系統識別號 U0007-2407201715395200
論文名稱(中文) 飲食中不同油脂合併補充枸杞多醣萃取物對四氯化碳誘發肝纖維化大鼠之影響
論文名稱(英文) Effects of different dietary oils combined with Lycium barbarum polysaccharide extract on liver fibrosis induced by CCl4 in rats
校院名稱 臺北醫學大學
系所名稱(中) 保健營養學研究所
系所名稱(英) Graduate Institute of Nutrition and Health
學年度 105
學期 2
出版年 106
研究生(中文) 江昀芸
研究生(英文) Yun-Yun Chiang
學號 M507104017
學位類別 碩士
語文別 中文
口試日期 2017-06-13
論文頁數 95頁
口試委員 指導教授-趙振瑞
中文關鍵字 橄欖油  枸杞多醣體  細胞凋亡  發炎反應  肝纖維化 
英文關鍵字 olive oil  Lycium barbarum polysaccharides  apoptosis  inflammation  liver fibrosis 
中文摘要 目前肝纖維化還未有有效的治療方式,而先前研究指出,橄欖油與枸杞多醣體皆具有保護肝臟的作用,本次實驗欲探討飲食中不同油脂合併枸杞多醣萃取物(Lycium barbarum polysaccharide extract, LBE)補充,對於四氯化碳(carbon tetrachloride, CCl4)誘發肝纖維化大鼠之發炎反應、細胞凋亡及纖維化的影響。雄性Sprague-Dawley 大鼠隨機分成七組:正常飲食組(4%大豆油飲食)、大豆油飲食組(4%大豆油飲食+ CCl4)、橄欖油飲食組(4%橄欖油飲食+ CCl4)、大豆油/橄欖油飲食組(2%大豆油/2%橄欖油飲食+ CCl4)、大豆油飲食+ LBE組(4%大豆油+ LBE + CCl4)、橄欖油飲食+LBE組(4%橄欖油+ LBE + CCl4)、大豆油/橄欖油飲食+ LBE組(2%大豆油/2%橄欖油飲食+ LBE + CCl4)。LBE給予量為每天50 mg/kg bw,預先給予油脂或/和LBE一週後,每週腹腔注射1-2 mL/kg bw誘發肝纖維化,共注射八週。結果顯示注射四氯化碳之六組有較嚴重的肝臟脂質堆積、細胞死亡、發炎反應及纖維增生,含有橄欖油的飲食可降低肝臟TGF-β1與TIMP-1濃度;給予LBE可降低肝臟caspase-9與caspase-3活性,降低肝臟TNF-α濃度,增加IL-10/TNF-α比值,降低肝臟TGF-β1與TIMP-1濃度;當橄欖油與LBE合併給予,可降低肝臟caspase-9與caspase-3活性、降低肝臟TNF-α濃度,增加肝臟IL-10濃度與IL-10/TNF-α比值,降低肝臟TGF-β1與hydroxyproline濃度。對於注射四氯化碳8週之大鼠,枸杞多醣萃取物可改善細胞凋亡、發炎反應及纖維化指標,而橄欖油合併枸杞多醣萃取物的改善效果更佳。
英文摘要 The effective therapy for liver fibrosis is still elusive. Olive oil and Lycium barbarum polysaccharides were considered to act as hepatoprotective activity. This study investigated anti-inflammatory, anti-apoptosis and anti-fibrotic effects of different dietary oils and Lycium barbarum polysaccharide extract (LBE) on CCl4-induced liver fibrosis in rats. Male Sprague-Dawley rats were divided into 7 groups: normal diet (4% soybean oil diet), soybean oil diet (4% soybean oil diet + CCl4), olive oil diet (4% olive oil diet + CCl4), soybean oil/olive oil diet (2% soybean oil / 2% olive oil diet + CCl4), soybean oil diet + LBE (4% soybean oil diet + LBE + CCl4), olive oil diet + LBE (4% olive oil diet + LBE + CCl4), and soybean oil/olive oil diet + LBE (2% soybean oil / 2% olive oil diet + LBE + CCl4). The LBE-treated groups were given 50 mg/kg/day LBE. Rats were injected with 1-2 ml/kg bw/wk CCl4 intraperitoneally for 8 weeks. The results showed that CCl4 caused more severe liver fatty change, cell death, inflammation, and collagen generation. The groups treated with olive oil reduced hepatic TGF-β1 and TIMP-1 concentrations. LBE reduced hepatic caspaspe-3 and caspase-9 activities, decreased the hepatic TNF-α level, increased IL-10/TNF-α ratio, and reduced hepatic TGF-β1 and TIMP-1 concentrations. The groups treated with olive oil combined with LBE reduced hepatic caspaspe-3 and caspase-9 activities, decreased hepatic TNF-α level, increased hepatic IL-10 level and IL-10/TNF-α ratio, as well as reduced hepatic TGF-β1 and hydroxyproline concentrations. Therefore, after CCl4 injection for 8 weeks in rats, LBE improves apoptotic, inflammatory and fibrotic markers, while olive oil combined with LBE has better effects on anti-apoptosis, anti-inflammation and anti-fibrosis.
論文目次 中文摘要 I
Abstract II
致謝 III
目錄 IV
圖目次 VI
表目次 VII
第一章 緒論 1
第二章 文獻回顧 5
第一節 肝臟功能與結構 5
第二節 肝臟纖維化之成因與演進 7
第三節 四氯化碳誘發肝纖維化之動物模式 10
一、 四氯化碳造成肝損傷之機制 10
二、 四氯化碳與細胞凋亡指標 12
三、 四氯化碳與發炎反應指標 12
四、 四氯化碳與肝纖維化指標 14
第四節 飲食中油脂成分對肝臟疾病之影響 18
一、 飲食中油脂成分對肝臟脂質代謝的影響 18
二、 橄欖油對肝臟纖維化的影響 20
第五節 枸杞多醣體對肝臟疾病之影響 21
一、 枸杞多醣體簡介 21
二、 枸杞多醣體之肝臟相關研究 23
第六節 研究目的與動機 25
第三章 實驗材料與方法 26
第一節 實驗設計 26
一、 實驗動物 26
二、 實驗設計 26
三、 飲食中的油脂 29
四、 枸杞多醣萃取物 32
第二節 測定項目與方法 35
一、 肝臟切片 35
二、 肝功能指標 36
三、 血漿與肝臟脂質濃度 37
四、 肝臟細胞凋亡指標 40
五、 肝臟發炎反應指標 41
六、 肝臟纖維化指標 43
第三節 統計分析 47
第四章 結果 48
第一節 大鼠體重、肝重與攝食量 48
第二節 肝功能指標 50
第三節 血漿與肝臟脂質成分 52
第四節 肝臟切片病理學檢查 54
第五節 肝臟細胞凋亡指標 58
第六節 肝臟發炎指標 60
第七節 肝臟纖維化指標 63
第五章 討論 69
第一節 以CCl4動物模式模擬人體之肝纖維化 69
第二節 CCl4對肝重與肝功能之影響 71
第三節 CCl4對脂質代謝之影響 72
第四節 橄欖油對肝臟細胞凋亡與發炎反應的影響 74
第五節 LBE對肝臟細胞凋亡與發炎反應的影響 76
第六節 橄欖油與LBE對肝臟纖維化指標的影響 78
第七節 綜合討論 83
第六章 結論 87
第七章 參考資料 88
附件一 實驗動物照護及使用委員會審查同意書 95
參考文獻 Adachi M, Osawa Y, Uchinami H, Kitamura T, Accili D, Brenner DA (2007) The forkhead transcription factor FoxO1 regulates proliferation and transdifferentiation of hepatic stellate cells. Gastroenterology 132: 1434-1446.
Afdhal NH (2004) Biopsy or biomarkers: is there a gold standard for diagnosis of liver fibrosis? Clin chem 50: 1299-1300.
Arthur MJ (2000) Fibrogenesis II. Metalloproteinases and their inhibitors in liver fibrosis. Am J Physiol Gastrointest Liver Physiol 279: G245-G249.
Assmann G, Schriewer H, Schmitz G, Hägele E (1983) Quantification of high-density-lipoprotein cholesterol by precipitation with phosphotungstic acid/MgCl2. Clin chem 29: 2026-2030.
Barcellos-Hoff M, Dix TA (1996) Redox-mediated activation of latent transforming growth factor-beta 1. Molecular endocrinology 10: 1077-1083.
Bataller R, Brenner DA (2005) Liver fibrosis. J Clin Invest 115: 209-218.
Beltrán G, del Rio C, Sánchez S, Martínez L (2004) Influence of harvest date and crop yield on the fatty acid composition of virgin olive oils from cv. Picual. J Agric Food Chem 52: 3434-3440.
Blouin A, Bolender RP, Weibel ER (1977) Distribution of organelles and membranes between hepatocytes and nonhepatocytes in the rat liver parenchyma. A stereological study. J Cell Biol 72: 441-455.
Boigk G, Stroedter L, Herbst H, Waldschmidt J, Riecken EO, Schuppan D (1997) Silymarin retards collagen accumulation in early and advanced biliary fibrosis secondary to complete bile duct obliteration in rats. Hepatology 26: 643-649.
Boll M, Weber LW, Becker E, Stampfl A (2001) Pathogenesis of carbon tetrachloride-induced hepatocyte injury bioactivation of CCl4 by cytochrome P450 and effects on lipid homeostasis. Z Naturforsch 56: 111-121.
Chao JC, Li B-Y (2017) Effects of Lycium Barbarum polysaccharides extract and betaine on cell activation and liver fibrosis in rat hepatic stellate HSC-T6 cells. FASEB J 31: 793-795.
Charlton M, Krishnan A, Viker K, Sanderson S, Cazanave S, McConico A, Masuoko H, Gores G (2011) Fast food diet mouse: novel small animal model of NASH with ballooning, progressive fibrosis, and high physiological fidelity to the human condition. Am J Physiol Gastrointest Liver Physiol 301: G825-G834.
Chen Z, Kwong Huat Tan B, Chan SH (2008) Activation of T lymphocytes by polysaccharide-protein complex from Lycium barbarum L. Int Immunopharmacol 8: 1663-1671.
Cheng D, Kong H (2011) The effect of Lycium barbarum polysaccharide on alcohol-induced oxidative stress in rats. Molecules 16: 2542-2550.
Cheng J, Zhou ZW, Sheng HP, He LJ, Fan XW, He ZX, Sun T, Zhang X, Zhao RJ, Gu L, Cao C, Zhou SF (2015) An evidence-based update on the pharmacological activities and possible molecular targets of Lycium barbarum polysaccharides. Drug Des Devel Ther 9: 33-78.
Cicerale S, Lucas L, Keast R (2010) Biological activities of phenolic compounds present in virgin olive oil. International journal of molecular sciences 11: 458-479.
Corless JK, Middleton HM (1983) Normal liver function: A basis for understanding hepatic disease. Arch Intern Med 143: 2291-2294.
Cui B, Liu S, Lin X, Wang J, Li S, Wang Q, Li S (2011) Effects of Lycium barbarum aqueous and ethanol extracts on high-fat-diet induced oxidative stress in rat liver tissue. Molecules 16: 9116-9128.
Domenicali M, Caraceni P, Giannone F, Baldassarre M, Lucchetti G, Quarta C, Patti C, Catani L, Nanni C, Lemoli RM (2009) A novel model of CCl 4-induced cirrhosis with ascites in the mouse. J Hepatol 51: 991-999.
Eitel K, Staiger H, Brendel MD, Brandhorst D, Bretzel RG, Häring H-U, Kellerer M (2002) Different role of saturated and unsaturated fatty acids in β-cell apoptosis. Biochem Biophys Res Commun 299: 853-856.
El–Serag HB, Rudolph KL (2007) Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 132: 2557-2576.
Fang HL, Lai JT, Lin WC (2008) Inhibitory effect of olive oil on fibrosis induced by carbon tetrachloride in rat liver. Clin Nutr 27: 900-907.
Fernandez M, Torres M, Gil A, Rios A (1997) Steatosis and collagen content in experimental liver cirrhosis are affected by dietary monounsaturated and polyunsaturated fatty acids. Scand J Gastroent 32: 350-356.
Ferramosca A, Savy V, Zara V (2008) Olive oil increases the hepatic triacylglycerol content in mice by a distinct influence on the synthesis and oxidation of fatty acids. Biosci Biotechnol Biochem 72: 62-69.
Ferramosca A, Zara V (2014) Modulation of hepatic steatosis by dietary fatty acids. World J Gastroenterol 20: 1746-1755.
Friedewald WT, Levy RI, Fredrickson DS (1972) Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin chem 18: 499-502.
Friedman SL (2003) Liver fibrosis – from bench to bedside. J Hepatol 38: 38-53.
Fulda S, Debatin KM (2006) Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25: 4798-4811.
Gao B, Jeong WI, Tian Z (2008) Liver: an organ with predominant innate immunity. Hepatology 47: 729-736.
Giannandrea M, Parks WC (2014) Diverse functions of matrix metalloproteinases during fibrosis. Dis Model Mech 7: 193-203.
Gressner AM, Weiskirchen R, Breitkopf K, Dooley S (2002) Roles of TGF-beta in hepatic fibrosis. Front Biosci 7: d793-807.
Guo X, Li H, Xu H, Halim V, Zhang W, Wang H, Ong KT, Woo S-L, Walzem RL, Mashek DG (2012) Palmitoleate induces hepatic steatosis but suppresses liver inflammatory response in mice. PLoS One 7: e39286.
Halfon P, Munteanu M, Poynard T (2008) FibroTest-ActiTest as a non-invasive marker of liver fibrosis. Gastroenterologie clinique et biologique 32: 22-39.
Hartland SN, Murphy F, Aucott RL, Abergel A, Zhou X, Waung J, Patel N, Bradshaw C, Collins J, Mann D, Benyon RC, Iredale JP (2009) Active matrix metalloproteinase-2 promotes apoptosis of hepatic stellate cells via the cleavage of cellular N-cadherin. Liver Int 29: 966-978.
Hashem RM, Mahmoud MF, El-Moselhy MA, Soliman HM (2008) Interleukin-10 to tumor necrosis factor-alpha ratio is a predictive biomarker in nonalcoholic fatty liver disease: interleukin-10 to tumor necrosis factor-alpha ratio in steatohepatitis. Eur J Gastroenterol Hepatol 20: 995-1001.
Hellerbrand C, Stefanovic B, Giordano F, Burchardt ER, Brenner DA (1999) The role of TGFβ1 in initiating hepatic stellate cell activation in vivo. J Hepatol 30: 77-87.
Hemmann S, Graf J, Roderfeld M, Roeb E (2007) Expression of MMPs and TIMPs in liver fibrosis - a systematic review with special emphasis on anti-fibrotic strategies. J Hepatol 46: 955-975.
Hernández R, Martínez-Lara E, Cañuelo A, del Moral ML, Blanco S, Siles E, Jiménez A, Pedrosa JÁ, Peinado MÁ (2005) Steatosis recovery after treatment with a balanced sunflower or olive oil-based diet: involvement of perisinusoidal stellate cells. World J Gastroenterol 11: 7480.
Hernandez-Gea V, Friedman SL (2011) Pathogenesis of liver fibrosis. Annu Rev Pathol 6: 425-456.
Hussein O, Grosovski M, Lasri E, Svalb S, Ravid U, Assy N (2007) Monounsaturated fat decreases hepatic lipid content in non-alcoholic fatty liver disease in rats. World J Gastroenterol 13: 361.
Inagaki Y, Okazaki I (2007) Emerging insights into transforming growth factor β Smad signal in hepatic fibrogenesis. Gut 56: 284-292.
Jump DB (2011) Fatty acid regulation of hepatic lipid metabolism. Curr Opin Clin Nutr Metab Care 14: 115-120.
Knittel T, Mehde M, Grundmann A, Saile B, Scharf J-G, Ramadori G (2000) Expression of matrix metalloproteinases and their inhibitors during hepatic tissue repair in the rat. Histochem Cell Biol 113: 443-453.
Larter CZ, Yeh MM, Van Rooyen DM, Teoh NC, Brooling J, Hou JY, Williams J, Clyne M, Nolan CJ, Farrell GC (2009) Roles of adipose restriction and metabolic factors in progression of steatosis to steatohepatitis in obese, diabetic mice. J Gastroenterol Hepatol 24: 1658-1668.
Leamy AK, Egnatchik RA, Young JD (2013) Molecular mechanisms and the role of saturated fatty acids in the progression of non-alcoholic fatty liver disease. Prog Lipid Res 52: 165-174.
Lee JY, Moon JH, Park JS, Lee BW, Kang ES, Ahn CW, Lee HC, Cha BS (2011) Dietary oleate has beneficial effects on every step of non-alcoholic Fatty liver disease progression in a methionine- and choline-deficient diet-fed animal model. Diabetes Metab J 35: 489-496.
Li ZZ, Berk M, McIntyre TM, Feldstein AE (2009) Hepatic lipid partitioning and liver damage in nonalcoholic fatty liver disease: role of stearoyl-CoA desaturase. J Biol Chem 284: 5637-5644.
Lin NC, Lin JC, Chen SH, Ho CT, Yeh AI (2011) Effect of Goji (Lycium barbarum) on expression of genes related to cell survival. J Agric Food Chem 59: 10088-10096.
Louis H, Van Laethem JL, Wu W, Quertinmont E, Degraef C, Van den Berg K, Demols A, Goldman M, Le Moine O, Geerts A (1998) Interleukin‐10 controls neutrophilic infiltration, hepatocyte proliferation, and liver fibrosis induced by carbon tetrachloride in mice. Hepatology 28: 1607-1615.
Nagaki M, Tanaka M, Sugiyama A, Ohnishi H, Moriwaki H (1999) Interleukin-10 inhibits hepatic injury and tumor necrosis factor-α and interferon-γ mRNA expression induced by staphylococcal enterotoxin B or lipopolysaccharide in galactosamine-sensitized mice. J Hepatol 31: 815-824.
Nigam P, Bhatt S, Misra A, Chadha DS, Vaidya M, Dasgupta J, Pasha QM (2014) Effect of a 6-month intervention with cooking oils containing a high concentration of monounsaturated fatty acids (olive and canola oils) compared with control oil in male Asian Indians with nonalcoholic fatty liver disease. Diabetes Technol Ther 16: 255-261.
Olaso E, Ikeda K, Eng FJ, Xu L, Wang L-H, Lin HC, Friedman SL (2001) DDR2 receptor promotes MMP-2–mediated proliferation and invasion by hepatic stellate cells. J Clin Investig 108: 1369-1378.
Pan X, Hussain FN, Iqbal J, Feuerman MH, Hussain MM (2007) Inhibiting proteasomal degradation of microsomal triglyceride transfer protein prevents CCl4-induced steatosis. J Biol Chem 282: 17078-17089.
Portillo MaP, Chávarri Ma, Durán D, Rodrı́guez VcM, Macarulla MT (2001) Differential effects of diets that provide different lipid sources on hepatic lipogenic activities in rats under ad libitum or restricted feeding. Nutrition 17: 467-473.
Potterat O (2010) Goji (Lycium barbarum and L. chinense): Phytochemistry, pharmacology and safety in the perspective of traditional uses and recent popularity. Planta Med 76: 7-19.
Rappaport A, Borowy Z, Lougheed W, Lotto W (1954) Subdivision of hexagonal liver lobules into a structural and functional unit. Role in hepatic physiology and pathology. Anat Rec 119: 11-33.
Roeb E, Purucker E, Breuer B, Nguyen H, Heinrich PC, Rose-John S, Matern S (1997) TIMP expression in toxic and cholestatic liver injury in rat. J Hepatol 27: 535-544.
Schuppan D, Ruehl M, Somasundaram R, Hahn EG (2001) Matrix as a modulator of hepatic fibrogenesis. Semin Liver Dis 21: 351-372.
Shackelford C, Long G, Wolf J, Okerberg C, Herbert R (2002) Qualitative and quantitative analysis of nonneoplastic lesions in toxicology studies. Toxicol Pathol 30: 93-96.
Shah R, Reyes-Gordillo K, Arellanes-Robledo J, Lechuga CG, Hernandez-Nazara Z, Cotty A, Rojkind M, Lakshman MR (2013) TGF-beta1 up-regulates the expression of PDGF-beta receptor mRNA and induces a delayed PI3K-, AKT-, and p70 (S6K)-dependent proliferative response in activated hepatic stellate cells. Alcohol Clin Exp Res 37: 1838-1848.
Shi J, Aisaki K, Ikawa Y, Wake K (1998) Evidence of hepatocyte apoptosis in rat liver after the administration of carbon tetrachloride. Am J Pathol 153: 515-525.
Simeonova PP, Gallucci RM, Hulderman T, Wilson R, Kommineni C, Rao M, Luster MI (2001) The role of tumor necrosis factor-α in liver toxicity, inflammation, and fibrosis induced by carbon tetrachloride. Toxicol Appl Pharmacol 177: 112-120.
Starkel P, Leclercq IA (2011) Animal models for the study of hepatic fibrosis. Best Pract Res Clin Gastroenterol 25: 319-333.
Tanaka N, Kono H, Ishii K, Hosomura N, Fujii H (2009) Dietary olive oil prevents carbon tetrachloride-induced hepatic fibrosis in mice. J Gastroenterol 44: 983-990.
Taniguchi M, Takeuchi T, Nakatsuka R, Watanabe T, Sato K (2004) Molecular process in acute liver injury and regeneration induced by carbon tetrachloride. Life Sci 75: 1539-1549.
Tsukamoto H, Towner SJ, Clofalo LM, French SW (1986) Ethanol‐induced liver fibrosis in rats fed high fat diet. Hepatology 6: 814-822.
Tuck KL, Hayball PJ (2002) Major phenolic compounds in olive oil: metabolism and health effects. J Nutr Biochem 13: 636-644.
Verspaget HW, Kuyvenhoven JP, Sier CF, van Hoek B. 2006. Matrix metalloproteinases in chronic liver disease and liver transplantation. Proteases in Gastrointestinal Tissues. Springer: 209-234.
Weber LW, Boll M, Stampfl A (2003) Hepatotoxicity and mechanism of action of haloalkanes: carbon tetrachloride as a toxicological model. Crit Rev Toxicol 33: 105-136.
Wisse E, Braet F, Luo D, De Zanger R, Jans D, Crabbe E, Vermoesen A (1996) Structure and function of sinusoidal lining cells in the liver. Toxicol Pathol 24: 100-111.
Wu H-T, He X-J, Hong Y-K, Ma T, Xu Y-P, Li H-H (2010) Chemical characterization of Lycium barbarum polysaccharides and its inhibition against liver oxidative injury of high-fat mice. Int J Biol Macromol 46: 540-543.
Wu S-J, Lin Y-H, Chu C-C, Tsai Y-H, Chao JC-J (2008) Curcumin or saikosaponin a improves hepatic antioxidant capacity and protects against CCl4-induced liver injury in rats. J Med Food 11: 224-229.
Xiao J, Liong E, Ching Y, Chang R, Fung M, Xu A, So K, Tipoe G (2013) Lycium barbarum polysaccharides protect rat liver from non-alcoholic steatohepatitis-induced injury. Nutr Diabetes 3: e81.
Xiao J, Liong EC, Ching YP, Chang RC, So KF, Fung ML, Tipoe GL (2012) Lycium barbarum polysaccharides protect mice liver from carbon tetrachloride-induced oxidative stress and necroinflammation. J Ethnopharmacol 139: 462-470.
Yang YM, Seki E (2015) TNFalpha in liver fibrosis. Curr Pathobiol Rep 3: 253-261.
Yuan Y, Wang YB, Jiang Y, Prasad KN, Yang J, Qu H, Wang Y, Jia Y, Mo H, Yang B (2016) Structure identification of a polysaccharide purified from Lycium barbarium fruit. Int J Biol Macromol 82: 696-701.
Zhang X, Zhou W, Zhang Y. 2015. Immunoregulation and Lycium Barbarum. Lycium Barbarum and Human Health. Springer: 27-44.
Zhou X, Hovell CJ, Pawley S, Hutchings MI, Arthur MJ, Iredale JP, Benyon RC (2004) Expression of matrix metalloproteinase-2 and -14 persists during early resolution of experimental liver fibrosis and might contribute to fibrolysis. Liver Int 24: 492-501.
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