| 摘要: | 背景：血管鈣化與心血管疾病之死亡率及發病率具有高度相關性，此一現象意指血管平滑肌細胞轉化為成骨細胞。血管鈣化普遍存在冠狀動脈疾病、慢性腎臟病、糖尿病及高血壓患者。血管鈣化為鈣離子沉積在動脈中，造成動脈硬化。近年來與血管鈣化相關的調節機制，主要都針對骨形成之相關基因。自體吞噬作用為一雙層膜結構形成之自噬小體到溶?體進行降解之過程。最近越來越多的證據顯示在動脈硬化斑中細胞會啟動自體吞噬的存活機制，透過消除有害的氧化修飾性蛋白和損壞的部份，以維護斑塊細胞對抗細胞壓力，尤其是氧化傷害、代謝壓力和發炎反應。因此，自體吞噬具有對抗細胞凋亡的作用，有助細胞從惡劣的環境中修復。目前越來越多的研究證實可透過特定藥物促進細胞的自體吞噬作用。山竹黃酮(α-mangostin) 為山竹果皮的主要成分，研究指出山竹黃酮具有抗氧化、抗腫瘤、抗發炎、抗過敏、抗菌、抗真菌及抗病毒之藥理活性；近期更有研究發現山竹黃酮能改善心臟再灌流之損傷與降低氧化壓力，但是山竹黃酮對於血管鈣化之相關功效仍不明確。因此本實驗目的為確認山竹黃酮是否抑制β-glycerophosphate在血管平滑肌細胞，以及高脂飲食造成之高膽固醇血症大鼠之血管鈣化並探討其作用機轉。 方法：第一?的研究中，我們將探討山竹黃酮在體外對β-glycerophosphate引起之血管鈣化的影響。關於山竹黃酮抑制血管鈣化的機制將進一步延伸到對細胞的自體吞噬作用和細胞凋亡。鈣離子沉積、Von Kossa染色、Alizarin red染色、反轉錄聚合?鏈式反應和Annexin V染色，以上方法用於定量細胞鈣化與凋亡。細胞的自體吞噬作用則利用穿透式電子顯微鏡、免疫螢光染色及西方墨點法偵測分析LC3、Beclin-1及Atgs相關蛋白質的表現。在第二年的研究中，我們將分析山竹黃酮在粥狀動脈硬化大鼠的血管保護作用及其機制。我們將測量大鼠血流動力學指標、鹼性磷酸?活性、主動脈組織的鈣沉積、粥狀動脈硬化病變的大小與病理變化及主動脈組織的蛋白質表現。 結果：根據預試驗的實驗結果，發現山竹黃酮作用於鈣化血管平滑肌細胞可以減少細胞鈣化的現象並增加自噬體的形成和內源性LC3-II和Atg7的蛋白表現。因此，經由細胞和動物實驗，我們將證實山竹黃酮可(1)防止血管鈣化與誘導細胞自體吞噬作用; (2)透過阻斷BMP-2/Smad1/5/8 和Wnt/β-catenin訊息傳遞路徑抑制成骨細胞分化的標記和鈣化表現型; (3)經由改善血管內皮功能與抑制發炎反應進而抑制高膽固醇大鼠的粥狀動脈硬化病變的進展。因此，經由上述之研究將支持我們的假設，山竹黃酮在血管疾病上可以作為新的血管保護劑。 結論：本研究將首次指出山竹黃酮為具有潛力的植物化學成分可用來預防或治療鈣化相關血管疾病。
Background: Vascular calcification is highly correlated with cardiovascular morbidity and mortality. It is characterized by phenotype transition from vascular smooth muscles (VSMCs) to osteoblast-like cells. Vascular calcification is prevalent in patients with coronary artery disease and chronic kidney disease, diabetes mellitus, and hypertension. Vascular calcification means the deposition of calcium minerals in the media of arteries, leading to the vascular stiffening. Recent studies on the regulatory mechanism of vascular calcification have been focused on the bone formation-related genes. Autophagy is a cellular process in which double-membrane vesicles deliver autophagosomes to lysosomes for degradation. A growing body of evidence suggests that autophagy in atherosclerotic plaques is a survival mechanism safeguarding plaque cells against cellular distress, in particular oxidative injury, metabolic stress and inflammation, by removing harmful oxidative modified proteins and damaged components. Recent report showed that basal autophagy can be intensified by specific drugs. α-Mangostin is a main constituent of the fruit hull of the mangosteen. Previous studies have shown that α-mangostin has pharmacological activities such as antioxidant, antitumor, anti-inflammatory, antiallergic, antibacterial, antifungal and antiviral effects. Recent studies have demonstrated that α-mangostin also improves cardiac reperfusion and decreases oxidative stress. However, the effects of α-mangostin on vascular calcification are still unclear. In this study, we investigated whether α-mangostin inhibits calcification induced by β-glycerophosphate in VSMCs and hypercholesterolaemia induced by high fat diet in rats, and the underlying mechanisms. Methods: In the first year study, we will investigate the in vitro effects of α-mangostin on β-glycerophosphate-induced vascular calcification. The mechanism underlying α-mangostin-mediated vascular calcification inhibition will further extend to autophagic and apoptotic response. Calcium deposition, Von Kossa, Alizarin red staining, RT-PCR and annexin V staining were measured applying quantification of calcification and apoptosis. Autophagy will be assessed by transmission electron microscope, immunofluorescence staining, and Western blotting of LC3, Beclin-1, and Atgs. In the second year study, we will examine the vasoprotective effects and mechanisms of α-mangostin in atherosclerotic rats. We will measure rat hemodynamic variables, ALP activity, calcium deposition, atherosclerotic lesion size, and pathological changes and proteins expression in aortic tissues. Results: According to the preliminary studies, α-mangostin reduces calcification and enhances autophagosome formation and the expression of endogeneous LC3-II and Atg7 in calcified VSMCs. From in vitro and in vivo studies, we may show that α-mangostin will (1) prevent vascular calcification and induce autophagy; (2) suppress osteoblast differentiation markers and calcification phenotype through the blockade of BMP-2/Smad1/5/8 and Wnt/β-catenin signalling pathways; (3) improve endothelial function, suppress the inflammatory reaction and inhibit the progression of atherosclerotic lesions in high fat diet rats. Conclusion: The present study will show for the first time that α-mangostin can act as a potential therapeutic phytochemical in preventing or treating calcification-associated vascular diseases. |
