川陳皮素調控脂肪細胞來源外泌體以改善高脂飲食誘導肥胖小鼠的脂質代謝異常

Abstract

全球肥胖盛行率持續攀升，對健康造成嚴重威脅，並引發多種代謝異常，其中非酒精性脂肪肝為常見併發症。川陳皮素 (Nobiletin) 為一種來源於柑橘類水果的類黃酮，因其具備抗氧化、抗發炎及改善代謝症候群與肥胖的潛力而備受關注。然而，川陳皮素的生物利用度有限，且其是否能透過調控細胞外囊泡 (Extracellular Vesicles, ELVs) 中特定微核醣核酸 (miRNA) 以改善高脂飲食誘發的代謝疾病，尚待進一步探討。本研究旨在闡明川陳皮素調控脂肪細胞來源 ELVs 中 miRNA，進而改善肥胖小鼠肝臟脂質代謝之機制。在體外實驗中，本研究證實川陳皮素(120 μM) 能有效抑制 3T3-L1 脂肪細胞的脂質堆積，且無明顯細胞毒性。進一步從未分化前脂肪細胞 (pre-ELVs)、分化成熟脂肪細胞 (ob-ELVs) 及經川陳皮素處理之脂肪細胞 (Nob-ELVs) 分別萃取 ELVs，並透過奈米粒子追蹤分析、穿透式電子顯微鏡及西方墨點法確認其尺寸 (40-160 nm)、碟狀結構及特異性蛋白標記 (CD63、CD9、TSG101)，確保 ELVs 成功分離及具內化能力。探討 ELVs 對脂質代謝之影響時發現，pre-ELVs 與 ob-ELVs 未能顯著抑制 3T3-L1 細胞脂質累積，惟 Nob-ELVs 在不影響細胞存活率下，顯著降低約 40% 脂質堆積。miRNA 微陣列分析顯示三組 ELVs 之 miRNA 表現模式存在顯著差異，尤以 ob-ELVs 中 miR-802-5p 表達顯著高於 pre-ELVs 與 Nob-ELVs，且 Nob-ELVs 顯著下調該 miRNA，暗示川陳皮素可能透過調節 ELVs 中 miRNA 組成，影響受體細胞生理功能。此外，本研究建立高脂飲食 (HFD) 誘導之 C57BL/6J 小鼠肥胖模型，並腹腔注射不同 ELVs 持續 10 週。結果顯示，HFD + ob-ELVs 組體重變化與 HFD 組相當；相較於 HFD 組，HFD + pre-ELVs 及 HFD + Nob-ELVs 組小鼠體重增加顯著受抑制，且食物轉換效率獲得改善。血液生化分析顯示，Nob-ELVs 顯著改善 HFD 誘發之總膽固醇、高密度脂蛋白膽固醇及低密度脂蛋白膽固醇異常；pre-ELVs 則對三酸甘油酯有正面影響；ob-ELVs 對血脂指標無顯著改善。組織學檢查發現，HFD + pre-ELVs 及 HFD + Nob-ELVs 組顯著減少內臟脂肪堆積及肝臟脂肪空泡之數量與大小，而 HFD + ob-ELVs 組則與 HFD 組相似。肝臟脂質代謝相關蛋白分析顯示，pre-ELVs 主要透過提升 AMPK 與 PPARα 表現促進脂質氧化；Nob-ELVs 則透過上調 SIRT1、PPARα 及 FGF21 等脂質氧化相關蛋白，同時下調 ACC、FASN 及 CD36 等脂肪酸合成與攝取蛋白，從而減少肝臟脂質累積並改善脂質代謝異常。值得注意的是，ob-ELVs 在活化 AMPK 方面無效，且對 FASN 與 CD36 調控與 HFD 組無顯著差異。本研究首次證實川陳皮素可誘導 3T3-L1 脂肪細胞釋放之 ELVs，其 miRNA 組成顯著改變，且能有效抑制脂肪細胞內脂質積累。在肥胖小鼠模型中，Nob-ELVs 展現全面改善肝臟脂質代謝異常之潛力，其分子機制涉及促進脂質氧化及抑制脂肪酸合成與攝取。此發現為基於川陳皮素調控 ELVs 之抗肥胖創新策略提供了重要理論基礎。

The global incidence of obesity is rising, presenting significant health challenges and leading to a range of metabolic disorders, among which non-alcoholic fatty liver disease (NAFLD) is a common complication. Nobiletin, a flavonoid compound extracted from citrus fruits, has attracted considerable interest due to its antioxidant and anti-inflammatory properties, as well as its potential to ameliorate metabolic syndrome and obesity. However, nobiletin’s limited bioavailability and its ability to modulate high-fat diet-induced metabolic disorders through the regulation of specific microRNAs (miRNAs) within extracellular vesicles (ELVs) remain poorly understood. This investigation aims to elucidate the mechanisms by which nobiletin modulates miRNA profiles in ELVs derived from adipocytes to enhance hepatic lipid metabolism in obese murine models. In vitro assays demonstrated that nobiletin, at a concentration of 120 μM, effectively inhibited lipid accumulation in 3T3-L1 adipocytes without inducing cytotoxic effects. ELVs were isolated from undifferentiated pre-adipocytes (pre-ELVs), fully differentiated mature adipocytes (ob-ELVs), and nobiletin-treated adipocytes (Nob-ELVs). Characterisation via nanoparticle tracking analysis, transmission electron microscopy, and Western blotting confirmed the ELVs’ size range (40–160 nm), characteristic disc-shaped morphology, and expression of specific protein markers (CD63, CD9, TSG101), thereby validating successful isolation and internalisation capacity. Functional assays assessing the impact of ELVs on lipid metabolism revealed that pre-ELVs and ob-ELVs did not significantly reduce lipid accumulation in 3T3-L1 cells. In contrast, Nob-ELVs induced a substantial reduction in lipid accumulation of approximately 40%, without compromising cell viability. miRNA microarray profiling identified distinct expression patterns among the three ELV groups, notably with miR-802-5p exhibiting significantly elevated levels in ob-ELVs compared to pre-ELVs and Nob-ELVs. The marked downregulation of miR-802-5p in Nob-ELVs suggests that nobiletin may modulate recipient cell physiology through alterations in the miRNA cargo of ELVs. In vivo, a high-fat diet (HFD)-induced obesity model was established in C57BL/6J mice, which received intraperitoneal injections of the respective ELVs over a 10-week period. Body weight trajectories in the HFD + ob-ELVs cohort paralleled those of the HFD control group. Conversely, mice treated with HFD + pre-ELVs and HFD + Nob-ELVs exhibited significantly attenuated weight gain and improved food conversion efficiency compared to HFD controls. Biochemical analyses revealed that Nob-ELVs significantly ameliorated HFD-induced dyslipidaemia, improving total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol levels; pre-ELVs favourably influenced triglyceride concentrations; ob-ELVs did not produce significant improvements in lipid profiles. Histopathological evaluation demonstrated that the HFD + pre-ELVs and HFD + Nob-ELVs groups had markedly reduced visceral adiposity and diminished hepatic lipid vacuole number and size, whereas the HFD + ob-ELVs group resembled the HFD control. Assessment of hepatic lipid metabolism-related proteins indicated that pre-ELVs primarily enhanced lipid oxidation through the upregulation of AMPK and PPARα expression. Nob-ELVs further increased the levels of lipid oxidation-associated proteins, including SIRT1, PPARα, and FGF21, while simultaneously downregulating proteins involved in fatty acid synthesis and uptake, such as ACC, FASN, and CD36. This resulted in the mitigation of hepatic lipid accumulation and the correction of lipid metabolic disturbances. Notably, ob-ELVs failed to activate AMPK and did not significantly alter FASN or CD36 expression compared with the HFD group. This study provides the first evidence that nobiletin induces 3T3-L1 adipocytes to secrete extracellular vesicles (ELVs) with significantly altered miRNA profiles, which effectively suppress lipid accumulation within adipocytes. In an obese mouse model, Nob-ELVs demonstrated comprehensive efficacy in ameliorating hepatic lipid metabolic dysfunction through mechanisms involving the promotion of lipid oxidation and the inhibition of fatty acid synthesis and uptake. These findings offer a crucial theoretical foundation for the development of novel anti-obesity interventions based on nobiletin-regulated ELVs.