School of Life Science, Zhejiang Chinese Medical University, Hangzhou, PR China.
College of Basic Medical Science, Zhejiang Chinese Medical University, Hangzhou, PR China.
Phytomedicine. 2022 Sep;104:154320. doi: 10.1016/j.phymed.2022.154320. Epub 2022 Jul 6.
After thrombosis, t-PA thrombolysis is the first choice, but the use of t-PA can easily lead to hemorrhagic injury and neurotoxicity. The combination of Danhong injection (DHI) and tissue plasminogen activator (t-PA) therapy may be a new strategy to find high-efficiency anti-thrombosis and low bleeding risk. However, nothing is about the effect of DHI plus t-PA on platelet activation.
The present research was to explore the optimal dose of DHI and t-PA in vivo and mechanisms involved with the treatment of combining DHI and t-PA for thrombotic disease and determined whether DHI plus t-PA affects thrombotic processes related to platelet activation.
Mice were induced by administering κ-carrageenan intraperitoneally, the ratio of different doses of DHI and t-PA in vivo, and the optimal dose effects on platelet aggregation, platelet adhesion, thrombosis formation, and platelet activation were determined. The effects of the αIIbβ3 signaling pathway were analyzed in mice.
In vitro, DHI (62% v/v), t-PA (1 mg/ml), and DHI + t-PA (62% v/v + 1 mg/ml) decreased rat platelet aggregation and adhesion, with a stronger effect from the combination as compared to t-PA monotherapy. In vivo, injections of κ-carrageenan were used to induce BALB/c mice. The optimal dose of DHI, t-PA, and DHI + t-PA is 12 ml/kg, 10 mg/kg, and 12 ml/kg + 7.5 mg/kg. The administration of DHI (12 ml/kg), t-PA (10 mg/kg), and DHI + t-PA (12 ml/kg + 7.5 mg/kg) decreased thrombi in mouse tissue vessels. Furthermore, the reduction of thrombosis formation by DHI, t-PA, and DHI + t-PA was related to lower collagen deposition, and lowered expressions of collagen I, matrix metalloproteinase 2 (MMP-2), and metalloproteinase 9 (MMP-9) in mouse tails, with increased efficacy in combination as compared to t-PA alone. The anti-thrombosis actions of DHI, t-PA, and their combination regulated the expression of CD41, purinergic receptor (P2Y), guanine nucleotide-binding protein G (q) subunit alpha (GNAQ), phosphatidylinositol phospholipase c beta (PLCβ), Ras-related protein 1 (Rap1), RIAM, talin1, fibrinogen alpha chain (FG), kindlin-3, and RAS guany1-releasing protein 1 (RasGRP1).
Based on expression, the mechanism responsible for thrombosis may be attributed to platelet activation via the αIIbβ3 signaling pathway. Combination therapy with DHI and t-PA exerted potent thrombolytic effects. Thus, our data can be used as a foundation for further clinical studies examining the efficacy of traditional Chinese medicines for the treatment of thrombosis.
血栓形成后，t-PA 溶栓是首选，但 t-PA 的使用容易导致出血损伤和神经毒性。丹红注射液（DHI）与组织型纤溶酶原激活剂（t-PA）联合治疗可能是寻找高效抗血栓形成和低出血风险的新策略。然而，目前还没有关于 DHI 加 t-PA 对血小板激活影响的研究。
本研究旨在探讨体内 DHI 和 t-PA 的最佳剂量及两者联合治疗血栓性疾病的作用机制，并确定 DHI 加 t-PA 是否影响与血小板激活相关的血栓形成过程。
通过腹腔注射 κ-卡拉胶诱导小鼠，比较不同剂量 DHI 和 t-PA 的体内比例及对血小板聚集、血小板黏附、血栓形成和血小板激活的最佳剂量效应。分析小鼠的 αIIbβ3 信号通路的作用。
体外，DHI（62%v/v）、t-PA（1mg/ml）和 DHI+t-PA（62%v/v+1mg/ml）均能降低大鼠血小板聚集和黏附，联合用药的效果强于 t-PA 单药治疗。体内，使用 κ-卡拉胶诱导 BALB/c 小鼠。DHI、t-PA 和 DHI+t-PA 的最佳剂量分别为 12ml/kg、10mg/kg 和 12ml/kg+7.5mg/kg。DHI（12ml/kg）、t-PA（10mg/kg）和 DHI+t-PA（12ml/kg+7.5mg/kg）可减少小鼠组织血管中的血栓形成。此外，DHI、t-PA 和 DHI+t-PA 降低血栓形成与胶原沉积减少有关，同时小鼠尾部的胶原 I、基质金属蛋白酶 2（MMP-2）和基质金属蛋白酶 9（MMP-9）表达降低，联合用药的效果强于 t-PA 单药。DHI、t-PA 及其联合用药的抗血栓作用通过调节 CD41、嘌呤能受体（P2Y）、鸟嘌呤核苷酸结合蛋白 G（q）亚单位α（GNAQ）、磷脂酰肌醇磷脂酶 Cβ（PLCβ）、Ras 相关蛋白 1（Rap1）、RIAM、talin1、纤维蛋白原α链（FG）、kindlin-3 和 Ras 鸟苷酸释放蛋白 1（RasGRP1）的表达来发挥作用。
基于表达，血栓形成的机制可能归因于通过 αIIbβ3 信号通路的血小板激活。DHI 和 t-PA 的联合治疗具有较强的溶栓作用。因此，我们的数据可以作为进一步研究中药治疗血栓形成的临床疗效的基础。
