Department of Pharmacology, School of Basic Medicine, Tongji Medical College of Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan, 430030, China; The Key Laboratory for Drug Target Research and Pharmacodynamic Evaluation of Hubei Province, 13 Hangkong Road, Wuhan, 430030, China.
Department of Pharmacology, School of Basic Medicine, Tongji Medical College of Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan, 430030, China; The Key Laboratory for Drug Target Research and Pharmacodynamic Evaluation of Hubei Province, 13 Hangkong Road, Wuhan, 430030, China; Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen, 518000, China.
Eur J Pharmacol. 2019 Nov 5;862:172626. doi: 10.1016/j.ejphar.2019.172626. Epub 2019 Aug 21.
Neferine has long been recognized as a medicinal herbal ingredient with various physiological and pharmacological activities. Although previous studies have reported its antithrombotic effect, the underlying mechanisms have not been thoroughly investigated. Since platelets play a key role in thrombosis, we investigated the effects of neferine on human platelet function and the potential mechanisms. Platelet aggregation, adhesion and spreading were performed to investigate the effect of neferine on inhibition of platelet function. Flow cytometry was used to determine platelet alpha granule secretion and integrin IIb/IIIa activation, as detected by CD62P (P-selectin) expression, PAC-1 and fibrinogen binding. Western blotting was utilized to investigate the effect of neferine on intracellular signaling of activated platelet. We found that neferine significantly suppressed platelet aggregation and remarkably promoted the dissociation of platelet aggregates induced by collagen, thrombin, U46619, ADP and adrenaline in a dose-dependent manner. Flow cytometry analysis showed that neferine inhibited thrombin-induced platelet P-selectin expression, PAC-1 and fibrinogen binding. In addition, neferine reduced the adhesion of human platelets on coated collagen under both static and shearing condition at an arterial shear rate of 40 dyne/cm. Neferine also inhibited the spreading of human platelets on immobilized fibrinogen. Western blot analysis showed that neferine inhibited PI3K activation, and decreased the levels of phosphorylation of Akt, GSK3β and p38 MAPK in platelets. In summary, neferine has the potential to be an antiplatelet and antithrombotic agent by inhibiting the PI3K-Akt-GSK3β/p38 MAPK signaling pathway.
荷叶碱长期以来被认为是一种具有多种生理和药理活性的药用草药成分。虽然以前的研究已经报道了其抗血栓作用，但潜在的机制尚未得到彻底研究。由于血小板在血栓形成中起着关键作用，我们研究了荷叶碱对人血小板功能的影响及其潜在机制。通过血小板聚集、黏附和铺展实验来研究荷叶碱抑制血小板功能的作用。采用流式细胞术检测血小板α颗粒分泌和整合素 IIb/IIIa 的激活，通过 CD62P（P-选择素）表达、PAC-1 和纤维蛋白原结合来检测。采用 Western blot 来研究荷叶碱对激活血小板细胞内信号的影响。我们发现荷叶碱显著抑制血小板聚集，并显著促进胶原、凝血酶、U46619、ADP 和肾上腺素诱导的血小板聚集物的解离，呈剂量依赖性。流式细胞术分析表明荷叶碱抑制凝血酶诱导的血小板 P-选择素表达、PAC-1 和纤维蛋白原结合。此外，荷叶碱在 40dyne/cm 的动脉剪切率下，在静态和剪切条件下均能抑制人血小板黏附在包被胶原上。荷叶碱还抑制人血小板在固定纤维蛋白原上的铺展。Western blot 分析表明荷叶碱抑制 PI3K 激活，并降低血小板中 Akt、GSK3β 和 p38 MAPK 的磷酸化水平。总之，荷叶碱通过抑制 PI3K-Akt-GSK3β/p38 MAPK 信号通路，具有成为抗血小板和抗血栓药物的潜力。
