3D血脑屏障类器官氧糖剥夺模型的构建及冠心宁注射液的保护作用探究

Construction of 3D blood-brain barrier organoid oxygen-glucose deprivation model and exploration of the protective effect of Guanxinning injection

血脑屏障(blood-brain barrier,BBB)在维持中枢神经系统稳态方面发挥着重要作用。在许多神经系统疾病如缺血性脑卒中、阿尔茨海默症等疾病中都会出现BBB损伤。中医药对于防治缺血性脑卒中潜力巨大,但临床相关模型的缺乏已成为其应用开发的瓶颈。本研究采用了由人脑微血管内皮细胞(HBMEC)、人脑星形胶质细胞(HA)和人脑血管周细胞(HBVP)组成的BBB类器官模型,并建立了细胞活力、屏障通透性及BBB标志物表达的氧糖剥夺/复氧(OGD/R)条件。然后在该类器官模型中研究了中药冠心宁注射液(GXNI)对OGD/R诱导的BBB功能障碍的保护作用。结果表明,OGD/R降低BBB类器官细胞活力,增加屏障通透性(渗漏),降低紧密连接蛋白闭塞带-1(ZO-1)、claudin-5、闭塞素和P-糖蛋白(P-gp)的水平。GXNI可显著阻止OGD/R诱导的BBB破坏,如细胞活力降低和通透性增加。本研究为中枢神经系统靶向药物的研发提供了一种新的人类细胞源性3D缺血性脑病模型,并证明了中药GXNI在体外可有效保护BBB功能障碍。

The blood-brain barrier(BBB)plays an important role in maintaining the homeostasis of the central nervous system.BBB is disrupted in many neurological disorders such as ischemic stroke and Alzheimer’s disease.Traditional Chinese medicine has great potential to prevent ischemic stroke,but the lack of clinically relevant models has been a challenge.We adapted a BBB organoid model formed by human brain microvascular endothelial cells(HBMEC),human astrocytes(HA),and human brain vascular pericytes(HBVP),and established conditions for oxygen-glucose deprivation/reoxygenation(OGD/R)on the cell vitality,barrier permeability,as well as BBB signature marker expression.The protective effect of Guanxinning injection(GXNI)on OGD/R-induced BBB dysfunction was then investigated in the organoid model.The results showed that OGD/R decreased BBB organoid cell viability,increased permeability(leakage),decreased the level of tight junction proteins zonula occludens-1(ZO-1),claudin-5,occludin and P-glycoprotein(P-gp).GXNI significantly prevented OGD/R-induced BBB disruption such as the decreased cell viability and increased permeability.This study provides a new human cellderived 3D ischemic brain disease model for central nervous system-targeted drug research and development and demonstrates that a Chinese injection medicine GXNI effectively protects BBB dysfunction in vitro.