Ankaflavin 調節高脂飲食倉鼠血漿中高密度脂蛋白膽固醇之可能機制

Abstract

本研究室實驗結果證實紅麴的黃色素 ankaflavin 及 monascin 具有降膽固醇及提升高密度脂蛋白膽固醇 (high density lipoprotein cholesterol, HDL-C) 之功效，且 ankaflavin 效果較 monascin 好，研究指出 monascin 能夠抑制活性氧自由基 (reactive oxygen species, ROS) 生成及降低胰島素抗性是透過過氧化體增生劑活化受體 (peroxisome proliferator-activated receptor gamma, PPAR gamma) 來調控，故推測和 monascin 結構相似的 ankaflavin 是透過 PPAR gamma 來調節高脂飲食倉鼠血漿之 HDL-C。實驗利用 30% 高脂飲食誘導倉鼠，同時餵食 ankaflavin，並利用腹腔注射 GW9662 (PPAR gamma antagonist; PPAR gamma 拮抗劑)，評估 ankaflavin 對於膽固醇及 HDL-C 之調控；在細胞實驗方面，以油酸 (oleic acid, OA) 誘導細胞產生氧化壓力，評估 ankaflavin 抗氧化能力，及對總膽固醇 (total cholesterol, TC) 和 HDL-C 之調控作用。動物實驗結果顯示，ankaflavin 能降低體重、血漿中 TC，提升 HDL-C/TC 比值，以及糞便中膽酸和膽固醇濃度，然而對於 LDL-C 並無明顯降低效果。在 TC 及 HDL-C 調控方面，實驗結果發現 ankaflavin 可能透過 PPAR gamma 的活化進一步提升 liver X receptor (LXR alpha)、cytochrome P450, family 7, subfamily A, polypeptide 1 (CYP7A1) 及 low density lipoprotein receptor (LDLr) 的表現，使膽固醇運送至肝臟中，再藉由 CYP7A1 將膽固醇轉成膽酸藉由糞便排出體外來達到降低血漿膽固醇之作用。此外 ankaflavin 可以促進 LXR alpha 轉錄 ATP-binding cassette subfamily A member 1 (ABCA1)，進而將膽固醇運輸至 apolipoprotein A1 (apo-A1) 增加 HDL-C 濃度；而 ankaflavin 也能夠促進 apo-A1 mRNA 的表現但對於蛋白表現量無顯著提升，實驗也發現油酸合併 ankaflavin 處理對提升 ABCA1 及 apo-A1 mRNA 能力較 ankaflavin 處理為低，顯示在高脂飲食中，ankaflavin 提升 HDL-C 之能力是避免 HDL-C 受氧化分解而達成。此外動物實驗顯示，血漿中巴拉松酶 (paraoxonase, PON) 活性在高脂誘導並餵食 ankaflavin 的組別和正常飲食組相比雖有提升，但在細胞試驗顯示 ankaflavin 對 PON1 並無提升其蛋白表現，顯示 ankaflavin 避免 HDL-C 氧化並非透過 PON1，此外經由試管試驗發現 ankaflavin 能夠抑制經由銅離子誘導造成的 HDL 之氧化，且效果較 monascin 及 quercetin 來的好。細胞實驗也發現 ankaflavin 能藉由 extracellular signal-regulated kinases (ERK) 途徑再活化 NF-E2-related factor 2 (Nrf2)，使 Nrf2 能夠進入核中進一步轉錄出二期抗氧化酵素第一型血紅素氧化酶 (heme oxygenase 1, HO-1) 保護 HDL 避免受到氧化壓力之傷害。綜合以上結果，ankaflavin 具有降低膽固醇及抑制 HDL-C 氧化之功效，具有開發成為降膽固醇及改善肥胖所造成的低濃度 HDL-C 之保健食品或藥物之潛力。

Our previous study has identified that Monascus yellow pigments monascin and ankaflavin could reduce concentration of cholesterol and elevate level of high density lipoprotein cholesterol (HDL-C). In addition, monascin could inhibit reactive oxygen species (ROS) and improve insulin resistance via regulation of NF-E2-related factor 2 (Nrf2) and activation of peroxisome proliferator-activated receptor gamma (PPAR gmma). In this study, the effect of ankaflavin on regulation of HDL-C by activating PPAR gamma was investigated. In the animal experiments, 30% high fat (HF) diet was used in hamsters which were recommended for hyperlipidemia and obesity. Ankaflavin and GW9662 (PPAR gamma antagonist) were introduced by oral gavage to estimate that whether ankaflavin reduces cholesterol and elevates HDL-C levels by PPAR gamma regulation. In cell experiments, we used oleic acid (OA)-treated HepG2 cells to find out the effect of ankaflavin on anti-oxidative ability. We also determined whether ankaflavin can regulate cholesterol and HDL-C level on OA-treated FL83B cells. In vivo results showed that ankaflavin reduced body weight and plasma TC level. Ankaflavin also elevated HDL-C/TC ratio, fecal cholesterol and bile acid concentration but, LDL-C level did not decrease significantly. We found that, ankaflavin facilitated conversion of cholesterol to bile acid by elevating liver X receptor (LXR alpha and cytochrome P450, family 7, subfamily A, polypeptide 1 (CYP7A1) expression, and subsequently decreasing TC level. Ankaflavin increased HDL-C level by cholesterol transporting via ATP-binding cassette subfamily A member 1 (ABCA1) which mediated the conversion of cholesterol to apo-A1 to increase HDL-C concentration. In vitro analysis, ankaflavin elevated apo-A1 mRNA level but not protein expression. PON1 is an HDL-associated enzyme with anti-oxidative properties. Although ankaflavin didn’t increase PON1 activity significantly, it could inhibit Cu2+-induced HDL oxidation. In addition, extracellular signal-regulated kinases (ERK) level was elevated by ankaflavin treatment to promote Nrf2 transcriptional activity. The increasing p-Nrf2 concentration in the cell then protected HDL-C via binding to ARE, which promoted the gene expression of an antioxidant enzyme heme oxygenase 1 (HO-1). These results obtained a conclusion that ankaflavin could reduce cholesterol and protect HDL-C from diminishing. In the future, we hope ankaflavin could serve as a functional food or drugs for decreasing cholesterol and improving low level of HDL-C from obesity.