| 摘要: | 芝麻酚是一種強效的酚類抗氧化物，具有抗癌、保護肝臟、和抗老化的作用。血小板活化與各種急性血栓性疾病和冠心病有關。然而芝麻酚對血小板的影響尚無人研究。因此，本研究的目的是要系統地經由體外和體內的研究，探討芝麻酚防止血小板活化的詳細機制。研究發現，在不同刺激劑所引發的血小板凝集反應中，芝麻酚（2.5-5 microM）較明顯地抑制膠原蛋白所刺激的血小板凝集反應。芝麻酚亦有效抑制膠原蛋白刺激血小板活化後所伴隨的內鈣離子上昇，血栓的產生，和PLCgamma2、PKC及ERK（MAPK）的磷酸化。此外，芝麻酚亦顯著促使cAMP和cGMP的產生，及內皮型一氧化氮合酶（eNOS）的活化，並釋放 NO以及刺激VASP的磷酸化。而在給予cAMP的抑制劑SQ22536後，明顯去除了芝麻酚導致的抑制血小板凝集作用和p38蛋白、VASP、eNOS的磷酸化，及NO的釋放。芝麻酚亦有效的抑制膠原蛋白刺激血小板所誘發的自由基（OH˙）產生。在動物實驗中，芝麻酚（5 mg/kg）亦可顯著延長小鼠血小板栓塞的形成時間。
雖然血小板是無核細胞，然而血小板中亦有細胞轉錄因子NF-kappaB的存在，亦可能產生非基因性調控所引發的血小板活化反應。因此，我們進一步探討芝麻酚對NF-kappaB引發血小板活化的調控作用。 NF-kappaB的訊息傳遞路徑，包括IKKbeta磷酸化，IkappaBalpha降解和p65磷酸化，而芝麻酚（2.5〜25 microM）可顯著的抑制膠原蛋白（1 microg/ml）所刺激人類血小板中NF-kappaB的訊息表現。此外，SQ22536和ODQ這兩個cAMP及cGMP的抑制劑，可有效的回復芝麻酚（25 microM）對IKKbeta磷酸化，IkappaBalpha降解和p65磷酸化的抑制作用。且PKA的抑制劑H89也扭轉芝麻酚對IkappaBalpha降解的抑制效果。NF-kappaB抑制劑BAY11-7082，亦可抑制血小板中膠原蛋白所刺激的IkappaBalpha降解、PLCgamma2磷酸化、PKC活化、細胞內鈣離子上昇、及血小板凝集反應。事先給予血小板SQ22536和H89，皆可有效地回復芝麻酚所導致的抗血小板凝集反應和抑制血小板內鈣離子效果。
本篇論文最重要的發現在於，首次證明芝麻酚具有強效的抗血小板活性，且與活化cAMP-eNOS/NO-cGMP，及抑制PLCgamma2-PKC-p38 MAPK-TxA 2訊息傳遞路徑有關，最終可抑制血小板凝集反應。芝麻酚可能是一種新的藥物可降低血栓栓塞相關的疾病風險或改善心血管功能。此外，我們進一步發現芝麻酚能激活cAMP-PKA路徑，並抑制了下游的NF-kappaB-PLC-PKC訊息傳遞，從而抑制細胞內鈣離子上昇及血小板凝集反應。由於血小板活化不僅與止血有關，亦與發炎性疾病及細胞轉移有關，我們的結果證明了芝麻酚可經由NF-kappaB而抑制血小板功能，此藥物對治療心血管疾病、癌症和發炎性疾病可能亦有其功效。
Sesamol is a potent phenolic antioxidant which possesses antimutagenic, antihepatotoxic, and antiaging properties. Platelet activation is relevant to a variety of acute thrombotic events and coronary heart diseases. There have been few studies on the effect of sesamol on platelets. Therefore, the aim of this study was to systematically examine the detailed mechanisms of sesamol in preventing platelet activation in vitro and in vivo. Sesamol (2.5-5 microM) exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists. Sesamol inhibited collagen-stimulated platelet activation accompanied by [Ca2+]i mobilization, thromboxane A2 (TXA2) formation, and phospholipase C (PLC)gamma 2, protein kinase C (PKC), and mitogen-activated protein kinase (MAPK) phosphorylation in washed platelets. Sesamol markedly increased cAMP and cGMP levels, endothelial nitric oxide synthase (eNOS) expression, and NO release as well as vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the sesamol-mediated inhibitory effects on platelet aggregation and p38 MAPK phosphorylation, and sesamol-mediated stimulatory effects on VASP and eNOS phosphorylation, and NO release. Sesamol also reduced hydroxyl radical (OH˙) formation in platelets. In an in vivo study, sesamol (5 mg/kg) significantly prolonged platelet plug formation of mice.
Although platelets are anucleated cells, they also express the transcription factor, NF-κB, that may exert non-genomic functions in platelet activation. Therefore, we further investigated the inhibitory roles of sesamol in NF-κB-mediated platelet function. NF-κB signaling events, including IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation, were markedly activated by collagen (1 μg/ml) in washed human platelets, and these signaling events were attenuated by sesamol (2.5~25 μM). Furthermore, SQ22536 and ODQ, inhibitors of adenylate cyclase and guanylate cyclase, respectively, strongly reversed the sesamol (25 μM)-mediated inhibitory effects of IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation stimulated by collagen. The protein kinase A (PKA) inhibitor, H89, also reversed sesamol-mediated inhibition of IκBα degradation. Moreover, BAY11-7082, an NF-κB inhibitor, abolished IκBα degradation, phospholipase C (PLC)γ2 phosphorylation, protein kinase C (PKC) activation, [Ca2+]i mobilization, and platelet aggregation stimulated by collagen. Preincubation of platelets with the inhibitors, SQ22536 and H89, both strongly reversed sesamol-mediated inhibition of platelet aggregation and [Ca2+]i mobilization.
The most important findings of this study demonstrate for the first time that sesamol possesses potent antiplatelet activity, which may involve activation of the cAMP-eNOS/NO-cGMP pathway, resulting in inhibition of the PLCγ2 -PKC-p38 MAPK-TXA2 cascade, and finally inhibition of platelet aggregation. Sesamol treatment may represent a novel approach to lowering the risk of or improving function in thromboembolism-related disorders. In addition, we further found could sesamol activate cAMP-PKA signaling, followed by inhibition of the NF-κB-PLC-PKC cascade, thereby leading to inhibition of [Ca2+]i mobilization and platelet aggregation. Because platelet activation is not only linked to hemostasis, but also has a relevant role in inflammation and metastasis, our data also demonstrating that inhibition of NF-κB interferes with platelet function may have a great impact when these types of drugs are considered for the treatment of cardiovascular disease, cancer and various inflammatory diseases. |
