柑橘皮萃取物改善肉鹼飲水誘導 ApoE-/- 小鼠 TMAO 生成及相關之動脈病灶

Abstract

根據美國心臟學會統計，全球心血管疾病死亡人數逐年攀升，至 2021 年已上升至 1991 萬人，因此全球應更密切關注心血管疾病相關議題。動脈硬化為心血管疾病前期進程，研究發現腸道菌代謝產物氧化三甲胺 (Trimethylamine-N-oxide, TMAO)，會藉由多條路徑促進動脈硬化形成。TMAO 形成來源，為人體攝取富含肉鹼、膽鹼或甜菜鹼等飲食，這些物質在大腸中被含有特定代謝酵素基因 cutC/D、cntA/B 及 yeaW/X 之菌種代謝為三甲胺 (Trimethylamine, TMA)，再至肝臟中被 Flavin monooxygenase 3 (FMO3) 酵素轉化為 TMAO。先前已有研究證實多甲氧基黃酮 (Polymethoxyflavone, PMF) 可降低 TMAO 形成，減緩動脈硬化。PMF 為柑橘皮中，主要具生物活性之成分。因此本實驗以柑橘皮萃取物作為樣品，目的為探討柑橘皮萃取物對肉鹼飲水誘導小鼠心血管疾病之影響，期望開發潛在的新型健康食品。實驗利用 ApoE-/- 小鼠探討樣品對腸道菌相及減緩斑塊形成之效果。研究結果顯示，低劑量樣品組 (CLPM) 可顯著減少 carnitine 代謝並降低血液 TMAO 含量、增加糞便 carnitine 的排出。而高劑量樣品組 (CHPM) 降低 TMAO 生成之效果較弱。在氧化低密度脂蛋白 (oxLDL) 與動脈脂質堆積部分，低高劑量樣品皆可顯著提升血清 oxLDL 及降低動脈脂質堆積。除此之外，低高劑量樣品也能顯著減緩肝臟 FMO3 酵素表現量。在泡沫細胞生成路徑、PERK 接受器及發炎因子 mRNA 表現量，CLPM 組同樣可顯著降低相關基因表現量，而 CHPM 效果未如 CLPM 顯著。最後腸道菌方面，低高劑量樣品可顯著減少糞便中，特定代謝基因 cntA 及 yeaW 之表現量。而誘導組與控制組相比，腸道菌相組成迥異。給予低劑量樣品會使腸道菌相改變，與誘導組相比，CLPM 減少與促進 TMAO 生成相關之 Prevotella，增加有益菌 Bacteroides、Lactobacillus、Akkermansia。而高劑量樣品無法有效改善腸道菌相組成，其腸道菌群與誘導組相近。總結上述結果，低劑量樣品的介入可顯著降低血液 TMAO、特定基因 mRNA 表現量、減緩動脈脂質堆積、調控腸道菌相並增加有益菌之豐富度，說明低劑量樣品可減緩 carnitine 誘導之動脈病灶形成。高劑量樣品介入之組別，其 TMAO 含量、特定 mRNA 表現量無顯著差異，且腸道菌相調控能力較弱，但在減緩動脈脂質堆積有顯著效果，推測可能原因為高劑量樣品抑制有益菌生長，但可能透過其他途徑影響脂質堆積，原因尚需進一步探討。本次實驗結果證實，柑橘皮萃取物可望作為預防心血管疾病健康食品之開發新導向。

According to American Heart Association statistics, deaths from cardiovascular disease (CVD) rose to 19.91 million in 2021, indicating that the mortality rate of CVD has climbed year by year. This trend highlights the need for a greater focus on cardiovascular health. Atherosclerosis, an inflammatory disease of the large arteries, is a major cause of CVD and stroke. Recent research has identified trimethylamine-N-oxide (TMAO) as a novel and independent risk factor for promoting atherosclerosis. Dietary choline, carnitine, and betaine are metabolized into trimethylamine (TMA) by gut microbiota, and TMA is subsequently converted to TMAO by the hepatic enzyme flavin monooxygenase 3 (FMO3). Studies suggest that polymethoxyflavone (PMF) can reduce TMAO formation. In this context, citrus peel extract, which contains the bioactive compound PMF, was investigated for its ameliorative effect on carnitine-induced CVD in mice. The experiment sought to investigate the regulatory effect of citrus peel on intestinal bacteria to mitigate cardiovascular disease in mice, focusing on arterial plaque formation. The results revealed that the low dosage PMF (CLPM) significantly reduced carnitine metabolise and decreased blood TMAO levels, and increased fecal carnitine levels. However, the high dosage PMF (CHPM) only showed weaker ability to reduce TMAO. Both groups demonstrated an increase in plasma oxLDL levels and a reduction in lipid accumulation in aortic arches. Also, reduced the expression of liver FMO3. Additionally, the CLPM group was able to attenuate foam cell formation by inhibiting the CD36/LYN/JNK pathway, PERK receptor, and inflammation-related gene expression in the aorta. And the expression of fecal specific metabolic genes cntA and yeaW, were significantly reduced in CLPM and CHPM group. In terms of gut microbiota composition, the induced group exhibited significant changes compared to the control group. The CLPM group inhibited the growth of the negative bacterium Prevotella and increased the presence of beneficial bacteria such as Bacteroides, Lactobacillus, and Akkermansia. However, the CHPM group displayed a gut microbiota composition similar to that of the induced group in β diversity. In summary, 1% PMF significantly attenuated plasma TMAO levels, specific gene relative mRNA expression, lipid accumulation in aortic arches, and promoted the abundance of healthy bacteria. The 2.5% PMF dosage had a lesser effect on plasma TMAO levels, mRNA expression, and gut microbiota composition but still reduced lipid accumulation in the aorta. It is speculated that a high dosage of PMF may inhibit beneficial bacteria or affect lipid accumulation through alternative pathways. These findings suggest that citrus peel extract may be a promising direction for developing functional foods to prevent cardiovascular diseases.