Honokiol 抑制血小板凝集作用之機轉探討

Mechanisms involved in the antiplatelet activity of honokiol

李姿穎(Tzu-Yin Lee)

指導教授 ： 周敦穗

共同指導教授 ： 許準榕(Joen-Rong Sheu)

臺北醫學大學/醫學院/醫學科學研究所/碩士(2014年)

摘要

Honokiol，木蘭科落葉喬木植物厚朴之樹皮所萃取，並運用在傳統中醫藥材，其主要功效有減緩發燒、頭痛、抗焦慮及止瀉等。近期研究指出honokiol具有抗腫瘤、抗發炎、抗氧化、抗菌、抗血管新生和神經保護活性。然而，在人類血小板中，honokiol是否參與的抗血小板活性機制仍不明。
在本篇研究中，我們證實honokiol (0.6-1 μM)會抑制由collagen (1 μg/ml)所誘導的血小板凝集反應和血小板內部鈣離子及ATP的釋放，而其他血小板活化劑，如arachidonic acid (AA, 60 μM)、thromboxane B2 analogue (U46619, 1 μM)及thrombin (0.05 U/ml)則不會影響血小板凝集。另外，使用??2??1刺激劑(aggretin, 1 μg/ml)和醣蛋白(GP) VI刺激劑(convulxin, 5 ng/ml)，發現honokiol具有專一性的抑制由醣蛋白(GP) VI參與的血小板凝集。並且，honokiol能減少由醣蛋白(GP) VI刺激劑(convulxin, 5 ng/ml)所誘導的Lyn、PLCγ2、PKC、p38 MAPK、ERK、JNK和Akt蛋白的磷酸化。除此之外，透過SPR、流式細胞儀和免疫沉澱法的實驗證實，honokiol會結合到GP VI上，進而影響GP VI訊息傳遞路徑下游中的Lyn的活性。另一方面，我們也利用血小板功能分析儀測試honokiol在全血中是否影響血小板凝集功能，發現honokiol (10 μM)有延長血栓形成時間的現象發生。
總結以上實驗結果，honokiol藉由干擾GP VI與Lyn之間的連結，進而抑制GP VI活化路徑中Lyn的活性，並且抑制GP VI訊息傳遞下游之路徑，包括：PLCγ2、PKC、p38 MAPK、ERK、JNK、Akt蛋白的活性。因此honokiol可藉由影響血小板GPVI訊息傳遞路徑，最終抑制血小板的活性及凝集作用。因此，honokiol可專一性的與血小板上的GPVI進行交互作用，可作為GPVI的抑制劑，並且能有助於抗血栓之形成，可望用於預防或治療血栓性疾病。

關鍵字

血小板 ； 厚朴 ； 醣蛋白(GP)VI ； convulxin ； collagen

並列摘要

Honokiol is the main biphenyl neolignan isolated from Magnolia officinalis Rehd. et Wils. Extract. The cortex of Magnolia officinalis has been used as a traditional Chinese medicine to treat fever, headache, anxiety, and diarrhea. Recent studies demonstrated that honokiol possesses anti-tumor, anti-inflammatory, anti-oxidant, anti-microbial, anti-angiogenic, and neuroprotective activities. However, the mechanisms involved in anti-platelet activity of honokiol remain unclear.
In the present study, our results revealed that honokiol (0.6-1 μM) inhibited platelet aggregation induced by collagen (1 μg/ml), but not arachidonic acid (AA, 60 μM), thromboxane B2 analogue (U46619, 1 μM), and thrombin (0.05 U/ml). We also found that honokiol inhibited collagen-induced Ca2+ and ATP release. These findings suggest that honokiol selectively inhibits collagen-induced platelet activation. Thus, convulxin (5 ng/ml), an agonist of glycoprotein (GP) VI, and aggretin (1 μg/ml), an agonist of integrin α2β1, were used to determine the effect of honokiol. The data showed that honokiol only inhibited convulxin-mediated platelet aggregation, indicating that honokiol may inhibit platelet activation mainly through the block of GP VI, but not integrin α2β1. Honokiol significantly inhibited the convulxin (5 ng/ml)-mediated activation of Lyn, phospholipase Cγ2, protein kinase C, p38 mitogen-activated protein kinases, ERK, JNK, and Akt. In addition, we found that honokiol may bind to GP VI detected by SPR, flow cytometry, and immunoprecipitation. Furthermore, we used platelet function analyzer as an ex vivo study to investigate the effects of honokiol on platelet function on human whole-blood sample. The results indicate that honokiol (10 μM) could prolong the platelet plug formation time of CEPI. Based on the above findings, we suggest that honokiol disturbs the association between GP VI and Lyn by competing with GP VI binding site, following by the inhibition of downstream of GPVI, including Lyn, PLCγ2, PKC, p38 MAPK, ERK, JNK, Akt. Finally, honokiol inhibits platelet activation and aggregation.
Honokiol could interact with GP VI receptor specifically, and could be used as an inhibitor of GP VI. It might have a great clinical potential as an anti-thrombotic drug. Thus, honokiol treatment may represent a novel approach to prevent or improving function in thromboembolism related disorders.

並列關鍵字

platelet ； honokiol ； glycoprotein VI ； convulxin ； collagen

參考文獻

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