5 位羥化多甲氧基黃酮透過減緩脂肪發炎與促進脂肪棕色化預防高脂飲食誘導小鼠肥胖之功效

Abstract

由於現代生活型態和飲食習慣的改變，肥胖人口與日俱增，已然成為現今社會健康與經濟議題。最近的研究表明，食品中的植化素可以透過不同途徑預防肥胖，包括促進脂肪組織棕色化、增加能量消耗、減輕發炎反應和氧化壓力。多甲氧基黃酮 (polymethoxyflavones, PMFs) 是柑橘皮中含量最豐富的黃酮類化合物之一，具有調節脂質代謝、降低氧化壓力和抗發炎等多種生物功能；而近年來，有多篇文獻指出羥化多甲氧基黃酮 (hydroxylated polymethoxyflavones, HPMFs) 有較好的生理活性，然而 HPMFs 是否對於脂肪棕色化和調節腸道菌群有所助益仍尚未被充分研究。因此本研究使用主成分為 HPMFs 之樣品粉末 (Citrus PMFs, CPs)，並將 C57BL/6 小鼠分為五組，普通飲食組 (ND)；高脂飲食組 (50% calories from fat，HFD)；高脂飲食混和低劑量 CPs (HPMFs含量 0.36 mg/g 飼料)；高脂飲食混和高劑量 CPs 粉末 (HPMFs 含量 1.45 mg/g 飼料) 以及普通飲食混和高劑量 CPs 粉末，飲食誘導 16 週後犧牲，藉此探討HPMFs對高脂飲食誘導小鼠是否具有改善肥胖之功效。本實驗結果顯示，與 HFD 組相比，高脂飲食合併給予高劑量 CPs 在不影響攝食量下顯著降低小鼠體重、肝臟重量和內臟脂肪重量；血清三酸甘油酯和動脈粥樣硬化指數 (LDL/HDL) 水平顯著低於 HFD 組。此外，CPs 顯著降低肝臟三酸甘油酯含量，切片結果也顯示脂肪細胞體積縮小與大小分佈的改變，且 CPs 可以有效減緩脂肪組織內的發炎反應和 M1 巨噬細胞浸潤。進一步研究分子機制，發現CPs 可以將脂聯素恢復至控制組水平並活化下游 AMPK 途徑以增加脂肪分解和脂肪酸 β-氧化。此外，高脂飲食合併給予高劑量 CPs 可以顯著提升 p-p38 MAPK、SIRT-1、PGC1-α 和 UCP-1 的蛋白質表現量，顯示 HPMFs 可以促進脂肪棕色化和能量消耗。此外，微生物群分析結果說明 HPMFs 可以預防腸道菌叢失調，並且根據 LefSe 分析，1% CPs 組別顯著增加乳酸菌與 Limosilactobacillus reuteri 相對豐度。綜上所述， 作為 CPs 中之主要成分，HPMFs 具有減輕發炎和調節脂質代謝之功效，並促進脂肪棕色化和增加益生菌豐度，有潛力作為一種天然抗肥胖劑，並為開發新型抗肥胖策略提供理論基礎。

Due to the changes in physical and dietary activity patterns, in 2022, more than 2.5 billion adults were overweight, with over 890 million of them being obese, according to the WHO. Obesity may lead to various health consequences, including diabetes, cardiovascular diseases, stroke, and cancer. Treatments for obesity are inevitably associated with reducing body weight, including lifestyle interventions, medications, and surgery. However, drugs and surgeries are often accompanied by side effects and high costs. Recent studies have demonstrated that certain natural dietary factors can effectively prevent obesity by serving as a valid strategy. Several bioactive compounds in the diet serve as antioxidants and anti-inflammatory agents, aiding progress in weight loss and/or the mitigation of metabolic disorders. This is achieved by boosting adipose tissue browning, thermogenesis and energy expenditure, as well as alleviating inflammation and oxidative stress. As a result, natural phytochemicals have become a novel choice for obesity. Polymethoxyflavones (PMFs), one of the most abundant flavonoids in citrus peel, have many biological functions, such as modulating lipid metabolism, reducing oxidative stress and anti-inflammation; however, their poor water solubility has become a challenge. Hydroxylated PMFs (HPMFs) are PMFs in which some methoxy groups are replaced by hydroxyl groups. This substitution leads to increased bioavailability and enhanced biological functions. However, the mechanisms of HPMFs in promoting fat browning and modulating gut microbiota haven’t been fully discussed. Therefore, we aimed to explore the potential effects of sample powder containing HPMFs on improving obesity, including activation of white adipose tissue browning, modulating lipid metabolisms, and alleviating inflammation in high-fat diet-induced mice. A 16-week animal experiment was conducted using C57BL/6 mice fed with high-fat diet (HFD) combining citrus PMFs sample powder (CPs) mainly composed of HPMFs to confirm the effects of HPMFs. Compared with HFD group, body weight, liver weight and visceral fat weight were significantly reduced in HFD+1% CPs group without affecting food intake. Serum TG and AI (LDL/HDL) levels were significantly lower in HFD+1% CPs group than HFD group. In addition, 1% HPMFs could reduce liver TG content, decrease adipocyte size and change the size distribution. Inflammatory cytokines and chemokine were significantly decreased in HFD+1% CPs group, and so was the M1 macrophage. Further investigating lipid metabolisms, 1% HPMFs could upregulate adiponectin to control level and activate downstream AMPK pathways, which increase lipolysis and fatty acid β-oxidation. Also, protein levels of p-p38 MAPK, SIRT-1, PGC1-α and UCP-1 were upregulated in HFD+1% CPs group, indicating that HPMFs could promote fat browning and energy expenditure. Furthermore, the results of microbiota analysis showed that CPs could prevent gut dysbiosis by increasing lactobacillus, and 1% CPs could significantly increase probiotic Lactobacillus and Limosilactobacillus reuteri based on LefSe. In conclusion, as the main compound in sample powder CPs, HPMFs not only alleviate inflammation and modulate lipid metabolisms, but also boost fat browning and probiotic abundance to prevent obesity, and could be a potent ingredient applied to the development of functional food for obesity prevention.