Cordyceps mycelia的多醣體萃取物CME-1抑制血小板凝集作用之機轉探討

Mechanisms involved in the antiplatelet activity of Cordyceps mycelia

廖君澄(Jiun-Cheng Liao)

指導教授 ： 許準榕

臺北醫學大學/醫學院/醫學科學研究所/碩士(2012年)

https://doi.org/10.6831/TMU.2012.00052

摘要

冬蟲夏草(Cordyceps sinensis)是在亞洲區域非常普遍的的中草藥，用於中醫及健康食品。目前已知的冬蟲夏草種類有400種之多，依不同分離的方法，以及鑑定後的分子量、醣基的組成比例、及結構等，來判斷彼此間的差異；本實驗使用的CME-1係一新穎冬蟲夏草菌絲體多醣體萃取物，首度的被分離與鑑定，其係一主要以半乳糖與甘露糖組成，分子量為27.6 kDa的低分子量的多醣萃取物。目前CME-1已被證實具有對抗過氧化氫誘發之細胞內ROS生成、粒線體膜極化、細胞死亡等功能(Wang et al., 2011)；然而，CME-1在血小板上的功用尚未被明確探討，因此本篇研究主要是想探討CME-1對於血小板活化過程的影響以及其訊息傳遞方面的抑制機轉。由本篇結果顯示，CME-1隨著濃度的增加 (125和210 μg/ml；換算濃度單位約為5和8 μg/ml)，能有效抑制collagen (1 μg/ml)所引發的血小板凝集反應，以及ATP釋放反應；且CME-1 (125和210 μg/ml)可以抑制由collagen所刺激細胞內鈣離子的流動、PLCγ2的磷酸化。血小板內p47 protein磷酸化，這是標記 protein kinase C活性的方法。在本實驗中我們分別使用collagen (1 μg/ml)和PDBu (150 nM)促進血小板 p47 protein磷酸化，發現CME-1只能抑制由collagen所活化的p47 protein；並且CME-1 (125和210 μg/ml)也會明顯抑制由collagen所活化的Akt磷酸化；而對於p38 MAPK、ERK2、JNK1的磷酸化，CME-1 (125和210 μg/ml)也有意義的抑制效果。
由上述結果證實，CME-1抑制血小板活性的作用可能會調節PLCγ2-PKC路徑進一步抑制鈣離子的流動；同時CME-1可減少MAPKs及 Akt的磷酸化，影響血小板凝集反應。
此研究結果意味著 CME-1 除了擔任抗氧化的功能之外，對於血小板凝集的作用可能也有抑制的作用，因此未來可能可以進一步應用在臨床上，以治療心血管方面之相關疾病。

關鍵字

冬蟲夏草 ； 冬蟲夏草菌絲體 ； 血小板 ； 多醣體 ； 凝集反應

並列摘要

Cordyceps sinensis (CS) is the Chinese herbs for Chinese medicine and health foods that is very common in the Asian. Known Cordyceps species have as many as 400 kinds, according to the different separation methods, and identified the molecular weight of the composition ratio of glycosylation, and structure, to determine the differences between each other; CME-1, a novel polysaccharides of Cordyceps sinensis mycelium ectract was first isolated and identified which is mainly compose of galactose and mannose with molecular weight of 27.6 kDa. Recent studies have shown that CME-1 protect cells against H2O2-induced cell death；Prevents H2O2-induce intracellular oxidative stress and mitochondrial membrane depolarization. However, the mechanisms involved in anti-platelet activity of CME-1 are unclear, and we are interested in investigating the effects on cellular signal transduction during the process of platelet activation. In this study, CME-1 concentration-dependently (125 and 210 μg/ml；is equal to 5 and 8 μg/ml)inhibited collagen (1 μg/ml)-induced human platelet aggregation and ATP release. CME-1 (125 and 210 ?慊/ml) inhibited intracellular Ca2+ mobilization, PLCγ2 activity stimulated by collagen (1 μg/ml)-induced human platelet aggregation and ATP release. Phosphorylation of p47 proteins is a marker of protein kinase C activation, and can be triggered by collagen (1 μg/ml) and PDBu (150 nM). In our experiments, we found that CME-1 (125 and 210 ?慊/ml)significantly inhibited p47 proteins phosphorylation stimulated by collagen (1 μg/ml) but not PDBu (150 nM). In addition, CME-1 (125 and 210 ?慊/ml) reduces p38 MAPK phosphorylation stimulated by collagen (1 μg/ml) in human platelets. In conclusion, our study suggested that the mechanisms of CME- (125 and 210 μg/ml) in anti-platelet activity maybe regulated the activity of PLCγ2-PKC-Ca2+ pathway, and CME-1 also reduces MAPKs and Akt phosphorylation.
Therefore, CME-1 not only has inhibitory action on, but also anti-platelet activity. CME-1 may be used as an effective tool in treating pathological disorder associated with platelet hyperaggregability clinically.

並列關鍵字

Cordyceps sinensis(CS) ； Cordyceps mycelia(CM) ； platelet ； polysaccharide ； aggregation

參考文獻

Adam, F, Kauskot, A, Rosa, JP, Bryckaert, M Mitogen-activated protein kinases in hemostasis and thrombosis. J Thromb Haemost, 2008, 6(12): 2007-2016.

Berridge, MJ Rapid accumulation of inositol trisphosphate reveals that agonists hydrolyse polyphosphoinositides instead of phosphatidylinositol. Biochem J , 1983, 212(3): 849-858.

Berridge, MJ, Bootman, MD, Roderick, HL Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol , 2003, 4(7): 517-529.

Borsch-Haubold, AG, Kramer, RM, Watson, SP Phosphorylation and activation of cytosolic phospholipase A2 by 38-kDa mitogen-activated protein kinase in collagen-stimulated human platelets. Eur J Biochem, 1997, 245(3): 751-759.

Canobbio, I, Reineri, S, Sinigaglia, F, Balduini, C, Torti, M A role for p38 MAP kinase in platelet activation by von Willebrand factor. Thromb Haemost , 2004, 91(1): 102-110.