Construction Of In Vitro Spheroids Model Of Blood-Brain Barrier And Its Application In Screening Of Neurotoxicity Of Traditional Chinese Medicine

Objective: Blood-brain barrier(BBB)is a highly selective and dynamic lipophilic barrier between brain and blood circulation.It can effectively prevent exogenous and endogenous toxicity from entering the brain and maintain the homeostasis of the central nervous system(CNS).BBB dysfunction is a pathophysiological marker of a variety of central nervous system diseases.The BBB is mainly composed of brain microvascular endothelial cells(BMVECs),pericytes,astrocytes and neurons.BMVECs are highly polarized cells whose main functions include expression of cell transporters,production of inflammatory mediators,transport of nutrients to brain tissue and prevention of drugs and toxins from entering the central nervous system.One way for toxic substances to reach the central nervous system is to cause BBB dysfunction.In recent years,the incidents of poisoning caused by taking traditional Chinese medicine have occurred frequently,which have attracted people’s attention to the study of its toxicity such as liver and kidney,but there are few reports of neurotoxicity.Therefore,the purpose of this study is to establish the blood-brain barrier model in vitro,and to screen the neurotoxic compounds of traditional Chinese medicine from the compounds bank of traditional Chinese medicine with definite toxicity of other organs,and to study the mechanism of BBB disorder of neurotoxic compounds CTD by using the established BBB model.Methods: 1.Making use of the characteristic that cells will spontaneously form spheroid under low adsorption culture,three kinds of primary cells HBMEC,HBVP and HA were co-cultured with three cells in order to form BBB spheroid-like organs with a structure similar to that in human body.firstly,the BBB spheroid cultured for 3,7 and 14 days were stained with living dead cells to verify whether BBB spheroid can be cultured for a long time.Secondly,IF technique was used to detect the relative spatial distribution of the three cellular components of BBB spheroid,and the characteristic proteins that marked the integrity of BBB(including tight junction associateed proteins,drug efflux transport proteins,extracellμlar matrix proteins)were characterrizeed.In addition,dextran permeability test and Rh123 efflux test were carried out on BBB spheroid to verify the physiological function of BBB spheroid.Finally,we used BBB spheroid for OGD/R modeling,and verified whether BBB spheroid coμld simulate the disease model by evaluating the expression of ZO-1 and the transcription level of hypoxia inducible factor HIF-1 α.2.By exploring the effects of traditional Chinese medicine compounds with other organ toxicity on nerve cells and BBB spheroid,combined with Discovery Studio software to study whether traditional Chinese medicine compounds have neurotoxicity.First of all,the BBB permeability properties of 44 compounds are obtained through the ADMET module of Discovery Studio software.Then,by interacting with PC12 cells and HT-22 cells,the viability of nerve cells was analyzed by Hoechst staining,44 kinds of toxic Chinese medicine compounds were screened for neurotoxicity,and the target drug(CTD)was obtained,and then the selected drugs were applied to BBB spheroid.High connotation imaging and RT-PCR techniques were used to detect their effects on cell viability,BBB characteristic protein and gene level of BBB spheroid.High connotation imaging and RT-PCR techniques were used to detect the effects of CTD on oxidative stress and inflammation-related gene transcription of BBB spheroid.H&E staining and Nissan staining were used to detect the effect of CTD on nerve damage in ICR mice.Results: 1.Through the co-culture of three primary human-derived cells HBMEC,HBVP and HA under low adsorption conditions,spheroid was successfully formed,which could be cultured for a long time(up to 14 days)and maintained high cell viability.Through the iconic protein characterization of the cellular components of the BBB spheroid,it is proved that the three kinds of cells in the BBB spheroid can coexist,and it can be seen from the fluorescence distribution that the three kinds of cells(HBMEC,HBVP,HA)are arranged from outside to inside,which simulates the structural distribution of the blood-brain barrier in the body(the structure of the sphere is very similar to that of the blood-brain barrier in the human body--the endodermis is wrapped in the outermost layer of the sphere.The external environment of the sphere is the interior of the microvascular lumen of the brain,and the internal environment of the sphere is the external lumen of the microvascular of the brain,and the interface between the blood system and the central nervous system is formed by endothelial cells.Then,in the experiment of characterizing the characteristic proteins of BBB integrity,it was proved that BBB spheroid highly expressed tight junction related proteins ZO-1,claudin5 and Occludin,as well as extracellular matrix proteins Laminin,efflux transporters P-gp and LRP-1,which proved that BBB spheroid had the key protein structure of barrier function.After verifying the physiological function of BBB spheroid,it was found that dextran influx was significant after histamine destroyed the blood-brain barrier,and P-gp transport substrate Rh123 influx was significantly after the use of P-gp activity inhibitor verapamil,which proved that BBB spheroid had similar physiological functions in vivo.Finally,through OGD/R modeling of BBB spheroid,IF and RT-PCR techniques were used to very-fy that OGD/ R could reduce the expression of tight junction protein ZO-1 and activate the transcription of HIF-1 α,which proved that BBB spheroid had the ability to simulate disease model.2.The BBB permeation properties of 44 compounds were predicted by computer simulation with Discovery Studio software.Then the neurotoxicity of 45 toxic compounds of traditional Chinese medicine was evaluated by Neuron Outgrowth Assay,and five drug candidates were screened.Then combined with the literature and HT-22 hippocampal neurons to verify the neurotoxicity of CTD,the results showed that CTD could significantly reduce the cell viability of HT-22 cells,and the damage to BBB spheroid coμld be observed under microscope.Through the quantitative analysis of cell viability and tight junction related protein ZO-1 in BBB spheroid,it was proved that CTD could destroy the structure of blood-brain barrier and did not affect the gene transcription level of BBB characteristic proteins ZO-1,Caudin-5,Occludin,P-gp,BCRP1 and GLUT1.Then high-connotation imaging and RT-PCR were used to detect the effects of CTD on the accumulation of reactive oxygen species and the transcription level of inflammatory factors in BBB spheroid.The results showed that CTD could significantly increase the intracellular ROS accumulation of BBB spheroid and significantly increase the gene transcription levels of IL-6,IL-8 and VEGF.H&E staining and Nissan staining showed that CTD can cause neuron loss,solubilization of Nissan bodies,and edema at the base of granular cells in the hippocampus of ICR mice.Conclusion: We co-cultured three primary human-derived cells HBMEC,HBVP and HA under low adsorption conditions to form spheroid spontaneously.Through the characterization of BBB characteristic proteins,physiological fun-ction verification and disease model simulation,it was proved that the spheroid self-assembled into modular organs similar to the blood-brain barrier structure and function in vivo.Then we used the comprehensive strategy of neuronal cytotoxicity test combined with BBB spheroid toxicity test to screen the neurotoxic CTD,from 44 kinds of toxic traditional Chinese medicine compounds and considered that CTD may destroy the blood-brain barrier by increasing the accumulation of intracellular reactive oxygen species and inducing the expression of pro-inflammatory factors.Finally,we used ICR mice to be infected,we found that CTD can cause nerve damage in mice,which verified the neurotoxic effect of CTD on the animal level.