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研究生:洪榆婷
研究生(外文):Yu-Ting Hung
論文名稱:以高脂飲食誘導肥胖大鼠模式探討Epigallocatechin gallate-quercetin-rutin複方對肥胖與腸道菌相之影響
論文名稱(外文):Effect of epigallocatechin gallate-quercetin-rutin formula on obesity and gut microbiota in a high-fat diet-induced obese rats
指導教授:徐慶琳徐慶琳引用關係
指導教授(外文):Chin-Lin Hsu
口試委員:張祐維鄭光成蔡宗佑張元衍
口試委員(外文):Yu-Wei ChangKuan-Chen ChengTsung-Yu TsaiYuan-Yen Chang
口試日期:2020-06-30
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:營養學系
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:中文
論文頁數:164
中文關鍵詞:EQR複方高脂飲食抗肥胖Wistar大鼠分子機轉腸道菌相
外文關鍵詞:EQR formulahigh-fat dietanti-obesityWistar ratmolecular mechanismsgut microbiota
DOI:10.6834/csmu202000016
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肥胖為重要的公共衛生議題,許多慢性疾病的發生都與肥胖密切相關。高油脂飲食會造成腸道菌相的失衡,進而加劇慢性發炎與肥胖之發生。先前文獻指出,表沒食子兒茶素沒食子酸酯 (epigallocatechin gallate, EGCG)、槲皮素 (quercetin) 和芸香苷 (rutin) 對肥胖具有改善作用,加上複方型式的產品具有協同作用之特性,因此本研究以組合複方的方式開發具抗肥胖作用之 EGCG-quercetin-rutin (EQR) 複方,並以高脂飲食預先誘導Wistar大鼠為肥胖症之動物模式,評估EQR複方改善肥胖與調控腸內菌相之影響,並釐清其分子作用機轉。本實驗將60隻6週齡Wistar雄性大鼠進行預養一週後,動物隨機分成正常飲食組 (normal diet, ND) 與高脂飲食組 (high-fat diet, HFD)。其中高脂飲食組大鼠以HFD誘導四週產生肥胖症 (肥胖大鼠之體重顯著高於正常飲食大鼠10%以上,即可進行後續介入試驗)。肥胖大鼠隨機分成五組 (n=10/group),包括:組別1:高脂飲食組 (HFD)、組別2:高脂飲食給予EQR複方低劑量組 (HFD + EQR formula-low dose, HFD + EQRF-LD)、組別3:高脂飲食給予EQR複方中劑量組 (HFD + EQR formula-medium dose, HFD + EQRF-MD)、組別4:高脂飲食給予EQR複方高劑量組 (HFD + EQR formula-high dose, HFD + EQRF-HD) 與組別5:高脂飲食給予市售健康食品正控制組 (HFD + positive control, HFD + PC) 為期四週。結果顯示
,給予肥胖大鼠EQR複方後可顯著抑制高脂飲食所誘導之體重和脂肪組織重量增加,並降低肝臟總脂質含量。由肝臟基因表現結果得知,給予EQR複方可提升高脂飲食誘導肥胖大鼠之PPAR-、ACO、CPT-1和HSL基因表現,並抑制其PPAR-和FATP-1基因表現。此外,在脂肪組織基因表現上,給予EQR複方可提升高脂飲食誘導肥胖大鼠之SIRT1和CPT-1基因表現,並抑制其FAS和FATP-1基因表現。在棕色脂肪基因表現上,給予EQR複方可提升高脂飲食誘導肥胖大鼠之AMPK和PGC-1基因表現。在腸道菌相的結果部分,EQR複方可提升菌群豐富度及多樣性;在菌門層面可降低Deferribacteres之相對豐富度,並增加Fusobacteria之相對豐富度;在菌科層面可增加Fusobacteriaceae和Christensenellaceae之相對豐富度;在菌屬層面可降低Mucispirillum之相對豐富度,並增加Enterorhabdus、Christensenellaceae R-7 group、Lachnoclostridium和Parvibacter之相對豐富度;在糞便短鏈脂肪酸的部分,其可增加乙酸、丙酸、丁酸和總短鏈脂肪酸的排出。綜合以上動物實驗得知,EQR複方可改善高脂飲食所誘導大鼠肥胖症,亦可逆轉高脂飲食之菌群組成,進而達到抗肥胖之作用。
Obesity is considered an important public health issue, and contributes to several chronic diseases. Meanwhile high-fat diet (HFD) is a inducer of gut dysbiosis which can result in the promotion of chronic inflammation and obesity. Previous publications have established that epigallocatechin gallate (EGCG), quercetin, and rutin have anti-obesity action. Based on the synergistic effect of the formula, the effect of EGCG-quercetin-rutin (EQR) formula on obesity, gut microbiota, and its molecular mechanisms in HFD-induced obese Wistar rats were investigated. After one week accommodation, sixty, and six-week-old male Wistar rats were divided into normal and obese groups, and then obese rats were prefed a HFD for four weeks. After the mean body weight of the obese rats (HFD groups) were over 10% heavier than that of the non-obese rats (normal diet, ND group). The obese rats were then divided into five groups (n=10/group) according to different doses of EQR formula for four weeks. Group 1: the HFD, Group 2: HFD + EQR formula-low dose (HFD + EQRF-LD), Group 3: HFD + EQR formula-medium dose (HFD + EQRF-MD), Group 4: HFD + EQR formula-high dose (HFD + EQRF-HD), and Group 5: HFD + positive control (HFD + PC). The results showed that, the weights of body, adipose tissues, and hepatic total lipid in HFD + EQR formula groups were significantly decreased as compared with the HFD group. In the gene expressions, EQR formula groups upregulated the gene expressions of PPAR-, ACO, CPT-1, and HSL, whereas it downregulated the gene expressions of PPAR- and FATP-1 in the liver of HFD-induced obese rats. In addition, EQR formula also upregulated the gene expressions of CPT-1, SIRT1, FAS, and FATP-1 in the perirenal adipose tissue of HFD-induced obese rats. Moreover, EQR formula could upregulate the gene expression of AMPK and PGC-1 in the brown adipose tissue of HFD-induced obese rats. Furthermore, EQR formula groups increased richness and diversity of the microbiota; decreased the relative abundance of the Deferribacteres, and increased the relative abundance of the Fusobacteria at the phylum level; increased the relative abundance of the Fusobacteriaceae and Christensenellaceae at the family level; decreased the relative abundance of the Mucispirillum, and increased the relative abundance of the Enterorhabdus, Christensenellaceae R-7 group, Lachnoclostridium, and Parvibacter at the genus level. In the contents of short chain fatty acids (SCFAs), EQR formula significantly increased the concentration of fecal acetic acid, propionic acid, butyric acid, and total SCFAs in HFD-induced obese Wistar rats. These results demonstrated that EQR formula is effective in preventing HFD-induced obesity and modulating gut microbiota.
摘要……………………………………………………………………………... Ⅰ
Abstract………………………………………………………………………….. Ⅲ
目錄………………………………………………………………………….….. Ⅴ
表次……………………………………………………………………………... XI
圖次……………………………………………………………………………... XIII
縮寫表…………………………………………………………………………... XV
前言……………………………………………………………………………... 1
壹、文獻回顧……………………………………………………………………. 3
一、肥胖…………………………………………………………………… 3
(一) 肥胖標準與定義……………………………………………..…. 3
(二) 肥胖盛行率……………………………………………………... 8
(三) 肥胖成因………………………………………………………... 10
1. 飲食………………………………………………………….. 10
2. 藥物………………………………………………………….. 11
3. 腸道菌群…………………………………………………….. 12
4. 老化………………………………………………………….. 12
5. 基因………………………………………………………..… 13
(四) 肥胖相關併發症………………………………………………... 13
1. 高血壓……………………………………………………….. 14
2. 高血糖……………………………………………………….. 14
3. 非酒精性脂肪肝病………………………………………….. 15
4. 血脂異常…………………………………………………….. 16
5. 癌症………………………………………………………….. 16
6. 骨關節炎…………………………………………………….. 17
(五) 預防及改善肥胖………………………………………………... 19
1. 行為矯正…………………………………………………….. 19
2. 飲食控制…………………………………………………….. 20
3. 體能活動…………………………………………………….. 20
4. 藥物治療…………………………………………………….. 21
5. 減肥手術…………………………………………………….. 22
6. 植物機能性成分…………………………………………….. 22
二、腸道菌群……………………………………………………………… 24
(一) 腸道菌群組成簡介……………………………………………... 24
(二) 腸道菌群與肥胖之相關性……………………………………... 24
(三) 腸道菌群與飲食之相關性……………………………………... 26
(四) 腸道菌群與多酚之相關性……………………………………... 27
三、單方與複方之比較……………………………………………………. 29
(一) 單方的特點……………………………………………………... 29
(二) 複方的特點……………………………………………………... 29
四、本研究所使用之EQR複方…………………………………………… 31
(一) EGCG表沒食子兒茶素沒食子酸酯…………………………… 31
1. EGCG簡介…………………………………………………… 31
2. EGCG生理功能……………………………………………… 32
(1) 抗肥胖…………………………………………………. 33
(2) 改善血脂異常…………………………………………. 34
(3) 抗糖尿病………………………………………………. 35
(4) 抗癌……………………………………………………. 35
(5) 抗氧化…………………………………………………. 36
(6) 改善高尿酸血症………………………………………. 37
(二) Quercetin槲皮素………………………………………………... 39
1. Quercetin簡介………………………………………………... 39
2. Quercetin生理功能…………………………………………... 39
(1) 抗肥胖…………………………………………………. 40
(2) 抗發炎…………………………………………………. 41
(3) 抗氧化…………………………………………………. 42
(4) 抗高血壓………………………………………………. 42
(5) 抗糖尿病………………………………………………. 43
(6) 抗癌……………………………………………………. 44
(三) Rutin芸香苷…………………………………………………….. 46
1. Rutin簡介…………………………………………………….. 46
2. Rutin生理功能……………………………………………….. 46
(1) 抗肥胖…………………………………………………. 47
(2) 抗高血脂症……………………………………………. 48
(3) 抗發炎…………………………………………………. 48
(4) 抗糖尿病………………………………………………. 49
(5) 保護心臟………………………………………………. 50
(6) 抗癌……………………………………………………. 51
貳、研究目的…………………………………………………………………… 54
參、材料方法…………………………………………………………………..... 55
一、實驗設計………………………………………………………………. 55
二、材料與設備…………………………………………………………… 56
(一) EQR複方之劑量選擇與換算…………………………………... 56
(二) 正控制組樣品之劑量換算……………………………………... 58
(三) 儀器……………………………………………………………... 59
(四) 藥品……………………………………………………………... 60
三、方法…………………………………………………………………… 61
(一) 動物、飲食與實驗設計……………………………………..…. 61
(二) 血清生化參數分析……………………………………….…….. 62
(三) 肝臟脂質分析…………………………………………….…...... 63
1. 總脂肪萃取………………………………………………….. 63
2. 三酸甘油酯測定…………………………………………….. 63
3. 總膽固醇測定………………………………………….......... 64
(四) 糞便脂質分析…………………………………………………... 64
(五) 肝臟與脂肪組織切片染色與油滴堆積之觀察……………...… 64
(六) 組織基因表現分析……………………………………………... 65
1. 組織RNA萃取…………………………………………...….. 65
2. RNA濃度測定……………………………………………….. 65
3. 反轉錄反應………………………………………………….. 65
4. 即時定量反轉錄聚合酶連鎖反應………………………...... 65
(七) 腸道菌相分析…………………………………………………... 66
1. 大鼠糞便收集……………………………………………….. 66
2. 腸道微生物菌相分析……………………………………….. 66
3. 糞便短鏈脂肪酸分析……………………………………….. 67
(八) 統計分析……………………………………………………...… 68
肆、結果……………………………………………………………………..….. 70
一、EQR複方對高脂飲食所誘導肥胖大鼠之體重、食物攝取量、能量
攝取與食物利用率之影響……………….………………………….. 70
二、EQR複方對高脂飲食誘導肥胖大鼠之臟器重量之影響……....…… 70
三、EQR複方對高脂飲食誘導肥胖大鼠之總脂肪量與脂肪組織重量之
影響…………………………………………………………………... 71
四、EQR複方對高脂飲食誘導肥胖大鼠之血清生化參數之影響…..… 71
五、EQR複方對高脂飲食誘導肥胖大鼠之肝臟總脂質、三酸甘油酯和
膽固醇含量之影響…………………………………………………... 72
六、EQR 複方對高脂飲食所誘導肥胖大鼠之糞便總脂質、三酸甘油
酯與膽固醇含量之影響………………………………….................. 72
七、EQR複方對高脂飲食誘導大鼠之肝臟和腎周脂肪組織切片形態
之影響………………………………………………………………... 72
八、EQR複方對高脂飲食誘導肥胖大鼠之肝臟組織基因表現之影響…. 73
(一) 脂肪酸氧化相關基因表現…………………………………….. 73
(二) 脂質生合成和脂質運輸相關基因…………………………….. 73
(三) 脂質分解相關基因…………………………………………….. 73
九、EQR複方對高脂飲食誘導肥胖大鼠之腎周脂肪組織基因表現之影
響…………………………………………………………………...... 74
(一) 脂肪酸氧化相關基因表現…………………………………...... 74
(二) 脂質生合成與脂質運輸相關基因…………………………...... 74
十、EQR複方對高脂飲食誘導肥胖大鼠之棕色脂肪組織基因表現之
影響…………………………………………………………….…….. 74
十一、EQR複方對高脂飲食誘導肥胖大鼠在腸道菌相之影響……...… 75
(一) EQR複方對高脂飲食誘導肥胖大鼠於糞便微生物多樣性
( diversity) 之影響……………………………………………. 75
(二) EQR複方對高脂飲食誘導肥胖大鼠於糞便菌微生物群聚組
成相似性之影響………………………………………………... 76
(三) EQR複方對高脂飲食誘導肥胖大鼠於糞便菌相組成之影響... 76
(四) EQR複方對高脂飲食誘導肥胖大鼠於糞便短鏈脂肪酸之影
響………………………………………………………………... 78
伍、討論………………………………………………………………………… 79
一、EQR複方對高脂飲食誘導肥胖大鼠之抗肥胖作用………………… 79
二、EQR複方對高脂飲食誘導肥胖大鼠之抗肥胖分子作用機轉……… 83
三、EQR複方對高脂飲食誘導肥胖大鼠之腸道菌相分析……………… 87
陸、總結論……………………………………………………………………… 93
柒、實驗圖表…………………………………………………………………… 94
捌、參考文獻…………………………………………………………………… 125
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