| 摘要: | 動脈粥狀硬化是心血管疾病的主因，亦為一種發炎反應，研究發現血小板在動脈粥樣硬化形成中扮演重要角色，不僅作為血栓形成的介質，也能透過血小板分泌的趨化因子(chemokine)使白血球數目增加並活化，與受損內皮細胞相互作用形成斑塊。因此發展有效的抗血栓以及抗發炎藥物是必須的。在本研究中，我們使用抗癌藥物norcantharidin (NCTD)以及四種ruthenium衍生物，[Ru(η6-cymene)(L1)Cl]BF4 (TQ-1), [Ru(η6-cymene)(L2)Cl]BF4 (TQ-2), [Ru(η6-cymene)(L3)Cl]BF4 (TQ-3), 和[Ru(η6-cymene)2-(1H-benzoimidazol-2-yl)-quinoline Cl]BF4 (TQ-6)，當作新抗血小板藥物，同時亦探討TQ-6在脂多醣體（LPS）誘導的巨噬細胞活化和小鼠肝損傷的抗發炎作用之分子機制。在本篇研究證明NCTD, TQ-3以及TQ-6具有抑制人類血小板凝集反應，在thrombin活化的血小板中，NCTD對於血小板內granule釋出影響並不顯著。而血小板凝集作用最後關鍵步驟為integrin αIIbβ3的活化，我們發現NCTD會干擾活化的αIIbβ3與PAC-1之間的連結，並減少貼附在纖維蛋白的血小板數目與單顆血小板展開的面積甚至是抑制血小板凝塊回縮，除此之外也發現NCTD會減少血小板integrin β3, Src, and FAK蛋白磷酸化。從以上這些結果顯示NCTD是透過integrin αIIbβ3所調控的outside-in訊息傳遞路徑進而抑制血小板凝集反應。另一方面，TQ-6抑制由collagen所誘導的血小板活化上ATP與鈣離子釋出反應、P-selectin與PAC-1表現量、自由基的形成、蛋白質(PLCγ2, protein kinase C, mitogen-activated protein kinases以及Akt)以及在不同刺激劑抑制Src與Syk磷酸化表現。顯示TQ-6透過抑制collagen調控的inside-out訊息傳遞如Src-Syk-PLCγ2和granule釋出而干擾integrin αIIbβ3調控的outside-in訊息傳遞並抑制血小板凝集作用。另外，利用血小板功能分析儀 (PFA-100)與動物實驗證明，NCTD與TQ-6皆會延長全血的阻塞時間、腸繫膜血栓形成與尾巴出血時間。在了解TQ-6抑制機轉後，比較其他三種ruthenium衍生物之結構-活性關係，發現TQ-3 (1-5 μM)抑制collagen和thrombin所誘導的血小板活化具有濃度依賴性，然而TQ-1或TQ-2並沒有影響，TQ-3亦明顯抑制collagen所誘導的顆粒釋出反應並且對血小板不具有細胞毒性。此外，TQ-3明顯抑制Lyn-Fyn-Syk, Akt-JNK以及p38 MAPK的磷酸化作用。從TQ-3結構可知其quinolone core上具有phenyl groups提供電子，可透過其疏水性來解釋具有抗血小板作用的原因並提供了抑制collagen引起的血小板凝集反應之分子基礎。另一方面，在抗發炎作用上，從體外實驗(in vitro)結果發現TQ-6抑制LPS所誘發的iNOS表現，其作用機轉可能經由抑制IκBα的降解、p65與p38之磷酸化，與減少TNF-α和IL-1β的表現。同時TQ-6也抑制了LPS誘導的急性肝損傷小鼠模型中iNOS，TNF-α和p65的表現，實驗結果與體外研究一致。本研究顯示，TQ-6可以預防LPS誘導的巨噬細胞體外發炎作用和小鼠體內肝損傷，也顯示NF-κB可能成為預防LPS誘導的發炎和肝損傷的治療目標。總結以上的結果，NCTD和ruthenium衍生物都擁有治療血栓疾病的潛力之外，TQ-6也可能是治療發炎疾病的潛在藥物。
Atherosclerosis, the major cause of CVD, actually involves an ongoing inflammatory response. Platelets are inflammatory anuclear cells with an important role in the development and manifestation of atherosclerosis. Platelet activation induces the release of chemokines, which mediate the recruitment of leukocytes to the vascular endothelium favoring atherogenesis and in later stages atheroprogression and atherothrombosis. Therefore, developing the more effective antithrombotic and anti-inflammatory agents are necessary. In this study, we used norcantharidin (NCTD), a promising anticancer agent in China from medicinal insect blister beetle, and ruthenium (II)-derived compounds, [Ru(η6-cymene)(L1)Cl]BF4 (TQ-1), [Ru(η6-cymene)(L2)Cl]BF4 (TQ-2), [Ru(η6-cymene)(L3)Cl]BF4 (TQ-3), and [Ru(η6-cymene)2-(1H-benzoimidazol-2-yl)-quinoline Cl]BF4 (TQ-6), as a new antiplatelet drug. We also used TQ-6 to investigate the molecular mechanisms underlying the anti-inflammatory effects against lipopolysaccharide (LPS)-induced macrophage activation and liver injury in mice. The evidences demonstrated that NCTD, TQ-3 and TQ-6 possess powerful antiplatelet activity in human platelets. In thrombin-activated platelets, NCTD had no effects on surface P-selectin expression and only slight inhibition on ATP-release reaction. During platelet activation, fibrinogen binds to integrin αIIbβ3, thus completing the final common pathway for platelet aggregation. NCTD markedly hindered integrin αIIbβ3 activation by interfering with the binding of FITC-labelled PAC-1. It also markedly reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen as well as clot retraction. Additionally, NCTD attenuated phosphorylation of proteins such as integrin β3, Src, and FAK in platelets spreading on immobilized fibrinogen. These results indicate that NCTD restricts integrin αIIbβ3-mediated outside-in signaling in human platelets. On the other hand, TQ-6 inhibited [Ca2+]i, granules secretion, FITC-PAC-1 binding, and hydroxyl radical formation, as well as the phosphorylation of phospholipase Cγ2, protein kinase C, mitogen-activated protein kinases, and Akt in collagen-activated platelets. It also exhibited inhibitory activity on Src, and Syk phosphorylation stimulated by agonists in platelets. Of the other three tested compounds, TQ-3 showed a concentration (1–5 μM) dependent inhibitory effect on platelet aggregation induced by collagen (1 μg/mL) and thrombin (0.01 U/mL) in washed human platelets; however, TQ-1 and TQ-2 had no response even at 250 μM of collagen and thrombin-induced aggregation. TQ-3 was effective with inhibiting collagen-induced ATP release, calcium mobilization ([Ca2+]i) and P-selectin expression without cytotoxicity. Moreover, TQ-3 significantly abolished collagen-induced Lyn-Fyn-Syk, Akt-JNK and p38 mitogen-activated protein kinases (p38 MAPKs) phosphorylation. The compound TQ-3 containing an electron donating amino group with two phenyl groups of the quinoline core could be accounted for by its hydrophobicity and this nature might be the reason for the noted antiplatelet effects of TQ-3. The present results provide a molecular basis for the inhibition by TQ-3 in collagen-induced platelet aggregation, through the suppression of multiple machineries of the signaling pathway. Comparatively, TQ-6 has a novel role in inhibiting platelet activation through the inhibition of the agonist receptors-mediated inside-out signaling such as Src-Syk-PLCγ2 cascade and subsequent suppression of granule secretion, leading to disturbing integrin αIIbβ3-mediated outside-in signaling, and ultimately inhibiting platelet aggregation. Besides, NCTD and TQ-6 substantially prolonged the closure time in human whole blood and increased the occlusion time of thrombotic platelet plug formation and prolonged the bleeding time in mice. Furthermore, we used TQ-6 to investigate the molecular mechanisms underlying the anti-inflammatory effects against lipopolysaccharide (LPS)-induced macrophage activation and liver injury in mice. Treating LPS-stimulated RAW 264.7 cells with TQ-6 suppressed nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in a concentration-dependent manner. The LPS-induced expression of tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) were reduced in TQ-6-treated cells. TQ-6 suppressed, LPS-stimulated p38 MAPK phosphorylation, IκBα degradation, and p65 nuclear translocation in cells. Consistent with the in vitro studies, TQ-6 also suppressed the expression of iNOS, TNF-α, and p65 in the mouse model with acute liver injury induced by LPS. In conclusion, NCTD has a dual activity, it can be a chemotherapeutic agent for cancer treatment. In addition, NCTD, TQ-3 and TQ-6 possess powerful antiplatelet activity for preventing or treating thromboembolic disorders. The present study also showed that TQ-6 could protect against LPS-induced in vitro inflammation in macrophage and in vivo liver injury in mice, and suggested that NF-κB could be a promising target for protecting against LPS-induced inflammation and liver injury by TQ-6, suggesting TQ-6 can also be a potential therapeutic agent for treating inflammatory diseases. |
