The Pharmacodynamic Investigation Of Baicalin Treatment Of Bronchial Asthma And Its Bitter Taste Receptor Mechanism

Objective: To investigate the pHarmacodynamics of Chinese medicinemonomer components baicalin in the treatment of bronchial asthma，and themechanism of baicalin to the bitter taste receptor.Methods:1Inhalation method for the determination of baicalin affecting latentperiod of asthma on guinea pig.2Determination on guinea pig tracheal relaxant effect of differentconcentrations of baicalin by isolated tracheal sheets.3Determination of the anti-inflammatory effects of baicalin by earswelling method.4The MTT assay was applied to detected the influence of baicalin onHBSMC cells activity.5HPLC Determination of the concentration of baicalin in mouse lungsand blood after aerosol inhalation10mg/ml at different time periods (0.5h,1h,2h,4h,8h,24h,48h)6Building pcDNA3.1（+）/GFP/hTAS2R14plasmid and transfects theHBSMC, the transfection efficiency was observed by fluorescence microscopy,using Agarose gel electropHoresis method to verify the expression ofhTAS2R14on HBSMC cell.7pcDNA3.1（+）/GFP/hTAS2R14plasmid transfected HEK293T cells,Luciferase assay was used to detect the influence of different concentrations ofbaicalin （10-9to10-4mol/L） on hTAS2R14expression.8Laser confocal calcium imaging method was used to to detect theinfluence of the calcium ion concentration on HEK293T cells with transfectedhTAS2R14gene after baicalin added (1mmol/ml).9Blood routine examination was perfomed to obtain the influence of different concentrations (5,10,25mg/ml) of baicalin on asthmatic rats bloodWBC, NEUT%, EO%BASO%.Results:1The impact of baical on latency of asthma in guinea pigs： Thedetermination of the asthma latent period by inhalation method was established.After administration，the latent period of asthma extend in varying degrees byeach group. However, latent period of the baicalin group and positive druggroup extended longer than the control group. Simultaneously, the baicalinprotection group (5mg/ml baicalin aqueous solution) extended longer thanpositive group (5mg/ml salbutamol solution).2Relaxant effect of different concentrations of baicalin on guinea pigtracheal：Trachea piece tension experimental determination of baicalin relaxanteffect on guinea pig tracheal was established.The relaxation rate of Baicalinhigh dose group is slightly lower than Doxofylline group on the contractedguinea pig tracheal sheets by acetylcholine stimulated. The relaxation rate ofBaicalin high dose group is slightly lower than Doxofylline group on thecontracted guinea pig tracheal sheet by acetylcholine stimulation（43.07%）, thediastolic trend is more gentlely than Doxofylline group, meanwhile theduration of diastolic action is longer than Doxofylline group. There’s norelaxation effect on normal tracheal by baicalin low dose group, but baicalinlow dose group can relax the guinea pig isolated tracheal tablets contraction byAcetylcholine stimulated. The max relaxation rate is24.63%, and the effectcan be superimposed after repeatedly added。3The anti-inflammatory effects of baicalin: By measuring the thicknessof the ear piece, we found that Baicalin (10mg/ml) can be effective againstxylene-induced ear edema in mice. At different time points after administration(1h,2h,6h),the baicalin group mice ear thickness were lower than the modelgroup. Compared the effect to the positive drug group (Pi Yan Ping Ointment),after dosing1and6hour，the baicalin group of anti-inflammatory effect wasslightly lower than the positive drug group. The anti-inflammatory effects ofbaicalin group and the positive drug group were essentially flat after dosing2 hour. After the organs index of the mice after administration in each group wasmeasured, we found that the ear piece index difference of the baicalin groupwere between the positive drug group and the group; the spleen index of thebaicalin group was flat with positive drug group，both the two groups werelower than the negative group; Thymus index among the three groups has nosignificant difference.4The influence of baicalin on the HBSMC cell activity: The MTTdetermination shows there’s no inhibition of baicalin to the HBSMC cellactivity（IC50＞100μM）5After inhalation of baicalin, concentration of baicalin in mouse lungsand blood at different time periods was evaluated. Baicalin and internalstandard compounds were well separated. There’s a linear relationship of areaand Baicalin concentration between0.5mg/L to95mg/L; the minimumdetectable concentration of baicalin was0.05mg/L. After nebulized inhalatedBaicalin (10mg/ml) for1hour, the metabolic curve in the lungs of mice within48hours was obtained. We found that after administration0.5hour, thebaicalin concentration reached the maximum for the first time(46.57±0.52mg/L).Then the concentration of baicalin was gradually decreasedwith time, at2hours the concentration increased gradually,t he second peak ofbaicalin concentration appeared at the time of4hours(21.67±0.35mg/L), andthe residues tend to zero after48hours. We found that the plasmaconcentration of baicalin in mice reached the peak (4.87±0.54mg/L) at2hours,then fell slightly, after4hours start to increased gradually and has stabilizedafter8hours(3.58~3.54mg/L), finally slightly increased at48hours(4.76±0.23mg/L).6Building pcDNA3.1(+)/GFP/hTAS2R14plasmid and transfects theHBSMC, Observed the transfection efficiency. Observed by fluorescencemicroscopy, the plasmid was able to transfected HBSMC cells successfully,the efficiency of transfection is greater than60%. Extracted the plasmid in thecells，the plasmid purity was well，the concentration is moderate under the UVspectropHotometer. PCR reaction results validated the plasmid with target gene-containing expressed well.The transfection efficiency was relatively highand the transfection conditions are available. Simultaneously found theHBSMC which have not been transfected also containing hTAS2R14genes.7pcDNA3.1(+)/GFP/hTAS2R14plasmid transfected HEK293T cells,Luciferase assay detected the influence of different concentrations of baicalin(10-9to10-4mol/L) on hTAS2R14expression. Determination the fluorescenceintensity changes in HEK293T cells which transfected target gene afterdifferent concentrations of baicalin stimulated(10-9to10-4mol/L).We foundthat the fluorescence intensity increased significantly within the concentrationrange (10-6to10-4mol/L). And the fluorescence intensity had a certaindose-dependent on the concentration of the drug. Suggested that baicalin mayact on the target gene hTAS2R14,and promoted its expression.8Laser confocal calcium imaging method was applied to detect theinfluence of the calcium ion concentration on HEK293T cells with transfectedhTAS2R14gene after baicalin added. After administration, we found that thecalcium ion concentration in the HBSMC cell increased rapidly within0s to5s,then slowly lowered to the initial concentration at40s.9The influence baicalin on asthmatic rats blood WBC, NEUT%, EO%BASO%by blood routine examination. By detecting WBC, NEUT%, EO%,BASO%in the blood of asthma model rats interfered with different doses ofbaicalin(5,10,25mg/ml),we found that various blood indicators of the negativegroup rats were presenced significant difference between the normal rats（p＜0.01), simultaneously various blood indicators of medium dose (10mg/ml)baicalin group rats were also presenced significant difference between thenegative group rats which prompted that the asthma model of rat successfullyestablished and the medium dose of baicalin have a certain protective effect tothe asthma model rats.Conclusions: In the treatment of bronchial asthma, baicalin showed agood effect of in vitro and in vivo anti-inflammatory and tracheal relaxation.There are no damage to the bronchial smooth muscle in the role of process. Itnot only showed a rapid metabolism in the lung after nebulized inhalation, but also could maintained a stable blood concentration for a long time after asingle dose and the residues in the lung were extremely low. Furthermore,baicalin showed a new mechanism through the bitter taste receptor, and couldregulate intracellular calcium ion channels indirectly, finally relaxed thebronchial smooth muscle。Thus，baicalin is a prospective novel Anti-asthma drag.