Ischemic stroke is a leading cause of death worldwide, and it remains an urgent task to develop novel and alternative therapeutic strategies for the disease. We previously reported the positive effects of Guhong injection (GHI), composed of safflower extract and aceglutamide, in promoting functional recovery in ischemic stroke mice. However, the active substances and pharmacological mechanism of GHI is still elusive. Aiming to identify the active anti-stroke components in GHI, here we conducted a multi-phenotypic screening in zebrafish models of phenylhydrazine-induced thrombosis and ponatinib-induced cerebral ischemia. Peripheral and cerebral blood flow was quantified endogenously in erythrocytes fluorescence-labeled thrombosis fish, and baicalein and rutin were identified as major anti-thrombotic substances in GHI. Moreover, using a high-throughput video-tracking system, the effects of locomotion promotion of GHI and its main compounds were analyzed in cerebral ischemia model. Chlorogenic acid and gallic acid showed significant effects in preventing locomotor dyfunctions. Finally, GHI treatment greatly decreased the expression levels of coagulation factors F7 and F2, NF-κB and its mediated proinflammatory cytokines in the fish models. Molecular docking suggested strong affinities between baicalein and F7, and between active substances (baicalein, chlorogenic acid, gallic acid, and rutin) and NF-κB p65. In summary, our findings established a novel drug discovery method based on multi-phenotypic screening of zebrafish, provided endogenous evidences of GHI in preventing thrombus formation and promoting behavioral recovery after cerebral ischemia, and identified baicalein, rutin, chlorogenic acid, and gallic acid as active compounds in the management of ischemic stroke.

缺血性中风是世界范围内导致死亡的主要原因，为该疾病开发新的替代治疗策略仍然是一项紧迫的任务。我们之前报道了由红花提取物和乙酰谷氨酰胺组成的谷红注射液 (GHI) 在促进缺血性中风小鼠功能恢复方面的积极作用。然而，GHI的活性物质和药理机制仍不清楚。为了鉴定 GHI 中的活性抗中风成分，我们在斑马鱼模型中进行了苯肼诱导的血栓形成和普纳替尼诱导的脑缺血的多表型筛选。在红细胞荧光标记的血栓形成鱼中，外周和脑血流被内源性量化，黄芩素和芦丁被确定为 GHI 中的主要抗血栓物质。而且，使用高通量视频跟踪系统，在脑缺血模型中分析了 GHI 及其主要化合物的运动促进作用。绿原酸和没食子酸在预防运动功能障碍方面显示出显着效果。最后，GHI 治疗大大降低了鱼类模型中凝血因子 F7 和 F2、NF-κB 及其介导的促炎细胞因子的表达水平。分子对接表明黄芩素和 F7 之间以及活性物质（黄芩素、绿原酸、没食子酸和芦丁）和 NF-κB p65 之间有很强的亲和力。综上所述，我们的研究结果建立了一种基于斑马鱼多表型筛选的新药物发现方法，提供了 GHI 在预防脑缺血后血栓形成和促进行为恢复方面的内源性证据，并鉴定了黄芩素，