Chang Yi, Hsia Chih-Wei, Chiou Kuan-Rau, Yen Ting-Lin, Jayakumar Thanasekaran, Sheu Joen-Rong, Huang Wei-Chieh
Department of Anesthesiology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei 111, Taiwan.
Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 110, Taiwan.
Biomedicines. 2024 Jul 29;12(8):1689. doi: 10.3390/biomedicines12081689.
Platelets, a type of anucleated cell, play a crucial role in cardiovascular diseases (CVDs). Therefore, targeting platelet activation is essential for mitigating CVDs. Endogenous agonists, such as collagen, activate platelets by initiating signal transduction through specific platelet receptors, leading to platelet aggregation. Eugenol, primarily sourced from clove oil, is known for its antibacterial, anticancer, and anti-inflammatory properties, making it a valuable medicinal agent. In our previous study, eugenol was shown to inhibit platelet aggregation induced by collagen and arachidonic acid. We concluded that eugenol exerts a potent inhibitory effect on platelet activation by targeting the PLCγ2-PKC and cPLA2-TxA2 pathways, thereby suppressing platelet aggregation. In our current study, we found that eugenol significantly inhibits NF-κB activation. This led us to investigate the relationship between the NF-κB and cPLA2 pathways to elucidate how eugenol suppresses platelet activation.
In this study, we prepared platelet suspensions from the blood of healthy human donors to evaluate the inhibitory mechanisms of eugenol on platelet activation. We utilized immunoblotting and confocal microscopy to analyze these mechanisms in detail. Additionally, we assessed the anti-thrombotic effect of eugenol by observing fluorescein-induced platelet plug formation in the mesenteric microvessels of mice.
For immunoblotting and confocal microscopy studies, eugenol significantly inhibited NF-κB-mediated signaling events stimulated by collagen in human platelets. Specifically, it reduced the phosphorylation of IKK and p65 and prevented the degradation of IκBα. Additionally, CAY10502, a cPLA2 inhibitor, significantly reduced NF-κB-mediated signaling events. In contrast, BAY11-7082, an IKK inhibitor, did not affect collagen-stimulated cPLA2 phosphorylation. These findings suggest that cPLA2 acts as an upstream regulator of NF-κB activation during platelet activation. Furthermore, both BAY11-7082 and CAY10502 significantly reduced the collagen-induced rise in intracellular calcium levels. In the animal study, eugenol demonstrated potential as an anti-thrombotic agent by significantly reducing platelet plug formation in fluorescein-irradiated mouse mesenteric microvessels.
Our study uncovered a novel pathway in platelet activation involving the cPLA2-NF-κB axis, which plays a key role in the antiplatelet effects of eugenol. These findings suggest that eugenol could serve as a valuable and potent prophylactic or therapeutic option for arterial thrombosis.
血小板是一种无核细胞，在心血管疾病（CVDs）中起关键作用。因此，靶向血小板活化对于减轻心血管疾病至关重要。内源性激动剂，如胶原蛋白，通过特定的血小板受体启动信号转导来激活血小板，导致血小板聚集。丁香酚主要来源于丁香油，以其抗菌、抗癌和抗炎特性而闻名，是一种有价值的药物。在我们之前的研究中，丁香酚被证明能抑制胶原蛋白和花生四烯酸诱导的血小板聚集。我们得出结论，丁香酚通过靶向PLCγ2-PKC和cPLA2-TxA2途径对血小板活化发挥强大的抑制作用，从而抑制血小板聚集。在我们目前的研究中，我们发现丁香酚能显著抑制NF-κB活化。这促使我们研究NF-κB与cPLA2途径之间的关系，以阐明丁香酚如何抑制血小板活化。
在本研究中，我们从健康人类供体的血液中制备血小板悬浮液，以评估丁香酚对血小板活化的抑制机制。我们利用免疫印迹和共聚焦显微镜详细分析这些机制。此外，我们通过观察荧光素诱导的小鼠肠系膜微血管中的血小板凝块形成来评估丁香酚的抗血栓作用。
对于免疫印迹和共聚焦显微镜研究，丁香酚显著抑制人血小板中胶原蛋白刺激的NF-κB介导的信号事件。具体而言，它降低了IKK和p65的磷酸化，并阻止了IκBα的降解。此外，cPLA2抑制剂CAY10502显著降低了NF-κB介导的信号事件。相比之下，IKK抑制剂BAY11-7082不影响胶原蛋白刺激的cPLA2磷酸化。这些发现表明，在血小板活化过程中，cPLA2作为NF-κB活化的上游调节因子。此外，BAY11-7082和CAY10502均显著降低了胶原蛋白诱导的细胞内钙水平升高。在动物研究中，丁香酚通过显著减少荧光素照射的小鼠肠系膜微血管中的血小板凝块形成，显示出作为抗血栓药物的潜力。
我们的研究揭示了血小板活化中一条涉及cPLA2-NF-κB轴的新途径，该途径在丁香酚的抗血小板作用中起关键作用。这些发现表明，丁香酚可作为动脉血栓形成的一种有价值且有效的预防或治疗选择。
