Study On Compatibility Mechanism Of Yuanhu Zhitong Prescription Based On Intestinal Barrier And Blood-Brain Barrier

ObjectiveTo study the effect of Angelica dahurica on trans-membrane transport of Corydalis yanhusuo from the perspective of transcription and expression level of related genes in intestinal absorption process and in blood-brain barrier process in vitro,and to clarify the compatibility mechanism of Corydalis yanhusuo and Angelica dahurica in Yuanhu Zhitong prescription;From the point of view of pharmacokinetics in vivo,the effects of Angelica dahurica on the blood concentration and brain tissue distribution of tetrahydropalmatine in rats were investigated,and the regulation of Angelica dahurica on tight junction protein in intestinal tract and brain tissue was studied,which provided the basis for the compatibility theory of Yuanhu Zhitong prescription.Methods1.Using human colon adenocarcinoma cell line Caco-2 as an in vitro model for studying intestinal barrier,a LC-MS/MS bioassay method for tetrahydropalmatine was established.By measuring the changes of apparent permeability coefficient(P_app)before and after compatibility of tetrahydropalmatine with imperatorin,isoimperatorin and Angelica dahurica,the effects of Angelica dahurica on tetrahydropalmatine were clarified RT-PCR and Western blot were used to detect the effects of imperatorin,isoimperatorin and angelica dahurica on the m RNA and protein expression of Occludin,Claudin-1,Claudin-5 and ZO-1 in Caco-2 cells,and the effects of Angelica dahurica on tetrahydropalmatine crossing intestinal barrier were clarified from gene transcription and expression level.2.In vitro BBB cell model was established by co-culture of immortalized human brain microvascular endothelial cells（h CMEC/D3）and human astrocytes（HA1800）.the apparent permeability coefficient of tetrahydropalmatine before and after compatibility with imperatorin,isoimperatorin and angelica dahurica was measured by LC-MS/MS,and the influence of Angelica dahurica on tetrahydropalmatine across blood-brain barrier was clarified through the change of apparent permeability coefficient of tetrahydropalmatine before and after compatibility.RT-PCR and Western blot were used to detect the effects of imperatorin,isoimperatorin and Angelica dahurica on the m RNA and protein of Occludin,Claudin-1,Claudin-5 and ZO-1 in blood-brain barrier model in vitro,and the mechanism of angelica dahurica on tetrahydropalmatine crossing blood-brain barrier was clarified from gene transcription and expression level.3.SD rats were used as the pharmacokinetic study model.Tetrahydropalmatine,imperatorin+tetrahydropalmatine,isoimperatorin+tetrahydropalmatine,Angelica dahurica+tetrahydropalmatine were given to different groups of SD rats by intragastric administration,and blood was collected from anesthetized eye vein plexus at 0,0.083,0.25,0.5,1,2,4,6,8,12h and 24h after administration.Another group of rats were anesthetized and decapitated after 4 hours of intragastric administration by the same method.The brain tissue was taken and the content of tetrahydropalmatine in the brain tissue was determined by LC-MS/MS.The pharmacokinetic parameters were fitted,and the pharmacokinetic changes of tetrahydropalmatine before and after compatibility with imperatorin,isoimperatorin and Angelica dahurica were compared.To provide data support for explaining the scientific compatibility of Corydalis yanhusuo and Angelica dahurica.4.The rats were killed after intragastric administration of Angelica dahurica and normal saline for 4 hours.The brain,duodenum and ileum were taken.The effect of Angelica dahurica on the m RNA expression of tight junction proteins Occludin,Claudin-1,Claudin-5 and ZO-1 in each tissue was determined by RT-PCR,and the penetration mechanism of Angelica dahurica on intestinal barrier and blood-brain barrier was clarified from the point of view of tight junction protein.Results1.The intestinal barrier model of Caco-2 in vitro was successfully established.The bidirectional transmembrane transport of tetrahydropalmatine on Caco-2 cells had good permeability,mainly in the way of passive diffusion.The efflux rate of tetrahydropalmatine in combination with imperatorin,isoimperatorin and Angelica dahurica extract decreased slightly,but there was no statistical difference.After extracting the total RNA of Caco-2 monolayer cells treated with different drugs,the RT-PCR results showed that imperatorin,isoimperatorin and Angelica dahurica could down-regulate the gene expression of Occludin,Claudin-1,Claudin-5 and ZO-1 in different degrees.The results of Western blot assay showed that the protein expression levels of Occludin and ZO-1 were significantly down-regulated,which indicated that Angelica dahurica could promote the intestinal absorption of tetrahydropalmatine by opening the tight junction between cells.2.Successfully established h CMEC/D3 and HA1800 co-cultured in vitro BBB model.The bidirectional transmembrane transport of tetrahydropalmatine on BBB model in vitro had good permeability,mainly in the way of passive diffusion.Compared with the blank control group,imperatorin,isoimperator,and Angelica dahurica could decrease of Occludin,Claudin-1,Claudin-5 and ZO-1 and the protein expression of Occludin and ZO-1 to different degrees,and loosen the tight junction between cells related to the BBB,thus increasing the crossing rate of tetrahydropalmatine across the BBB.3.Pharmacokinetic studies have shown that the AUC_（0-t）and C_max of tetrahydropalmatine can be significantly increased after the compatibility of tetrahydropalmatine with imperatorin,isoimperatorin,and Angelica dahurica extracts（P<0.05）,the amount of distribution in the brain tissue of rats increased significantly（P<0.05）.4.After oral administration of Angelica dahurica in SD rats,the m RNA expressions of Occludin,Claudin-1,Claudin-5 and ZO-1 in duodenum,ileum and brain were significantly decreased.ConclusionTetrahydropalmatine had good permeability in Caco-2 cell model,h CMEC/D3 and HA1800 co-culture model,and its transmembrane transport was mainly passive diffusion.Imperatorin,isoimperatorin and Angelica dahurica could decrease of Occludin,Claudin-1,Claudin-5 and ZO-1 and the protein expression of Occludin and ZO-1 to different degrees.Tissue distribution experiment showed that imperatorin,isoimperatorin and Angelica dahurica extract combined with tetrahydropalmatine could significantly increase the concentration of tetrahydropalmatine in plasma and brain group of rats.The results showed that in Yuanhu Zhitong prescription,Angelicae dahuricae could decrease the expression of Occludin,Claudin-1,Claudin-5 and ZO-1 in different ways,which could loosen the tight connection between the intestinal barrier and the blood-brain barrier epidermal cells,and thus improve the ability of tetrahydropalmatine to cross the intestinal barrier and BBB.