Wang Guang-fa, Wu Shao-yu, Rao Jin-jun, Lü Lin, Xu Wei, Pang Jian-xin, Liu Zhong-qiu, Wu Shu-guang, Zhang Jia-jie
School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, China.
Acta Pharmacol Sin. 2009 May;30(5):589-96. doi: 10.1038/aps.2009.31. Epub 2009 Apr 6.
Exocytosis of endothelial Weibel-Palade bodies, which contain von Willebrand factor (VWF), P-selectin and other modulators, plays an important role in both inflammation and thrombosis. The present study investigates whether genipin, an aglycon of geniposide, inhibits endothelial exocytosis.
Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords and cultured. The concentration of VWF in cell supernatants was measured using an ELISA Kit. P-selectin translocation on the cell surface was analyzed by cell surface ELISA. Cell viability was measured using a Cell Counting Kit-8. Mouse bleeding times were measured by amputating the tail tip. Western blot analysis was used to determine the amount of endothelial nitric oxide synthase (eNOS) and phospho-eNOS present. Nitric oxide (NO) was measured in the cell supernatants as nitrite using an NO Colorimetric Assay.
Genipin inhibited thrombin-induced VWF release and P-selectin translocation in HUVECs in a dose- and time-dependent manner. The drug had no cytotoxic effect on the cells at the same doses that were able to inhibit exocytosis. The functional study that demonstrated that genipin inhibited exocytosis in vivo also showed that genipin prolonged the mouse bleeding time. Furthermore, genipin activated eNOS phosphorylation, promoted enzyme activation and increased NO production. L-NAME, an inhibitor of NOS, reversed the inhibitory effects of genipin on endothelial exocytosis.
Genipin inhibits endothelial exocytosis in HUVECs. The mechanism by which this compound inhibits exocytosis may be related to its ability to stimulate eNOS activation and NO production. Our findings suggest a novel anti-inflammatory mechanism for genipin. This compound may represent a new treatment for inflammation and/or thrombosis in which excess endothelial exocytosis plays a pathophysiological role.
内皮细胞的魏尔-帕拉德小体可分泌血管性血友病因子（VWF）、P-选择素及其他调节因子，其胞吐作用在炎症和血栓形成过程中均发挥着重要作用。本研究旨在探究京尼平（栀子苷的苷元）是否能抑制内皮细胞胞吐作用。
从脐带中分离并培养人脐静脉内皮细胞（HUVECs）。使用酶联免疫吸附测定试剂盒检测细胞上清液中VWF的浓度。通过细胞表面酶联免疫吸附测定分析细胞表面P-选择素的转位情况。使用细胞计数试剂盒-8检测细胞活力。通过截断小鼠尾尖测量出血时间。采用蛋白质免疫印迹分析确定内皮型一氧化氮合酶（eNOS）和磷酸化eNOS的含量。使用一氧化氮比色测定法检测细胞上清液中亚硝酸盐形式的一氧化氮（NO）含量。
京尼平以剂量和时间依赖性方式抑制凝血酶诱导的HUVECs中VWF释放和P-选择素转位。在能够抑制胞吐作用的相同剂量下，该药物对细胞无细胞毒性作用。体内实验表明京尼平抑制胞吐作用，同时也延长了小鼠的出血时间。此外，京尼平激活eNOS磷酸化，促进酶激活并增加NO生成。一氧化氮合酶抑制剂L-NAME可逆转京尼平对内皮细胞胞吐作用的抑制效果。
京尼平抑制HUVECs中的内皮细胞胞吐作用。该化合物抑制胞吐作用的机制可能与其刺激eNOS激活和NO生成的能力有关。我们的研究结果提示了京尼平一种新的抗炎机制。该化合物可能代表一种针对炎症和/或血栓形成的新治疗方法，其中内皮细胞过度胞吐发挥着病理生理作用。
