探討黃豆發酵萃取物對於 Apoe−/− 小鼠腸道菌相的影響與心血管疾病的關聯性

Investigation of fermented soybean extract on cardiovascular disease through gut microbiota in Apoe−/− mice

黃熠埼(Yi-Chee Ng)

指導教授 ： 沈立言

國立臺灣大學/生物資源暨農學院/食品科技研究所/碩士(2020年)

https://doi.org/10.6342/NTU202004039

摘要

根據世界衛生組織 (World Health Organization, WHO) 最新的統計，心血管疾病 (Cardiovascular disease, CVD) 是全球人口死亡的主要原因。而根據台灣衛生福利部所公佈的 108 年國人十大死亡原因中，心血管相關疾病所造成的死亡總人數已超過位於榜首的癌症，可見預防 CVD 之必要性。隨著近年來腸道菌相相關研究的興起，腸道菌及其代謝產物如氧化三甲胺 (Trimethylamine-N-oxide) 、短鏈脂肪酸 (Short chain fatty acids, SCFA) 等被證實與 CVD 的發生有關。其中，腸道菌透過生成 TMAO 導致人類 CVD 的發現是目前科學理論架構完整且最具臨床應用價值之一的研究方向。許多研究指出動脈粥狀硬化 (Atherosclerosis) 是造成 CVD 的主要原因，並證實 TMAO 具有致動脈粥狀硬化 (Pro-atherogenic) 及血栓形成的能力，因此人體血液中 TMAO 的濃度是促使動脈粥狀硬化的重要且獨立的危險因子。TMAO 的產生主要來自於飲食中的肉類及蛋類所含的肉鹼 (Carnitine) 和膽鹼 (Choline)，在腸道中被腸道菌代謝利用並透過肝臟酵素氧化而形成 TMAO。動脈粥狀硬化是一種慢性發炎性疾病，低密度脂蛋白 (Low-density lipoprotein, LDL) 氧化後具有細胞毒性，會引起血管內皮發生發炎反應，最終導致動脈粥狀硬化斑塊的生成而逐漸阻塞動脈。因此，減輕發炎反應可以有效降低主動脈斑塊的生成。黃豆經過發酵會產生黃酮類 (Flavones)、異黃酮 (Isoflavones)、黃頑酮類 (Flavanones) 等，這些化合物具有許多生理活性如抗發炎、抗氧化、抗過敏等活性。此外，根據本實驗室初步 in vitro study 結果發現，黃豆發酵萃取物可以有效抑制特定菌株的生長，進而抑制 carnitine 及 choline 轉換成 TMA子路徑。於短期 C57BL/6J 小鼠實驗中也發現黃豆發酵萃取物的介入可顯著降低小鼠血液中 TMA 及 TMAO 的濃度，減緩血小板凝集的發生。然而，前述 C57BL/6J 小鼠模式無法提供動脈粥狀硬化的主要表徵，如主動脈斑塊的生成。因此，本實驗將該用 Apoe-/- 小鼠動物模式探討黃豆發酵萃取物是否能透過調節小鼠腸道菌相，降低 TMAO 的產生，進而減少小鼠主動脈斑塊的生成，達到心血管保護的效果。實驗結果顯示， 黃豆發酵萃取物可顯著降低 carnitine 介入後小鼠血液中 TMAO 的濃度，進而減少血管斑塊之生成，同時降低了小鼠血液中 TG 的濃度。此外，黃豆發酵萃取物也改善了 choline 介入下小鼠的肝功能指數並提升了小鼠血液中高密度脂蛋白 (HDL-C) 的濃度。根據小鼠菌相分析的結果顯示，黃豆發酵萃取物顯著改變 carnitine 組別小鼠整體腸道菌相的組成，且提高腸道菌之多樣性及豐富度。而主座標分析 (PCoA) 結果顯示黃豆發酵萃取物不會改變給予 choline 的小鼠整體腸道菌相之組成，但對於特定菌群的相對豐富度仍造成顯著的改變。綜上所述，黃豆發酵萃取物可透過改善血脂及肝功能，調節腸道菌相，降低 TMAO 的產生，進而減少主動脈斑塊的生成，達到心血管保護之功效。

關鍵字

心血管疾病 ； 氧化三甲胺 ； 肉鹼 ； 膽鹼 ； 腸道菌相 ； 黃豆發酵萃取物

並列摘要

According to the statistic announced by the World Health Organization (WHO) in 2018, the cardiovascular disease (CVD) is the main cause of death worldwide and have remained the leading causes of death globally. For the prevalence in Taiwan, Ministry of Health and Welfare (MOHW) stated that the mortality of CVD-related diseases is comparable to those of cancer. Thus, the prevention of CVD is increasingly important as CVD is considered as the health burden worldwide. Atherosclerosis is the main underlying cause of CVD and TMAO plasma levels have been proved to be associated with atherosclerosis. Studies recently have identified TMAO as an essential proatherogenic and prothrombotic compound that contribute to the progression of atherosclerosis. Studies have proved that the specific diet-gut microbiota interaction plays a critical role in the CVD pathogenesis. TMAO is produced mainly from the gut microbiota-dependent metabolism of dietary carnitine and choline. L-carnitine and choline, the abundant nutrient in red meat, eggs and milk, are hydrolysed and are converted to trimethylamine (TMA) by microbial lyases in the colon. TMA is carried via the portal circulation to the liver where it is rapidly converted by host hepatic flavin-containing monooxygenases (FMO3) into trimethylamine N-oxide (TMAO). Therefore, modification of gut microbiota may contribute to the reduced formation of serum TMAO. The main compounds identified in fermented soybean products were flavanons, isoflavones and flavonols, which possess many biological activities such as anti-inflammatory, antioxidant and anti-allergy. Our laboratory previous in vitro study showed that the fermented soybean extracts inhibited the growth of specific bacteria responsible for the transformation of choline and carnitine into TMA and further reduced the production of serum TMAO. Besides, the fermented soybean extracts prevented the elevation of serum TMAO from carnitine and choline diet in a short-term C57BL/6J mice model and resulted in decreased platelet aggregation. Therefore, my present study aimed to investigate the cardiovascular protective effect of the fermented soybean extract against the formation of aortic lesions and CVD-related biomarkers by using Apoe-/- mice. The result showed that the fermented soybean product significantly reduced the concentration of serum TMAO and triglyceride (TG), and the formation of aortic lesions in mice fed with carnitine diet. Furthermore, fermented soybean extracts decreased the
serum AST and ALT, and increased the serum HDL-C in mice fed with choline diet. Gut microbiota analysis of carnitine-fed mice showed that the fermented soybean extracts significantly altered the overall gut microbiota composition and increased the diversity and richness of gut microbiota. The PCoA showed no significant differences of overall gut microbiota composition after consuming fermented soybean extracts in choline-fed mice. However, the relative abundance of specific gut bacteria such as Erysipelatoclostridium and Coprococcus 2 in genus level decreased significantly after fermented soybean extracts intervention. In, conclusion, intervention of fermented soybean extracts exhibited cardio-protective effects by reducing the production of serum TMAO and formation of aortic lesion, improving the serum biochemical through modulation of gut microbiota.

並列關鍵字

CVD ； TMAO ； carnitine ； choline ； gut microbiota ； fermented soybean extract

參考文獻

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