| 摘要: | 背景：鈣離子通道阻斷劑是治療心血管疾病的重要藥物。先前已有研究報告指出多種鈣離子通道阻斷劑，如 amlodipine，能減少心臟病事件的發生率並且抑制頸動脈粥狀動脈硬化的病程進展，藥物主要的血管保護作用有一部分是被歸於抑制氧化壓力與抗發炎的作用。Labedipinedilol-A 與lercanidipine是新一代長效型二氫吡啶類鈣離子通道阻斷劑，於先前的研究已證明二者對於培養的老鼠血管平滑肌細胞有抑制細胞增生、抗氧化與抗血管粥狀化的作用，但其抗發炎功能尚未被確定。因此，本研究主題目的為釐清labedipinedilol-A於預防及治療小鼠內毒素血症所引起的血管發炎作用之效益並與lercanidipine做比較。 方法: 在第一年度的研究計畫，我們將使用以LPS誘發內毒素血症的動物模式與LPS/IFN-刺激血管平滑肌細胞的離體模式 (IFN-γ在發炎反應過程中也扮演一個重要角色，因為它可以加強LPS 誘發iNOS的基因表現，所以同時使用LPS/IFN-γ刺激代表血管發炎的狀態) 評估 labedipinedilol-A 與lercanidipine之抗發炎活性。為了瞭解labedipinedilol-A 與 lercanidipine 對內毒素血症老鼠包括細胞激素的釋放、免疫組織染色與存活率的影響，動物實驗分成預處理藥物組與藥物治療組，每日觀察小鼠的存活率，連續觀察2週。另外，評估2種藥物對投予LPS 24小時後小鼠血液中cytokines、活性氧屬與HMGB1釋放的影響及組織切片的變化，在注射LPS 24小時後，麻醉動物後採取血液與組織樣本，將肺臟 (均質液分析MPO activity) 與胸主動脈取下，部分組織做組織切片，剩下的組織則進行蛋白質表現分析。第二年度的研究計畫，為了瞭解 labedipinedilol-A 與 lercanidipine 抗發炎的分子機轉，我們將測量2種藥物對MAP kinases、PI3K/Akt、AMPK、MMPs 和已知在培養細胞與動脈粥樣化的斑塊中可以被大量活化並在粥狀動脈硬化損害上扮演重要角色之轉錄因子NF-B之作用。最後，我們將探討2種藥物對LPS引起的內皮細胞功能障礙的保護作用機轉，內皮細胞表面黏著分子的表現在發炎反應的致病機轉中也扮演一個關鍵角色，我們擬利用人類臍靜脈內皮細胞來評估2種藥物對以 LPS 刺激後內皮細胞表面黏著因子 (E-selectin, VCAM-1 and ICAM-1) 的表現，以及THP-1細胞與內皮細胞之間的黏著作用來進行分析，為了更進一步瞭解2種藥物與 LPS 誘導出來的內皮細胞表面黏著因子之間的作用機轉，我們將進一步偵測內皮細胞中 NF-κB 轉位到細胞核的情況及 NF-B 與 DNA 結合的能力。 結果：從體內及體外試驗的結果，我們想要呈現labedipinedilol-A與 lercanidipine這2個第三代二氫吡啶類鈣離子通道阻斷劑 (1) 將顯著改善致死內毒素血症小鼠的存活率並降低血清中細胞激素、自由基、HMGB1的含量、胸主動脈檢體中MMPs的活性與表現與肺臟中MPO的活性；(2) 減少LPS/IFN-刺激血管平滑肌細胞中一氧化氮、前發炎性細胞激素與HMGB1的釋放；(3) 抑制LPS誘發的MAP kinases與NF-B的活化，另外，透過活化AMPK來抑制LPS刺激細胞所產生的發炎反應；(4) 減弱LPS所誘發的黏著因子表現與降低單核細胞黏附至內皮細胞的作用。因此這些實驗支持設定的假說，labedipinedilol-A可當作血管功能異常相關的慢性發炎性疾病如粥狀動脈硬化的新治療化合物。 結論：在本研究中我們期望能證實新的二氫吡啶類鈣離子通道阻斷劑labedipinedilol-A於LPS誘發發炎的動物與細胞模式中具有減少氧化壓力、抗發炎的作用與減少黏著因子生成的效果，其作用機制是透過減弱LPS誘發之MAP kinase和NF-B的活性，從這個研究當中我們希望找到新證據顯示labedipinedilol-A具有抗氧化與抗發炎的特性能應用在血管發炎性疾病如粥狀動脈硬化的預防與治療上，以及lercanidipine抑制粥狀動脈硬化形成預防心血管疾病發生的可能作用機轉。
Background: Calcium channel blockers (CCBs) are important drugs in the treatment of cardiovascular diseases. Previous studies have shown that several CCBs, such as amlodipine could reduce the rate of cardiac events and inhibited the progression of carotid artery atherosclerosis. Such vasoprotective effects of CCBs are mediated, at least in part, by the inhibitory effect on oxidative stress and inflammatory response. Labedipinedilol-A and lercanidipine are new dihydropyridine CCBs with long duration. We have previously demonstrated that labedipinedilol-A and lercanidipine have the anti-proliferative and the anti-atherogenic effects in vascular smooth muscle cells (VSMCs) through their anti-oxidative effects; however, the anti-inflammatory effects of labedipinedilol-A and lercanidipine are still not determined. Therefore, the objectives of the present study are thus to clarify the efficacy of labedipinedilol-A in preventing vascular inflammation, not only when administrated before, but also after endotoxaemia, to compare its efficacy with lercanidipine. Methods: In the first year study, we will investigate the anti-inflammatory activities of labedipinedilol-A and lercanidipine on the LPS-treated animals in vivo and LPS/interferon-IFN-treated VSMCs in vitro. To elucidate the effects of labedipinedilol-A and lercanidipine on survival rate in endotoxaemic mice, mice were both pretreated and post-treated with labedipinedilol-A or lercanidipine. Survival was monitored daily for up to 2 weeks. In addition, to evaluate the cytokines, HMGB1 and ROS, the blood was taken 24 h after LPS treatment. Subsequently, hearts, lungs and thoracic aorta were excised and parts of tissue samples were fixed in buffered formalin, and then embedded in paraffin for immunohistochemical studies. The rest tissue samples were stored for determination the proteins expression by Western blot assay. In the second year study, to elucidate the action mechanisms of labedipinedilol-A and lercanidipine on the activation of mitogen-activated protein (MAP) kinases, PI3K/Akt, AMP-activated protein kinase (AMPK), matrix metalloproteinases (MMPs) and transcription factors, such as NF-B, known to play a major role in atherogenesis, and its activation has been reported in atherosclerotic plaques and in cultured cells. Finally, we will investigate the vasoprotective mechanisms of labedipinedilol-A and lercanidipine on LPS-stimulated expression of adhesion molecules and THP-1 cells adhesion to human umbilical vein endothelial cells (HUVECs). Results: From in vivo and in vitro studies, we may show that the third generation dihydropyridine CCBs labedipinedilol-A and lercanidipine will (1) significantly improved survival of animals during lethal endotoxaemia, decreased serum levels of cytokines, free radicals, HMGB1, MMPs expression in thoracic aortas and myeloperoxidase activity in lungs; (2) attenuate the production of NO, pro-inflammatory cytokines and HMGB1 release in LPS/interferon-IFN-treated VSMCs; (3) suppress the LPS-induced MAP kinases, NF-B activation, and activated AMPK in VSMCs; (4) inhibit LPS-induced vascular cell adhesion molecule-1 (VCAM-1) expression, and reduced monocyte adhesion in HUVECs. Therefore, these studies support the hypothesis that labedipinedilol-A could be used as a new therapeutic compound against vascular diseases such as atherosclerosis. Conclusion: In this research, we expect to demonstrate that labedipinedilol-A and lercanidipine could attenuate the oxidative stress, inflammation and the production of adhesive molecules induced by LPS both in vivo and in vitro by inhibiting MAP kinases and NF-B activation. From these studies, we hope to provide new evidence of the anti-oxidative and anti-inflammatory properties of labedipinedilol-A that may have a potential therapeutic use for the prevention and treatment of vascular diseases such as atherosclerosis and suggest a possible mechanism by which lercanidipine inhibits the onset of cardiovascular events in patients with atherosclerosis. |
