Aristolochic Acid 抑制血小板凝集作用之機轉探討 Mechanisms Involved in the Antiplatelet Activity of Aristolochic Acid

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Aristolochic Acid 抑制血小板凝集作用之機轉探討

Mechanisms Involved in the Antiplatelet Activity of Aristolochic Acid

中文摘要

馬兜鈴酸是一種來自馬兜鈴科(Aristolochiaceae)的生物鹼，已被證實有致癌性，且長期使用下將造成腎衰竭。最近的研究報告指出馬兜鈴酸可以抑制由蛇毒所引起的水腫現象、抗發炎，且有抑制血小板的活性的功效。然而，馬兜鈴酸在血小板上的藥理學功效尚未明確，因此我們有意探討馬兜鈴酸在血小板活化過程中，對於訊息傳遞方面的抑制機轉。由本研究結果顯示，馬兜鈴酸隨著濃度的增加(75-150 micromolar)，能有效地抑制 collagen (1 microgram/ml) 所引起的人類血小板凝集反應以及ATP 釋放反應；且馬兜鈴酸(115 和 150

micromolar)可以抑制由 collagen 所刺激細胞內鈣離子的流動、

phosphoinositide 的增加和 thromboxane A2 的形成。此外，馬兜鈴酸(115 和150 micromolar)可以增加細胞內 nitrate 的含量及 vasodilator-

stimulated phosphoprotein (VASP)的磷酸化。對於血小板內 47 kDa 蛋白質磷酸化，這是一個標記protein kinase C 活性的方法。在本實驗中我們分別使用collagen (1 microgram/ml)和 PDBu (150 nano molar)促進血小板 47 kDa 蛋白質磷酸化，發現馬兜鈴酸只能抑制由 collagen 所活化 47 kDa 蛋白質磷酸化。另外，馬兜鈴酸(115 和 150 micromolar)可以抑制由

collagen (10 microgram/ml)所引起 p38 MAPK 的磷酸化反應但不能清除由collagen (1 microgram/ml)刺激血小板所導致的自由基。

由結果證實，馬兜鈴酸抑制血小板活性的作用可能涉及下列路徑：(一)馬兜鈴酸可以抑制PLC 的活性，接著進一步抑制 phosphoinositide breakdown、鈣離子的流動、以及47 kDa 蛋白質的磷酸化(二)馬兜鈴酸可經由抑制 p38 MAPK 磷酸化來調控 phospholipase A2 的活性而使 TXA2 的含量減少而抑制血小板的活化。 (三)馬兜鈴酸會影響 eNOS 的活性，增加血小板細胞內 NO 的產生，可能進一步影響guanylate cyclase 的活性，增加 cyclic GMP 的含量

以誘發VASP 磷酸化而抑制血小板的活化

英文摘要

Aristolochic acid (AsA) is an alkaloid from the plant Aristolochiaceae. The naturally occurring herbal toxin that can cause cancer and end-stage kidney failure. Recently, it had been reported that AsA could reduce edema induced by snake venom and possess anti-inflammation and anti-platelet activity of AsA. However, the mechanisms

involved in anti-platelet activity of AsA is still unclear, and we are interested in investigating the effects on cellular signal transduction during the process of platelet activation. In this study, AsA concentration-dependently (75-150 micromolar) inhibited collagen (1 microgram/ml) induced human platelet aggregation and ATP

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release reaction. AsA (115 and 150 micromolar) inhibited intracellular Ca2+

mobilization, phosphoinositide breakdown, and thromboxane A2 formation

stimulated by collagen (1 microgram/ml) in human platelets. In addition, AsA (115 and 150 micromolar) increased levels of nitrate and induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP). Phosphorylation of 47 kDa proteins is a marker of protein kinase C activation, and can be triggered by collagen (1

microgram/ml) and PDBu (150 nano molar). In our experiments, we found AsA (115 and 150 micromolar) could inhibit phosphorylation of 47 kDa proteins by collagen (1 microgram/ml). Besides, AsA (115 and 150 micromolar) reduced p38 MAPK phosphorylation induced by collagen (10 microgram/ml), and had no effects on scavenging collagen (1 micro gram/ml)-induced hydroxyl radicals in platelets. In conclusion, our study suggested that the pathways of AsA (115 and 150

micromolar) about anti-platelet activity maybe involved the following: (1) AsA could regulate the activity of PLC and then inhibit phosphoinositide breakdown, intracellular Ca2+ mobilization and 47 kDa protein phosphorylation. (2) AsA significantly reduced thromboxane A2 formation maybe through inhibition of p38 MAPK phosphorylation and Ca2+ mobilization, which are responsibe for PLA2 activation. (3) AsA inhibted the activity of platelet by increasing the amount of NO and VASP phosphorylation .