Ali Enas Hussein, Sharifpanah Fatemeh, Wartenberg Maria, Sauer Heinrich
Department of Physiology, Faculty of Medicine, Justus Liebig University Giessen, Germany.
Department of Internal Medicine I, Division of Cardiology, Angiology, Pneumology and Intensive Medical Care, University Hospital Jena, Friedrich-Schiller-University Jena, Germany.
Planta Med. 2018 Jul;84(11):768-778. doi: 10.1055/s-0044-100043. Epub 2018 Jan 12.
Silibinin, the bioactive compound of milk thistle (), exerts tissue protective and regenerative effects that may include stem cell differentiation toward vascular cells. The purpose of the present study was to investigate whether silibinin stimulates blood vessel formation from mouse embryonic stem (ES) cells and to unravel the underlying signaling cascade. Vascular branching points were assessed by confocal laser scanning microscopy and computer-assisted image analysis of CD31-positive cell structures. Protein expression of vascular markers and activation of protein kinases were determined by western blot. Nitric oxide (NO) generation was investigated by use of the fluorescent dye 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. Silibinin dose-dependently increased CD31-positive vascular branching points in embryoid bodies cultivated from ES cells. This was paralleled by increase of protein expression levels for the endothelial-specific markers vascular endothelial cadherin (VE-cadherin), vascular endothelial growth factor receptor 2, and hypoxia-inducible factor-1α. Moreover, silibinin increased activation of endothelial nitric oxide synthase (eNOS), which boosted generation of NO in embryoid bodies and enhanced phosphorylation of signal transducer and activator of transcription 3 (STAT3) as well as phosphoinositide 3-kinase (PI3-K) and AKT. Vasculogenesis, VE-cadherin expression, STAT3 and AKT phosphorylation, NO generation, and eNOS phosphorylation were inhibited by the small molecule STAT3 inhibitor Stattic, AKT inhibitor VIII, the PI3-K inhibitor LY294002, or the NOS inhibitor N-Nitro-L-arginine methyl ester hydrochloride. In conclusion, our findings indicate that silibinin induces vasculogenesis of ES cells via activation of STAT3, PI3-K, and AKT, which regulate NO generation by eNOS.
水飞蓟宾是水飞蓟的生物活性化合物，具有组织保护和再生作用，可能包括促使干细胞向血管细胞分化。本研究旨在探讨水飞蓟宾是否能刺激小鼠胚胎干细胞形成血管，并揭示其潜在的信号级联反应。通过共聚焦激光扫描显微镜和计算机辅助图像分析CD31阳性细胞结构来评估血管分支点。采用蛋白质印迹法测定血管标志物的蛋白表达和蛋白激酶的激活情况。使用荧光染料4-氨基-5-甲基氨基-2',7'-二氟荧光素二乙酸酯研究一氧化氮（NO）的生成。水飞蓟宾能剂量依赖性地增加由胚胎干细胞培养的拟胚体中CD31阳性血管分支点。与此同时，内皮特异性标志物血管内皮钙黏蛋白（VE-钙黏蛋白）、血管内皮生长因子受体2和缺氧诱导因子-1α的蛋白表达水平也有所增加。此外，水飞蓟宾增强了内皮型一氧化氮合酶（eNOS）的激活，这促进了拟胚体中NO的生成，并增强了信号转导和转录激活因子3（STAT3）以及磷酸肌醇3激酶（PI3-K）和AKT的磷酸化。小分子STAT3抑制剂Stattic、AKT抑制剂VIII、PI3-K抑制剂LY294002或NOS抑制剂盐酸N-硝基-L-精氨酸甲酯可抑制血管生成、VE-钙黏蛋白表达、STAT3和AKT磷酸化、NO生成以及eNOS磷酸化。总之，我们的研究结果表明，水飞蓟宾通过激活STAT3、PI3-K和AKT诱导胚胎干细胞的血管生成，这些因子通过eNOS调节NO的生成。
