ABSTRACT Background: The florets of Carthamus tinctorius L. (Safflower) is an important traditional medicine for promoting blood circulation and removing blood stasis. However, its bioactive compounds and mechanism of action need further clarification. Objective: This study aims to investigate the effect and possible mechanism of 6-hydroxykaempferol 3,6-di-O-glucoside-7-O-glucuronide (HGG) from Safflower on endothelial injury in vitro, and to verify its anti-thrombotic activity in vivo. Methods: The endothelial injury on human umbilical vein endothelial cells (HUVECs) was induced by oxygen-glucose deprivation followed by reoxygenation (OGD/R). The effect of HGG on the proliferation of HUVECs under OGD/R was evaluated by MTT, LDH release, Hoechst-33342 staining, and Annexin V-FITC apoptosis assay. RNA-seq, RT-qPCR, Enzyme-linked immunosorbent assay and Western blot experiments were performed to uncover the molecular mechanism. The anti-thrombotic effect of HGG in vivo was evaluated using phenylhydrazine (PHZ)-induced zebrafish thrombosis model. Results: HGG significantly protected OGD/R induced endothelial injury, and decreased HUVECs apoptosis by regulating expressions of hypoxia inducible factor-1 alpha (HIF-1α) and nuclear factor kappa B (NF-κB) at both transcriptome and protein levels. Moreover, HGG reversed the mRNA expression of pro-inflammatory cytokines including IL-1β, IL-6, and TNF-α, and reduced the release of IL-6 after OGD/R. In addition, HGG exhibited protective effects against PHZ-induced zebrafish thrombosis and improved blood circulation. Conclusion: HGG regulates the expression of HIF-1α and NF-κB, protects OGD/R induced endothelial dysfunction in vitro and has anti-thrombotic activity in PHZ-induced thrombosis in vivo.

摘要背景：红花（红花）小花是一种重要的活血化瘀中药。然而，其生物活性化合物和作用机制需要进一步阐明。目的：本研究旨在探讨红花中6-羟基山奈酚3,6-二-O-葡萄糖苷-7-O-葡萄糖苷酸（HGG）对体外内皮损伤的作用及可能的机制，并验证其抗血栓形成活性。体内。方法：人脐静脉内皮细胞（HUVECs）的内皮损伤是通过氧葡萄糖剥夺再氧合（OGD/R）诱导的。通过MTT、LDH释放、Hoechst-33342染色和Annexin V-FITC凋亡试验评估HGG对OGD/R下HUVECs增殖的影响。RNA-seq、RT-qPCR、进行酶联免疫吸附测定和蛋白质印迹实验以揭示分子机制。使用苯肼（PHZ）诱导的斑马鱼血栓形成模型评估了HGG在体内的抗血栓形成作用。结果：HGG通过在转录组和蛋白质水平上调节缺氧诱导因子-1α（HIF-1α）和核因子κB（NF-κB）的表达，显着保护OGD/R诱导的内皮损伤，减少HUVECs凋亡。此外，HGG 逆转了包括 IL-1β、IL-6 和 TNF-α 在内的促炎细胞因子的 mRNA 表达，并减少了 OGD/R 后 IL-6 的释放。此外，HGG 对 PHZ 诱导的斑马鱼血栓形成具有保护作用，并改善了血液循环。结论：HGG调节HIF-1α和NF-κB的表达，