穿心蓮內酯抑制人類血小板活化與凝集之分子機轉探討

Molecular mechanisms of andrographolide-inhibited human platelet activation and aggregation

呂婉榕(Wan-Jung Lu)

指導教授 ： 蕭哲志

共同指導教授 ： 許準榕

臺北醫學大學/醫學院/醫學科學研究所/博士(2011年)

https://doi.org/10.6831/TMU.2011.00132

摘要

Andrographolide是由Andrographis paniculata (Burm. f.) Nees (Acanthaceae)所分離出來，一種新型NF-κB抑制劑。另外，血小板活化廣泛的發生在血栓疾病中。然而andrographolide在血小板上的藥理學功效尚未明確的被探討，因此我們有意探討andrographolide抑制血小板活化過程中所扮演的角色，以及其對於訊息傳遞方面所參與的抑制機轉。研究結果顯示，andrographolide隨著濃度的增加(25-75 μM)，能有效地抑制collagen (1 μg/ml)所引起的人類血小板凝集反應以及ATP釋放反應；且在人類血小板中，andrographolide (35與75 μM)可以抑制由collagen (1 μg/ml)所刺激的細胞內鈣離子流動、thromboxane A2的形成和PLCγ2、PKC、MAPK以及Akt蛋白的磷酸化。此外，andrographolide (35與75 μM)同時刺激eNOS活化、NO和cyclic GMP增加以及VASP蛋白磷酸化，並且andrographlide (35與75 μM)並無參與cyclic AMP路徑。另外，使用guanylate cyclase抑制劑可以回復andrographolide抑制由collagen (1 μg/ml)所刺激的血小板凝集反應、p38 MAPK以及Akt蛋白的磷酸化並且抑制andrographolide所刺激的VASP蛋白磷酸化。此外，分別使用PI3 kinase、PKC及p38 MAPK抑制劑，分析出PI3-kinase/Akt-p38 MAPK上下游關係。 Andrographolide 同時也抑制自由基(OH●)的生成。在 In vivo實驗中證實，andrographolide (22 和 55 μg/kg) 可延長腸繫膜血栓快形成和增加ADP引起的急性肺栓塞之小鼠存活率。
雖然血小板為無核細胞，但仍有轉錄因子在血小板活化中扮演著non-genomic的功能，如、NF-κB。所以，我們有意探討andrographolide在血小板對其所扮演的角色。研究結果顯示，collagen (1 μg/ml)可活化血小板中的IKKβ、p65、及IκBα，並有時間上的依賴；並且，andrographolide (35與75 μM)有意義抑制其活化。此外，guanylate cyclase抑制劑和cyclic GMP-dependent kinase抑制劑皆可以回復andrographolide抑制由collagen (1 μg/ml)所刺激的血小板凝集反應、鈣離子生成、IKK?狴H及p65蛋白的磷酸化；並且利用p38 MAPK和ERK抑制劑，證實p38 MAPK在NF-κB上遊。然而，我們有意再進一步探討自由基對此路徑所扮演的角色，對此分別使用p38 MAPK、自由基、NF-κB和ERK抑制劑，證實NF-κB和ERK抑制劑不會影響自由基的生成。另外，cyclic GMP-dependent kinase抑制劑可以回復andrographolide抑制由collagen (1 μg/ml)所刺激的p38 MAPK和ERK2蛋白的磷酸化和自由基的生成。
由上述結果證實，andrographolide (35與75 μM)抑制血小板活性的作用可能涉及下列路徑：1.活化eNOS-NO-cyclic GMP pathway及調節PI3-kinase/Akt-p38 MAPK和PLCγ2-PKC cascades；2. 活化cyclic GMP-PKG pathway；及調節p38 MAPK-OH●-NF-κB-ERK2 cascade，而進一步抑制血小板凝集作用。
未來的研究我們將再進一步去定義，andrographolide在心血管疾病中或是治療cancer及發炎疾病等，可以當作治療藥物 ，並尋求解決之道。

關鍵字

穿心蓮內酯 ； eNOS ； cyclic GMP ； PLCγ2 ； p38 MAPK ； NF-κB ； hydroxyl radical ； 血小板凝集

並列摘要

Andrographolide is a novel NF-κB inhibitor from the leaves of Andrographis paniculata. Platelet activation is relevant to a variety of thrombotic diseases. However, no data are available concerning the effects of andrographolide in platelet activation. The aim of this study was to examine the mechanisms of andrographolide in preventing platelet activation. Andrographolide (25-75 μΜ) exhibited more potent activity of inhibiting platelet aggregation stimulated by collagen. Andrographolide inhibited collagen-stimulated platelet activation accompanied by relative Ca2+ mobilization, thromboxane A2 (TxA2) formation, and phospholipase C (PLC) γ2, protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and Akt phosphorylation. Andrographolide markedly increased cyclic GMP, but not cyclic AMP levels. Andrographolide also stimulated endothelial nitric oxide synthase (eNOS) expression, NO release, and vasodilator-stimulated phosphoprotein (VASP) phosphorylation. ODQ, an inhibitor of guanylate cyclase, markedly reversed the andrographolide-mediated inhibitory effects on platelet aggregation, p38 MAPK and Akt phosphorylation, and the andrographolide-mediated stimulatory effect on VASP phosphorylation. Furthermore, a PI3 kinase inhibitor (LY294002) but not a PKC inhibitor (Ro318220) significantly diminished p38 MAPK phosphorylation; nevertheless a p38 MAPK inhibitor (SB203580) and LY294002 diminished PKC activity stimulated by collagen. Andrographolide also reduced collagen-triggered hydroxyl radical (OH●) formation. In vivo studies revealed that andrographolide (22 and 55 μg/kg) is effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism, and significantly prolonged platelet plug formation in 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 andrographolide in NF-κB-mediated events in platelets. In this study, NF-κB signaling events, including IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation, were time-dependently activated by collagen in human platelets, and these signaling events were attenuated by andrographolide (35 and 75 μM). ODQ and KT5823, respective inhibitors of guanylate cyclase and cyclic GMP-dependent kinase (PKG), strongly reversed andrographolide-mediated inhibition of platelet aggregation, relative [Ca2+]i mobilization, and IKKβ, and p65 phosphorylation. In addition, SB203580 (an inhibitor of p38 MAPK), but not PD98059 (an inhibitor of ERKs), markedly abolished IKKβ and p65 phosphorylation. SB203580, NAC (a free-radical scavenger), and BAY11-7082 (an inhibitor of NF-κB) all diminished ERK2 phosphorylation, whereas PD98059, BAY11-7082, and NAC had no effects on p38 MAPK phosphorylation. Furthermore, SB203580, but not BAY11-7082 or PD98059, reduced collagen-induced hydroxyl radical (HO●) formation. KT5823 also markedly reversed andrographolide-mediated inhibition of p38 MAPK and ERK2 phosphorylation, and hydroxyl radical formation in platelets.
In conclusion, we are the first to demonstrate that (1) andrographolide possesses a novel role of antiplatelet activity, which may involve activation of the eNOS-NO-cyclic GMP pathway, resulting in inhibition of the PI3 kinase-Akt-p38 MAPK and PLCγ2-PKC cascades, thereby leading to inhibition of platelet aggregation; and that (2) andrographolide may involve an increase in cyclic GMP/PKG, followed by inhibition of the p38 MAPK/OH●-NF-κB-ERK2 cascade in activated platelets.
Further studies are needed to define the specific role of andrographolide may have a high therapeutic potential to treat such thromboembolic disorders or be also considered for treating cancer and various inflammatory diseases.

並列關鍵字

Andrographolide ； cGMP ； eNOS ； PLCγ2 ； p38 MAPK ； NF-κB ； hydroxyl radical ； platelet activation

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