The Study On Mutual Effects On Peameability Of Blood-brain Barrier Among Compounds In Prscription Of Rhizoma Chuanxiong And Gastrodia Elata

ObjectivePrescription of Rhizoma Chuanxiong and Gastrodia Elata was in common used from ancient times to modern times.Dose of two herbs change on account of various pathogenesis,and chemical structure of compounds in the prescription account for different permeabilities of Blood-brain Barrier(BBB).This paper used human microvascular enthothelial cells(HCMEC/D3)to build up models of BBB in vitro,and 4 evaluation were included to identify.In order to survey permeabilities of bioactive compounds in the prescription and their mutual effects,after models were built,Tetramethylpyrazine(TMP),Gastrodin(GAS)and P-hydroxybenzyl alcohol(HBA)were added to the models and had their permeabilities of BBB determined.Abilities of penetrating BBB of compounds above were appraised and their mutual effects were estimated,references to clinical use and drug development about the prescription could be provide from this dissertation.Methods(1)Culture of HCMEC/D3 HCMEC/D3 were seeded on transwell inserts(Polyethylene terephthalate membrane)and experiments began at which time microscopy shown few blank area.(2)Determination of transepithelial electrical resistance(TEER)After HCMEC/D3 seeded,the integrity of cell monolayers were evaluated by measuring TEER every two days,then time-TEER curve was traced.(3)Expression of tight-junction proteins Cell suspension of HCMEC/D3 from different culturing times were collected and had proteins extracted.Expression of Claudin-5,ZO-1 and Occludin were determined by western blotting,then ensureing whether BBB models were accomplished by comparing with contradistinctive cells(human umbilical vascular endothelial cell,HUVEC).(4)Primary evaluation of BBB models The BBB models were evaluated by 4 h liquid interview leak test,and comparation of liquid levels in different times(5)The BBB restriction of hydrophilia micromolecule Fluorescein disodium(FLU)was added in apical side(AP)of transwell and had receiving solution collected from basolateral side(BL),then calculated apparent permeability coefficients(Papp)according to the concentration of FLU.(6)Permeability of TMP and effects of GAS and HBA on permeability of BBB One unchanged concentration of TMP and different concentration of GAS or HBA were added in AP side of transwell and had receiving solution collected from BL side.A LC/MS/MS method was established to determine concentration of TMP,then calculated permeability of TMP.The permeability of different group was compared.(7)Permeability of HBA and effects of TMP on permeability of BBB One unchanged concentration of HBA and different concentration of TMP were added in AP side of transwell and had receiving solution collected from BL side.A HPLC method was established to determine concentration of HBA,then calculated permeability of HBA.The permeability of different group was compared.Result(1)Morphology observation pointed out that spindle HCMEC/D3 showed with well-culturing.Cells concentrated in 3 or 4 days after seeded on transwell inserts and no apparent blank area shown under microscopy,which proved that monolayers completion on membrane.(2)According to TEER determination,TEER improved with times,then remained stable from the 6th day to 10th,this phenomenon implied that BBB models accomplished after six days.(3)Expression of Claudin-5,ZO-1 and Occludin in di fferent group revealed that three proteins were expressed in both HCMEC/D3 and HUVEC,and expression levels of three proteins were higher in HCMEC/D3 than in HUVEC.Expression levels of Claudin-5 and ZO-1 improved with times in HCMEC/D3,then remained stable from the the 6th day to 10th.The western blotting results asserted that BBB models were built successfully with six days cultured.(4)HCMEC/D3 qualified in 4 h liquid interview leak test with six days cultured,which means BBB models accomplished in macroscopy.(5)FLU was restricted effectively by BBB models and Papp in different times kept lower than 1X10-6 cm/s.(6)Method development and validation were described in terms of calibration curves,extraction yield,lower limit of quantification(LLOQ),precision,accuracy,intra and inter-day variability,which were in accordance with the requirements.(7)Both high concentration of GAS and HBA were capable to increase BBB permeability of TMP but no statistic significance were shown among other groups.(8)Different concentration of TMP were capable to increase BBB permeability of HBA,statistic significance between high concentration group and control group occurred in 20 min,which showed more rapid affection than other groups.Conclusion(1)Stability,expression of proteins,liquid leak test,and restriction of hydrophilia micromolecule,typical characters of BBB in vitro established of HCMEC/D3 showed generality with BBB in vivo,which proved that BBB models built of HCMEC/D3 were suitable for permeability research.(2)Both GAS and HBA were able to improve BBB permeability of TMP,but only when concentration of compounds above become higher than TMP could show effects.Moreover,GAS affects more quickactingly than HBA.(3)TMP was able to improve BBB permeability of HBA.