长根姜黄鞭茎（CLR）是一种安全的天然草药，已经被广泛用作功能性食品和保健品数个世纪。然而，它对血管生成及其相关的基础机制的影响仍不清楚。异常的血管生成与各种疾病密切相关，因此精确控制血管生成非常重要。广为人知的血管生成因子血管内皮生长因子（VEGF）通过结合VEGF受体（VEGFR）介导血管生成并诱导多种信号通路。减弱VEGF引发的血管生成相关信号通路可能通过抑制血管生成来缓解各种疾病。因此本研究旨在阐明CLR提取物在体外和体内可产生强烈的抗血管生成活性。我们通过LDH和MTT实验检测了人脐静脉内皮细胞（HUVEC）的存活率。通过创伤愈合和Transwell实验独立评估了内皮细胞的迁移和侵袭能力。通过管形成实验测试CLR提取物在体外血管生成上的作用。在CLR提取物的存在下，通过使用斑马鱼胚胎模型确定了体内血管化。应用西方印迹法确定VEGFR2、PI3K、AKT和eNOS的磷酸化水平。此外，我们通过Griess实验和2'7'-二氯荧光素醋酸酯反应分别评估硝酸盐（NO）和反应性氧（ROS）的水平。此外，通过使用培养的人结肠癌细胞（HT-29细胞系）评估了癌细胞的迁移能力，并应用免疫荧光法评估CLR提取物对VEGF处理下HT-29培养物中NF-κB p65亚单位的核转位的影响。CLR提取物显着抑制了一系列VEGF介导的血管生成反应，包括内皮细胞增殖、迁移、侵袭和管形成。此外，CLR提取物减少了斑马鱼胚胎模型中体内亚肠血管形成。在机制上，CLR提取物减少了VEGF引起的信号传导，如VEGFR2的磷酸化水平下降且随之下游的调节因子被失活，例如磷酸化的PI3K、AKT和eNOS。HUVEC中的NO产生和ROS的形成明显受到抑制。此外，CLR提取物抑制了HT-29细胞的迁移和NF-κB转位。这些预临床研究结果表明CLR提取物显著减弱了血管生成，并具有作为天然药物候选药物的巨大潜力，具有极好的抗血管生成活性。版权所有©2023 Elsevier B.V.出版。

Curcumae Longae Rhizoma (CLR) is a safe natural herbal medicine, and which has been widely used for centuries as functional food and health products, but its effects on angiogenesis and related underlying mechanism remain unclear.The abnormal angiogenesis is closely related with various diseases, and therefore the precise control of angiogenesis is of great importance. The well-known angiogenic factor, vascular endothelial growth factor (VEGF), mediates angiogenesis and induces multiple signalling pathways via binding to VEGF receptor (VEGFR). The attenuation of VEGF-triggered angiogenic-related signalling pathways may relieve various diseases through suppression of angiogenesis. Here, we aimed to elucidate that CLR extract could exert striking anti-angiogenic activities both in vitro and in vivo.The viability of human umbilical vascular endothelial cell (HUVEC) was examined by LDH and MTT assays. Migrative and invasive ability of the endothelial cells were independently evaluated by wound healing and transwell assays. The activities of CLR extract on in vitro angiogenesis was tested by tube formation assay. In vivo vascularization was determined by using zebrafish embryo model in the present of CLR extract. Western blotting was applied to determine the phosphorylated levels of VEGFR2, PI3K, AKT and eNOS. Besides, the levels of nitric oxide (NO) and reactive oxygen species (ROS) were separately evaluated by Griess assay and 2'7'-dichlorofluorescein diacetate reaction. In addition, the cell migrative ability of cancer cell was estimated by using cultured human colon carcinoma cells (HT-29 cell line), and immunofluorescence assay was applied to evaluate the effect of CLR extract on nuclear translocation of NF-κB p65 subunit in the VEGF-treated HT-29 cultures.CLR extract significantly suppressed a series of VEGF-mediated angiogenic responses, including endothelial cell proliferation, migration, invasion, and tube formation. Moreover, CLR extract reduced in vivo sub-intestinal vessel formation in zebrafish embryo model. Mechanistically, the extract of CLR attenuated the VEGF-triggered signalling, as demonstrated by decreased level of phosphorylated VEGFR2 and subsequently inactivated its downstream regulators, e.g. phospho-PI3K, phospho-AKT and phospho-eNOS. The production of NO and formation of ROS were markedly inhibited in HUVECs. Furthermore, CLR extract suppressed cell migration and NF-κB translocation in cultured HT-29 cells.These preclinical findings demonstrate that the extract of CLR remarkably attenuates angiogenesis and which has great potential as a natural drug candidate with excellent anti-angiogenic activity.Copyright © 2023. Published by Elsevier B.V.