Fan Siwen, Du Hongying, Yao Yixin, Wang Huanyi, Zhang Min, Shi Xuliu, Yan Jiankun, Peng Li, Xiao Guangxu, He Shuang, Lyu Ming, Zhu Yan
State Key Laboratory of Component-Based Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Beihua South Road, Jinghai District, Tianjin 301617, China; Haihe Laboratory of Modern Chinese Medicine, Tianjin 301617, China.
School of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China.
Phytomedicine. 2025 Aug 16;147:157162. doi: 10.1016/j.phymed.2025.157162.
The blood-brain barrier (BBB) is essential for central nervous system (CNS) homeostasis, yet neuroinflammatory mechanisms driving BBB disruption remain poorly understood.
To explore the oxygen-glucose deprivation/reoxygenation (OGD/R)-induced BBB dysfunction and evaluate the therapeutic effects of Guanxinning injection (GXNI), a Danshen-Chuanxiong herbal combination, targeting neuroinflammatory pathways.
A 3D-BBB organoid composed of human brain microvascular endothelial cells, human astrocytes, and primary human brain microvascular pericytes was constructed, and conditions for OGD/R that simulate ischemic stroke were established. Structure and function of the in vitro BBB were evaluated by morphology, paracellular permeability, and tight junction proteins ZO-1, claudin-5, and occludin expression. In vivo, infarct volume and BBB leakage were measured in a mid-cerebral artery occlusion-induced cerebral ischemia-reperfusion injury model. RNA-seq and network pharmacology analysis were used to identify key genes and pathways for ischemic BBB disruption. HPLC-MS was performed to identify and quantify active components. Molecular docking, SPR, and molecular dynamics were performed to predict and confirm the interaction of active compounds and target proteins.
A Danshen-Chuanxiong double herbal medicine, GXNI, mitigated these effects, restoring transport capacity, reducing oxidative stress (ROS), and enhancing basement membrane components (laminin, collagen IV). In vivo, GXNI alleviated cerebral ischemia-reperfusion injury (CIRI), decreasing BBB leakage, infarct volume, and neurological deficits. The pivotal role of TLR4/NF-κB/MMP9 neuroinflammatory axis for GXNI BBB protection was identified through transcriptomic analysis and validated via immunofluorescence in BBB spheroids. Molecular docking revealed Danshen-derived salvianolic acid B (SAB) as a high-affinity MMP9 binder, confirmed by quantitative binding assays. The SAB and Chuanxiong-derived senkyunolide I (SI) combination achieved more prominent upregulation of tight junction proteins and suppression of MMP9.
Our findings further confirm neuroinflammation as a central driver of ischemic BBB damage and demonstrate that GXNI preserves BBB integrity by targeting TLR4/NF-κB/MMP9 signaling in 3D models and CIRI mice, with SAB-SI synergistically contributing to enhanced therapeutic efficacy.
血脑屏障（BBB）对中枢神经系统（CNS）的稳态至关重要，但驱动BBB破坏的神经炎症机制仍知之甚少。
探讨氧糖剥夺/复氧（OGD/R）诱导的BBB功能障碍，并评估丹参-川芎草药组合冠心宁注射液（GXNI）针对神经炎症途径的治疗效果。
构建由人脑微血管内皮细胞、人星形胶质细胞和原代人脑微血管周细胞组成的3D-BBB类器官，并建立模拟缺血性中风的OGD/R条件。通过形态学、细胞旁通透性以及紧密连接蛋白ZO-1、claudin-5和occludin表达来评估体外BBB的结构和功能。在体内，在大脑中动脉闭塞诱导的脑缺血再灌注损伤模型中测量梗死体积和BBB渗漏。使用RNA测序和网络药理学分析来确定缺血性BBB破坏的关键基因和途径。进行高效液相色谱-质谱分析以鉴定和定量活性成分。进行分子对接、表面等离子体共振（SPR）和分子动力学以预测和确认活性化合物与靶蛋白的相互作用。
丹参-川芎双草药制剂GXNI减轻了这些影响，恢复了转运能力，降低了氧化应激（ROS），并增强了基底膜成分（层粘连蛋白、IV型胶原）。在体内，GXNI减轻了脑缺血再灌注损伤（CIRI），减少了BBB渗漏、梗死体积和神经功能缺损。通过转录组分析确定了TLR4/NF-κB/MMP9神经炎症轴对GXNI保护BBB的关键作用，并在BBB球体中通过免疫荧光进行了验证。分子对接显示丹参衍生的丹酚酸B（SAB）是一种高亲和力的MMP9结合剂，定量结合试验证实了这一点。SAB与川芎衍生的藁本内酯I（SI）的组合实现了更显著的紧密连接蛋白上调和MMP9抑制。
我们的研究结果进一步证实神经炎症是缺血性BBB损伤的核心驱动因素，并表明GXNI通过在3D模型和CIRI小鼠中靶向TLR4/NF-κB/MMP9信号通路来维持BBB完整性，SAB-SI协同作用增强了治疗效果。
