| 摘要: | 背景：血管鈣化普遍存在動脈粥樣硬化、慢性腎臟病、糖尿病及高血壓的患者，更與心血管疾病之死亡率及發病率具有高度相關性。血管鈣化是一個活躍的細胞介導過程，關於心血管鈣化調控機制的研究主要集中在成骨細胞分化、基質囊泡、細胞外基質分解和礦化以及細胞凋亡等方面。黃腐醇是啤酒花和啤酒中具有生物活性的異戊烯化類黃酮在傳統醫學中長期用作鎮靜劑和抗微生物劑。最近，在體外和體內的研究指出其具有抗發炎、抗增生、降膽固醇、抑制血小板凝集與降血糖活性等藥理活性，學者已經將注意力集中在對心血管和代謝疾病的保護作用上。然而，黃腐醇在血管鈣化中的作用尚不清楚。在106年度科技部專題研究計畫裡，已由體外試驗證實細胞經β-GP培養14天後的確有礦物化的現象產生，給與不同濃度的黃腐醇與β-GP組相比顯著減少鈣含量和鈣化結節的形成。然而，這些體外研究數據仍需要利用臨床前動物模型進一步驗證。因此，本研究將使用兩種已確立的血管鈣化和動脈粥樣硬化的臨床前動物模型來評估黃腐醇在體內的潛在作用。方法：在本研究中，我們將評估維生素D3加尼古丁誘導的體內大鼠血管鈣化模型來探討黃腐醇是否對體內動脈血管鈣化具有抑制作用。另外，我們還使用ApoE-剔除小鼠來評估黃腐醇對動脈粥樣硬化的血管鈣化影響。實驗中，將測量實驗動物的血液動力學指標、鹼性磷酸?活性、主動脈和心臟組織的鈣沉積以及組織的病理變化及相關蛋白質的表現。結果：在106年度研究計畫的初步數據顯示，黃腐醇濃度相關性地減少β-GP誘導大鼠血管平滑肌細胞的成骨分化和鈣化作用，包括減少鹼性磷酸?活性、鈣沉積和相關蛋白Pit-1、Runx2、β-catenin與BMP-2的表現以及礦化結節的形成。此外，與以β-GP刺激鈣化組相比較，黃腐醇明顯抑制MMP-2/9和caspase 3/9蛋白質的表現。以上的結果顯示黃腐醇透過影響血管平滑肌細胞轉分化和抑制MMPs和caspases的活化來影響血管鈣化的病理生理學。因此，經由細胞和動物實驗，我們將證實黃腐醇可 (1)防止血管鈣化; (2) 透過阻斷BMP-2/ Runx2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) 影響血管平滑肌細胞轉分化和抑制基質金屬蛋白?活性和細胞凋亡。結論：這一新穎的活性為黃腐醇的心血管保護作用提供了新的見解，並有可能是透過減少血管鈣化來降低心血管風險。
Background: Vascular calcification (VC) is prevalent in patients with atherosclerosis, chronic kidney disease, diabetes mellitus, and hypertension, is a strong independent predictor of increased cardiovascular morbidity and mortality. VC is an active, cell-regulated process. Recent studies on the regulatory mechanism of cardiovascular calcification have been focused osteogenic dedifferentiation, matrix vesicles, extracellular matrix degradation & mineralization and cell apoptosis. Xanthohumol, a bioactive prenylated flavonoid in hops (Humulus lupulus L.) and beer, has long been used in traditional medicine as a sedative and antimicrobial agent. More recently, attention has been devoted to the protective effects on the cardiovascular and metabolic diseases. However, the role of xanthohumol in VC is still unclear. Our preliminary data of last-year research project shows that xanthohumol significantly attenuated the β-glycerophosphate (β-GP)-induced osteoblastic differentiation and mineralization of vascular smooth muscle cells (VSMCs). However, these in vitro studies should by further validated in preclinical animal models. Therefore, the aim of this study is to evaluate the potential effects of xanthohumol in two well-established preclinical models of vascular calcification and atherosclerosis, and the molecular mechanisms involved.Methods: A variety of stimulants, such as hypercholesterolemia, vitamin D, and nicotine, alone or in combination have been used to induce arterial calcification in a variety of different animals. In this study, we will evaluate the inhibitive effect of xanthohumol on VC in an in vivo rat VC model induced by vitamin D3 plus nicotine. In addition, we also used apolipoprotein E-knockout mice to identify the effects of xanthohumol on the calcification of atherosclerotic lesions. In experiments, we will measure hemodynamic variables, alkaline phosphate (ALP) activity, calcium deposition, pathological changes and proteins expression in myocardium and aorta of experimental animals. Results: Our preliminary data of last-year research project showed that xanthohumol concentration-dependently reduced β-GP-induced osteoblastic differentiation and calcification of VSMCs including ALP activity, calcium content and sodium-phosphate cotransporter Pit-1, Runx2, β-catenin, and bone morphogenetic protein-2 (BMP-2) expression as well as the formation of mineralized nodule. Furthermore, xanthohumol was shown to inhibit the β-GP-induced protein expressions of MMP-2/9 and caspase 3/9, which are known contributors to vascular calcification. Our study demonstrates the potential of xanthohumol to impact the pathophysiology of vascular calcification by affecting VSMC transdifferentiation and to inhibit MMPs and caspases activation. From in vitro and in vivo studies, we may show that xanthohumol will (1) prevent vascular calcification; (2) suppress osteoblast differentiation markers and calcification phenotype through the blockade of BMP-2/ Runx2 and Wnt/β-catenin signalling pathways; (3) inhibit MMPs and apoptosis in VSMCs.Conclusion: This novel activity provides new insights of cardiovascular protection of xanthohumol and may have the potential to decrease cardiovascular risk by reducing vascular calcification. |
