| 摘要: | 本篇論文的主要目的為研究咖啡醇(Cafestol)在血管內皮細胞和心臟心肌細胞的作用，以及其相關的分子機制。根據大型的研究調查，適量的飲用咖啡的確會帶給身體不少健康的益處，其中包含了降低心血管疾病的發生及併發症；然而，目前對其原因及機轉並不甚清楚。也因此，咖啡中有許多具有生物活性的成份便引起了許多的注意。咖啡醇，是一種雙萜(diterpene)存在於咖啡中，就有許多的生物活性，其中之一為抗發炎反應的能力。但是，在心血管疾病中，咖啡醇到底是扮演著什麼角色目前也仍然未明。因此，我們首先研究咖啡醇在內皮細胞，對週期性展延引起發炎分子生成的作用及其作用機轉。此外，心臟心肌肥厚也是一個心血管疾病的重要因子，因此，我們也研究了是否咖啡醇和心肌細胞肥大的現象有所關連及其背後可能的生理分子機轉。我們將內皮細胞培養在衡定的條件下或是接受週期性展延的環境下，利用酵素免疫吸附法測量所釋放出來的發炎反應因子。咖啡醇對促分裂原活化蛋白激酶、血鐵質氧化酶-1、sirtuin 1 (Sirt1)訊息傳導路徑的影響，則是藉由西方方墨點法及相關的抑制劑來試驗。咖啡醇處置後會顯著地抑制週期性展延處置下IL-8、ICAM-1、和MCP-1的分泌。咖啡醇是經由阻止活性含氧物(reactive oxygen species, ROS)生成而抑制了週期性展延誘發IL-8、ICAM-1、和MCP-1的產生。在人類臍靜脈內皮細胞，週期性展延會誘發ERK、JNK、和p38的磷酸化；用咖啡醇處置則會顯著地預防週期性展延所誘發的ERK和p38磷酸化。咖啡醇也會增強血鐵質氧化酶-1 (heme oxygenase-1, HO-1)蛋白及Sirt1蛋白含量的表現；使用zinc protoporphyrin IX (ZnPP, 一種血鐵質氧化酶-1強效競爭型抑制劑)、Sirt1抑制劑Sirtinol、和短干擾核糖核酸(siRNA)轉染技術則會將咖啡醇抑制週期性展延誘發IL-8、ICAM-1、和MCP-1釋放的作用消除掉。大鼠的新生幼鼠心臟心肌細胞單純接受尾加壓素Ⅱ(Urotension II)(1 nM)處置或是先以咖啡醇(1-10 μM)處置後再接受尾加壓素Ⅱ(1 nM)處理，咖啡醇(3-10 μM)的處置可以防止尾加壓素Ⅱ刺激心臟心肌細胞肥大且抑制尾加壓素Ⅱ刺激的氧化還原訊號傳遞。尾加壓素Ⅱ的處置會顯著地增加心臟心肌細胞表皮生長因子受體(EGFR)和細胞外信號調節激酶(ERK)的磷酸化，而預先以咖啡醇前處理則會明顯地抑制尾加壓素Ⅱ誘使表皮生長因子受體和細胞外信號調節激酶磷酸化的形成。咖啡醇同時也會消除尾加壓素Ⅱ藉由活性含氧物對SHP-2去活性的作用，咖啡醇亦會提昇核E2相關因子-2(nuclear factor erythroid-2-related factor 2, Nrf2)的轉位及血鐵質氧化酶-1蛋白的表現。利用鴉膽子苦素(brusatol, a specific inhibitor of Nrf2)或是以核E2相關因子-2短干擾核糖核酸(Nrf2 siRNA)轉染，則會顯著地減緩咖啡醇抑制尾加壓素Ⅱ刺激活性含氧物的生成及心肌細胞肥大的作用。這些發現是目前第一個證明咖啡醇會抑制週期性展延處置下發炎反應因子的分泌，且其機轉可能是藉由活化內皮細胞的血鐵質氧化酶-1蛋白及Sirt1蛋白；而咖啡醇抑制尾加壓素Ⅱ刺激心肌細胞肥大的作用則是藉由核E2相關因子-2及血鐵質氧化酶-1的活化及氧化還原訊息傳導路徑的抑制，進而達到心臟的保護作用。這些結果提供了非常具有價值性的觀點，使得我們對咖啡醇作用的分子生物機轉有了更進一步的瞭解，而這樣嶄新的發現也意味著咖啡醇可能是一個新的治療方向且可以藉此研發出新的藥物來治療心臟及心血管相關的疾病。
The aim of this thesis is to investigate the effect of cafestol on endothelial cells and cardiomyocytes, and the underlying molecular mechanisms. According to population-based studies, moderate coffee consumption is associated with numerous health benefits, including reducing cardiovascular disease; however the mechanisms underlying this causal effect remains obscure. Therefore, active ingredients of coffee have gained abundantly attention. Cafestol, a diterpene found in coffee, has various properties, including an anti-inflammatory property. The role of cafestol in cardiovascular disease is still unclear. Thus, we first investigated the effect of cafestol on cyclic-strain-induced inflammatory molecule secretion and the mechanisms in vascular endothelial cells. Besides, cardiac hypertrophy is an important risk factor of cardiovascular events, we also examined whether cafestol inhibits urotensin II (UII) –induced cardiomyocyte hypertrophy. Endothelial cells were cultured under static or cyclic strain conditions, and the secretion of inflammatory molecules was determined using enzyme-linked immunosorbent assay. The effects of cafestol on mitogen-activated protein kinase (MAPK), heme oxygenase-1 (HO-1), and sirtuin 1 (Sirt1) signaling pathways were examined using Western blotting and specific inhibitors. Cafestol attenuated cyclic-strain-stimulated intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein- (MCP-) 1, and interleukin- (IL-) 8 secretion. Cafestol inhibited the cyclic-strain-induced phosphorylation of extracellular signal-regulated kinase, p38, and MAPKs. By contrast, cafestol upregulated the expression of cyclic-strain-induced HO-1 and Sirt1. The addition of zinc protoporphyrin IX, sirtinol, or Sirt1 silencing (transfected with Sirt1 siRNA) significantly suppressed cafestol-mediated modulatory effects on cyclic-strain-stimulated ICAM-1, MCP-1, and IL-8 secretion. Neonatal rat cardiomyocytes were treated only with U-II (1nM) or with U-II (1nM) following 12-h pretreatment cafestol (1-10μM). Cafestol (3-10μM) pretreatment significantly inhibited U-II-induced cardiomyocyte hypertrophy with an accompanying decrease in U-II-induced reactive oxygen species (ROS) production. Cafestol also suppressed U-II-stimulated phosphorylation of redox-sensitive extracellular signal-regulated kinase (ERK) and epidermal growth factor receptor (EGFR) transactivation. Moreover, cafestol pretreatment upregulated Src homology region 2 domains-containing phosphatase-2 (SHP-2) activity, suggesting that cafestol prevents ROS-induced SHP-2 inactivation. Furthermore, cafestol also enhanced the translocation of nuclear factor erythroid-2-related factor 2 (Nrf2) and the expression of heme oxygenase-1 (HO-1). Adding brusatol (a specific inhibitor of Nrf2) or Nrf2 siRNA significantly attenuated cafestol-mediated inhibition on U-II-induced ROS production and cardiomyocyte hypertrophy. This is the first study to demonstrate that cafestol suppressed cyclic-strain-stimulated inflammatory molecule secretion, possibly through the activation of HO-1 and Sirt1 in endothelial cells. Cafestol prevented U-II-induced cardiomyocyte hypertrophy through Nrf2/HO-1 activation and inhibition of redox signaling, leading to cardioprotection. The results provide valuable insights into molecular pathways that may contribute to the effects of cafestol and these novel findings indicate that cafestol could be a new strategy and applied in pharmacological therapy for cardiac diseases. |
