Transport Of Diazepam In Hypoxia Based On HCMEC/D₃ Cell Monolayer As A Blood Brain Barrier Model

Hypoxia,as a characteristic state of many central nervous system diseases,is not only related to Alzheimer's disease,traumatic brain injury,cerebral ischemia,and stroke,but also to high altitude cerebral edema,intracranial infection,and asphyxia after long-term epilepsy.Plateau-prone diseases are also closely related.The effects of hypoxia on the central nervous system are more serious.Because the brain has strong metabolism and large oxygen consumption,the cerebral cortex is less tolerant to hypoxia.Drug delivery for central nervous system diseases mainly through the blood-brain barrier and blood-cerebrospinal fluid barrier.The broader blood-brain barrier eliminates toxic substances and metabolites through the brain microvascular endothelial cells,and ingests the nutrients needed for the brain.Barriers are important physiological structures that protect the brain and regulate the stability of the environment within the brain.However,a large number of experimental data prove that hypoxia may affect the structure of the blood-brain barrier,interfere with its function,and then affect the drug in and out of the brain tissue,causing patients to have various adverse reactions.In a previous study,the research team found that the expression of drug transporters in the blood-brain barrier changed under hypoxic conditions.However,previous studies used rat brain tissue,which is difficult to reflect the differences in drug transporter expression in the human blood-brain barrier.Therefore,based on related literature,the human brain microvascular endothelial cells were used as the representative structure of the blood-brain barrier,and the following experiments were designed to investigate the effect of hypoxia on drug transport in the blood-brain barrier.First,the effect of hypoxia on the transport of diazepam on the hCMEC/D₃monolayer was investigated.The results showed that the time and concentration dependence of diazepam transport is affected by hypoxia,and the efflux ratio of hypoxic transport decreased significantly.Second,the effect of hypoxia on the drug transporter P-gp in hCMEC/D₃ cells was studied.The results showed that the expression of the drug transporter P-gp at the mRNA and protein levels decreased after hypoxia.Rho123 efflux experiments showed that hypoxia reduced the efflux capacity of P-gp.Finally,the effect of hypoxia on tight junction proteins in hCMEC/D₃ cells was studied.Immunofluorescence staining results showed that the cytoplasmic localization of ZO-1,claudin-5 and Occludin proteins in the hypoxic group decreased,and obvious delocalization occurred.In summary,this study mainly reached the following conclusions:Hypoxia can affect the expression and transport function of the drug transporter P-gp in hCMEC/D₃.In turn,it affects the transmembrane transport of diazepam mediated by P-gp on the hCMEC/D₃ single cell layer of the blood-brain barrier model in vitro.This study can provide a reference for the treatment of hypoxic central nervous system diseases,and may also provide directions for the study of the mechanism of central diseases.