS can be detected in the blood.To explore the possible effect components,we detected the serum containing YPFS.Serum containing YPFS in mice:The ICR mice were administrated with YPFS by i.g.twice a day for 3 days.1 hour after the last administration the mice blood were collected.Then the YPFS containing serum was analyzed by HPLC-MS,the method was same as 16HBE-HPLC-MS.There were three components in the serum(a,b,c),they were detected as Claycosin,Formononetin and Cimifugin.Ⅴ、Effect of Formononetin and Claycosin on the model of Th2 allergic inflammationMany pharmacokinetic studies have proved that most glycosides in Chinese medicine would be decomposed to the aglycone,then entering into the blood circulation and playing their role.Therefore we examined the effects of flavonoid aglycone on the model of Th2 allergic inflammation in vivo,referred as Formononetin and Claycosin.1.Establishing a murine model of TSLP,IL-25,IL-33 production in AD-like inflammation at the initial stage,exploring the effect of Formononetin and Claycosin on TSLP,IL-25 and IL-33.Mice were administrated with these components by i.p.for 5 days.On day 3,4,mice were treated with 0.6%FITC on both ears to produce lare amounts of TSLP,IL-25 and IL-33.Results showed that Formononetin and Claycosin could inhibit TSLP,IL-25 or IL-33.2.Effect of administration with Calycosin and Formononetin at the initial stage of sensitization phase on ACD mice:Mice were topically sensitized on the shaved abdomens with 1.5%FITC solution on abdominal skin on day 1 and day 2 and elicited on the right ear with 0.5%FITC solution on day 6.Mice were treated with either YPFS or normal saline before sensitized for 2 days and then treated with YPFS from day 1 to day 3(initiation phase).Ear thickness was measured by a calibrated thickness gauge 24 h after the elicitation.Results showed that Formononetin and Claycosin administrated at the initial stage of sensitization could affect the Th2 cell cytokines,decrease the ear thickness and release the inflammatory cell infiltration.3.Effect of components on NF-κB promoter active:16HBE cells were seeded at a density in 24-well plate and transfected with pNFκB-TA-luc plasmid containing the response element that drives transcription of the luciferase reporter gene and Renilla the next day with LipofectamineTM 2000 according to the manufacturer’s instructions for 6 hours.Cells were further incubated with fresh medium for 24 hours.After 24 hours,cells were administrated with components.The induction of the reporter was reduced in cells treated with Formononetin and Calycosin significantly.4.Effect of components on NF-κB translocation:Administrating the components for 2 hours after 16HBE cells were starved overnight,then stimulated with TNF-α for 1 h.After 1h treatment of the 16HBE cells with 100ng/ml of TNF-α,the immunofluorescence analysis revealed that NF-κB(p65)was predominantly presented in the nucleus compared to the control cells.Formononetin and Claycosin could inhibit translocation of NF-κB to the nucleus.