Table1_Effects and mechanisms of 6-hydroxykaempferol 3,6-di-O-glucoside-7-O-glucuronide from Safflower on endothelial injury in vitro and on thrombosis in vivo.XLS

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.

背景：红花（Carthamus tinctorius L.）的小花是一种重要的传统活血化瘀中药材。然而其活性成分及作用机制仍有待进一步阐明。
目的：本研究旨在探讨红花来源的6-羟基山奈酚3,6-二-O-葡萄糖苷-7-O-葡萄糖醛酸苷（6-hydroxykaempferol 3,6-di-O-glucoside-7-O-glucuronide, HGG）对体外内皮损伤的作用及其潜在机制，并验证其体内抗血栓活性。
方法：采用氧糖剥夺再灌注（oxygen-glucose deprivation followed by reoxygenation, OGD/R）模型诱导人脐静脉内皮细胞（human umbilical vein endothelial cells, HUVECs）发生内皮损伤。通过MTT法、乳酸脱氢酶（LDH）释放实验、Hoechst-33342染色及Annexin V-FITC凋亡检测，评估HGG对OGD/R处理下HUVECs增殖的影响。通过RNA测序（RNA-seq）、实时定量聚合酶链反应（RT-qPCR）、酶联免疫吸附实验（Enzyme-linked immunosorbent assay, ELISA）及蛋白质印迹（Western blot）实验阐明其分子机制。采用苯肼（phenylhydrazine, PHZ）诱导的斑马鱼血栓模型，评估HGG的体内抗血栓作用。
结果：HGG可显著减轻OGD/R诱导的内皮损伤，并通过在转录组和蛋白水平调控缺氧诱导因子-1α（hypoxia inducible factor-1 alpha, HIF-1α）与核因子κB（nuclear factor kappa B, NF-κB）的表达，降低HUVECs凋亡。此外，HGG可逆转包括白细胞介素1β（IL-1β）、白细胞介素6（IL-6）及肿瘤坏死因子α（TNF-α）在内的促炎细胞因子的mRNA表达，并减少OGD/R处理后IL-6的释放。另外，HGG对PHZ诱导的斑马鱼血栓具有保护作用，并可改善血液循环。
结论：HGG可通过调控HIF-1α与NF-κB的表达，在体外减轻OGD/R诱导的内皮功能障碍，并在体内对PHZ诱导的血栓发挥抗血栓活性。