Effect of camellia oil on alleviation of acetic acid-induced colitis rats by modulating the gut microbiota

隨著微生物與人體健康的關係研究不斷深入，飲食對腸道微生物的影響逐漸成為人們關注的話題。近年研究證實苦茶油具有胃腸保護及抗發炎的功效，然尚少有與腸道菌相相關之研究。因此本試驗先以體外模擬人體腸道菌相試驗，評估苦茶油對益生菌的利用性，結果顯示苦茶油能提升腸道內 Lactobacillus spp. 及 Bifidobaterium spp.，具改善腸道菌相之潛力。進而利用Sprague-Dawley (SD) 品系大鼠進行試驗，探討苦茶油是否可經由調節腸道菌相減緩醋酸誘導之腸炎。以管餵方式投予大鼠苦茶油三週後利用4% 醋酸 (2 mL) 進行腸炎誘導，誘導後給予 sulfasalazine 做為正控藥物，三天後犧牲。動物實驗結果顯示，4% 醋酸 (2 mL) 會造成腸炎，但對肝臟不會造成任何損傷。然而，預先餵食苦茶油 (2 mL/kg b.w.) 能有效減緩醋酸誘導大鼠腸道損傷與出血之現象，提升抗氧化酵素麩胱甘肽 (GSH)、超氧化物歧化酶 (SOD) 之活性，降低脂質過氧化物 (MDA)、骨髓過氧化酵素 (myeloperoxidase) 含量，減緩醋酸誘導腸組織發炎因子 TNF-α、 IL-6 及 IL-1β 之蛋白表現。而在血清分析結果顯示，預先餵食苦茶油對醋酸誘導大鼠其抗體 IgG1、IgG2a 含量並無顯著性差異。另外，利用選擇性培養基進行腸道菌相分析試驗。餵食苦茶油後能提高腸道內益生菌Lactobacillus spp.、Bifidobaterium spp.，降低Clostridium peringens。腸道菌相定序分析部分，結果顯示苦茶油能提升厚壁菌與擬桿菌比例、 α 多樣性及益生菌 Akkermansia muciniphila、Bifidobacterium adolescentis 豐富度。綜合上述，苦茶油能增加腸道內益生菌，降低有害菌生成及提升腸道菌相多樣性及益生菌豐富度，達到預防及降低醋酸所誘導的氧化傷害及減緩發炎作用。此外，本研究亦利用Int407與RGM-1細胞建立一細胞快速篩選平台，評估對於腸胃道保護具有功效的油品，以減少實驗動物之使用。With the increase in studies of relationship between gut microbiota and human health, the impact of diet on intestinal microorganism has become the hot issue around the world. Recent study has demonstrated that camellia oil shows great bioactivities, including gastrointestinal protection and anti-inflammation. However, there is still little known about the effect of camellia oil on gut microbiota. In this study, the effect of camellia oil on gut microbiota was first evaluated using an ex vivo model. The result showed that camellia oil displayed great potential on improvement of gut microbiota as evidenced by increased number of Lactobacillus spp. and Bifidobaterium spp. Moreover, Sprague-Dawley (SD) rat model of acetic acid-induced colitis was developed to confirm whether camellia oil can alleviate colitis through regulation of gut microbiota. 4% Acetic acid (2 mL) can lead to colitis in SD rats, but no lesion was found in their livers. However, pretreatment with camellia oil (2 mL/kg b.w.) can attenuate acetic acid-induced colonic injury and hemorrhage, enhance antioxidant enzyme activities including glutathione (GSH) and superoxide dismutase (SOD), and decrease malondialdehyde (MDA), myeloperoxidase (MPO), and inflammatory cytokines (TNF-α, IL-6, and IL-1β). Serum analysis indicated that pretreatment with camellia oil did not affect the amount of IgG1 and IgG2a, while camellia oil can increase probiotics (Lactobacillus spp. and Bifidobaterium spp.) and decrease Clostridium peringens in the gut microbiota. The result of next generation sequencing (NGS) further indicated that camellia oil can elevate Firmicutes/Bacteroidetes ratio, α-diversity indexes of gut microbiota, commensal bacteria, and biodiversity of Akkermansia muciniphila and Bifidobacterium adolescentis. In conclusion, camellia oil could increase probiotics/pathogen ratio and alpha-diversity in the acetic acid-induced SD rats, of which oxidative damage and inflammation in the gut may be ameliorated. In addition, Int407 and RGM-1 cell models were established to assess gastrointestinal protection by camellia oil, which may lead to reduction of experimental animals.全文摘要 i 英文摘要 ii 目次 iv 圖次 vi 表次 ix 縮寫表 x 壹、前言 1 貳、文獻整理 3 一、發炎性腸道疾病 (Inflammatory bowel disease) 4 (一) 腸道生理功能 4 (二) 發炎性腸道疾病 5 (三) 發炎性腸道疾病之治療 7 (四) 腸炎動物誘導模式 8 (五) 天然化合物改善發炎性腸道疾病之相關研究 11 二、腸道微生物 (Gut microbiome) 17 (一) 腸道微生物 17 (二) 人體腸道微生物的組成 19 (三) 腸道微生物與發炎性腸道疾病之間的影響 22 (四) 飲食對發炎性腸道疾病腸道微生物之影響 28 三、苦茶油 (Camellia oil) 29 (一) 油茶樹 29 (二) 苦茶油壓榨及保存方式 31 (三) 苦茶油之機能性成分與相關研究 33 四、研究目的 39 五、研究架構 40 參、材料方法 41 肆、結果 56 一、苦茶油經由調節腸道菌相減緩醋酸誘導之研究 56 (一)、體外模擬人體腸道菌相試驗 56 (二)、 苦茶油對Int-407 細胞之保護效應 57 (三)、苦茶油對醋酸誘導 Sprague-Dawley 大鼠腸炎之減緩效應 58 1. 腸炎相關生理生化參數 58 2. 腸道菌相分析 64 3. 次世代腸道菌相分析 67 4. 苦茶油成分分析 71 二、苦茶油對胃腸潰瘍保護效果之細胞快速篩選平台建立 72 (一)、不同榨油條件之苦茶油對 Indomethacin 誘導 Int-407 細胞存活率及活性氧生成之保護功效 72 (二)、不同榨油條件之苦茶油對乙醇誘導 RGM-1 細胞存活率及活性氧生成之保護功效 73 (三)、 不同榨油條件之苦茶油快速篩選之細胞平台積分結果 74 伍、討論 76 一、苦茶油經由調節腸道菌相減緩醋酸誘導腸炎之研究 76 二、苦茶油對胃腸潰瘍保護效果之細胞快速篩選平台建立 86 陸、總結論 88 柒、參考文獻 13