臺灣博碩士論文加值系統

非酒精性脂肪肝疾病(Nonalcoholic fatty liver disease，NAFLD)是因脂肪囤積而引起廣泛性肝臟細胞損傷疾病的統稱，此疾病已成為全球重要議題。研究指出NAFLD的發生與肥胖、第二型糖尿病及心血管疾病有關。當肝臟堆積過多脂肪時，產生胰島素阻抗及氧化壓力，使得單純性脂肪肝進展為肝炎、肝纖維化、肝硬化及肝癌，故減少脂肪合成及氧化壓力將有助於預防NAFLD的發生及進展。紅麴(Monascus)及葫蘆巴(Trigonella foenum-graecum)皆被證實有降血脂、改善胰島素阻抗及抗氧化的功效。本研究之目的為探討紅麴及葫蘆巴水萃混合物(MFW)延緩高脂飼料誘導NAFLD的效用。實驗採用雄性C57BL/6J小鼠，每天管餵去離子水或低、中或高劑量MFW。一週後開始配對餵食標準飼料(13%熱量為脂肪)或高脂飼料(60%熱量為脂肪)。管餵18週後，低及中劑量MFW顯著降低高脂飼料所增加的血清三酸甘油酯(TG)、游離脂肪酸(NEFA)及麩丙酮酸轉胺酶(ALT)活性，並降低肝臟TG含量。中及高劑量MFW進一步降低高脂飼料所增加的血膽固醇及肝臟膽固醇與NEFA含量，但僅有中劑量MFW顯著降低肝臟脂肪堆積及發炎指標p-NF-κB蛋白表現。然而，低劑量MFW顯著增加肝臟脂質合成酵素Acetyl-CoA carboxylase蛋白表現，而低、中及高劑量MFW未顯著影響高脂飼料改變之肝臟脂肪合成及脂肪氧化相關酵素、能量恆定代謝感受器腺苷單磷酸活化蛋白激酶(AMPK)的活化及細胞凋亡相關蛋白的表現。綜上所述，紅麴及葫蘆巴水萃混合物可藉由減少肝臟脂肪堆積及肝臟細胞發炎的情形以延緩高脂飲食引起之脂肪肝。

Nonalcoholic fatty liver disease (NAFLD), a spectrum of diseases with fat accumulation-induced liver damage, has become an important global issue. Studies showed that NAFLD is closely related to obesity, type 2 diabetes mellitus and cardiovascular disease.With excess fat accumulation, the hepatocytes develop insulin resistance followed by increased free fatty acids, oxidative stress, and steatosis, which subsequently result in the progression of NAFLD from simple steatosis to nonalcoholic steatohepatitis, and further to liver fibrosis, cirrhosis, and hepatocellular carcinoma. Monascus and fenugreek (Trigonella foenum graecum) have been demonstrated to have beneficial effects on reducing hyperlipidemia, improving insulin resistance and decreasing oxidative stress. Therefore, the aim of this study was to investigate the effects of Monascus and Trigonella foenum-graecum water extracted mixture alleviates high fat diet-induced nonalcoholic fatty liver in mice. Male C57BL/6J mice were intragastrically administered with deionized water (the NC and HF groups) or with a low, medium or high dose (the WL, WM, or WH groups) of Monascus and fenugreek water extracted mixture (MFW). After treated for 1 week, mice were started to pair-fed with a standard chow diet (13% calories from fat) for the NC group and a high-fat diet (60% calories from fat) for the other 4 groups. After 18 weeks of feeding, the results showed that the WL and WM MFW group had significantly decreased high fat diet-induced serum levels of triglyceride (TG), non‐esterified fatty acids (NEFA), alanine aminotransferase (ALT) activity and liver TG content. The WM and WH MFW group further had significantly decreased high fat diet-induced serum levels of cholesterol, liver cholesterol and NEFA content. But, only the WM MFW group had significantly decreased lipid accumulation status and the protein expression of phosphate nuclear factor kappa-light-chain-enhancer of activated B cell (p-NF-κB) of inflammatory responses in liver. However, the WL MFW group had significantly increased the protein expression of acetyl CoA carboxylase (ACC) of lipogenic enzymes; the WL, WM and WH MFW group had no significantly difference in high fat diet-induced imbalance of lipogenic and lipolysis enzymes, activation of AMP-activated protein kinase (AMPK) is a crucial cellular energy sensor and apoptotic proteins. In summary, Monascus and Trigonella foenum-graecum water extracted mixture may via decreased lipid accumulation status and liver inflammation to alleviate high fat diet-induced nonalcoholic fatty liver.

中文摘要 2
Abstract4
致謝6
目錄7
表目錄11
圖目錄12
縮寫13
第一章 序言15
第二章 文獻回顧17
第一節 非酒精性脂肪肝臟疾病17
一、致病機轉假說19
二、肝臟胰島素阻抗22
三、肝臟內生性脂肪合成(De novo lipogenesis, DNL)22
四、脂肪酸氧化(β-oxidation)23
五、能量恆定代謝25
六、脂質代謝異常25
七、脂毒性(Lipotoxicity)26
八、細胞凋亡(apoptosis)26
第二節 非酒精性脂肪肝之預防及治療27
一、藥物治療27
二、生活型態介入28
第三節 紅麴29
一、來源及有效成分29
二、生理活性30
第四節 葫蘆巴31
一、 來源及有效成分31
二、 生理活性31
第三章 目的與假說33
第四章 材料與方法35
第一節 實驗動物35
第二節 實驗設計35
一、實驗分組35
二、實驗流程36
三、實驗動物與人體間試驗劑量之換算36
第三節 實驗動物樣本採集37
一、體重與攝食量37
二、血液樣本37
三、肝臟及脂肪組織37
第四節 分析項目38
一、肝臟功能指標(Liver Function Index)38
二、血脂檢測39
三、肝臟組織病理分析41
四、肝臟脂肪含量分析41
五、肝臟組織蛋白質萃取42
六、肝臟組織細胞質及細胞核蛋白質定量43
七、西方墨點法(western blot)44
八、統計分析46
第五章 實驗結果48
第一節 動物體重、攝食和臟器重量分析48
一、體重與攝食量48
二、肝臟及副睪脂肪49
第二節 血液生化數值分析52
第三節 肝臟脂質含量58
第四節 肝臟組織型態60
第五節 肝臟中脂質代謝與能量恆定相關蛋白63
一、脂質與能量代謝相關蛋白63
二、肝臟中能量恆定相關蛋白63
第六節 肝臟中發炎及細胞凋亡相關蛋白67
一、肝臟中發炎相關蛋白67
二、肝臟中細胞凋亡相關蛋白67
第六章 討論 69
第一節高脂飼料誘導NAFLD的小鼠模式 69
第二節MFW對NAFLD小鼠體重及血液生化值的影響 71
第三節 MFW對NAFLD小鼠血液中肝損傷指標的影響 72
第四節 MFW對NAFLD小鼠肝臟脂肪堆積及脂質代謝的影響 73
一、肝臟脂肪堆積73
二、肝臟脂質代謝74
第五節 MFW對於NAFLD小鼠肝臟發炎反應及肝細胞凋亡的影響76
第六節 紅麴及葫蘆巴水萃混合物人類建議攝取量 77
第七章 結論78
第八章 參考資料79
附錄85

Abu-Elheiga, L., Matzuk, M. M., Abo-Hashema, K. A. & Wakil, S. J. (2001). Continuous fatty acid oxidation and reduced fat storage in mice lacking acetyl-CoA carboxylase 2. Science 291, 2613-2616.
Ahmadian, M., Suh, J. M., Hah, N., Liddle, C., Atkins, A. R., Downes, M. & Evans, R. M. (2013). PPARgamma signaling and metabolism: the good, the bad and the future. Nat Med 19, 557-566.
Ahmed, M. (2015). Non-alcoholic fatty liver disease in 2015. World J Hepatol 7, 1450-1459.
Ameer, F., Scandiuzzi, L., Hasnain, S., Kalbacher, H. & Zaidi, N. (2014). De novo lipogenesis in health and disease. Metabolism 63, 895-902.
Banini, B. A. & Sanyal, A. J. (2016). Nonalcoholic Fatty Liver Disease: Epidemiology, Pathogenesis, Natural History, Diagnosis, and Current Treatment Options. Clin Med Insights Ther 8, 75-84.
Baquer, N. Z., Kumar, P., Taha, A., Kale, R. K., Cowsik, S. M. & McLean, P. (2011). Metabolic and molecular action of Trigonella foenum-graecum (fenugreek) and trace metals in experimental diabetic tissues. J Biosci 36, 383-396.
Bellentani, S. (2017). The epidemiology of non-alcoholic fatty liver disease. Liver Int 37 Suppl 1, 81-84.
Berlanga, A., Guiu-Jurado, E., Porras, J. A. & Auguet, T. (2014). Molecular pathways in non-alcoholic fatty liver disease. Clin Exp Gastroenterol 7, 221-239.
Boden, G., She, P., Mozzoli, M., Cheung, P., Gumireddy, K., Reddy, P., Xiang, X., Luo, Z. & Ruderman, N. (2005). Free fatty acids produce insulin resistance and activate the proinflammatory nuclear factor-kappaB pathway in rat liver. Diabetes 54, 3458-3465.
Broca, C., Gross, R., Petit, P., Sauvaire, Y., Manteghetti, M., Tournier, M., Masiello, P., Gomis, R. & Ribes, G. (1999). 4-Hydroxyisoleucine: experimental evidence of its insulinotropic and antidiabetic properties. Am J Physiol 277, E617-623.
Browning, J. D. & Horton, J. D. (2004). Molecular mediators of hepatic steatosis and liver injury. J Clin Invest 114, 147-152.
Buzzetti, E., Pinzani, M. & Tsochatzis, E. A. (2016). The multiple-hit pathogenesis of non-alcoholic fatty liver disease (NAFLD). Metabolism 65, 1038-1048.
Carr, R. M., Oranu, A. & Khungar, V. (2016). Nonalcoholic Fatty Liver Disease: Pathophysiology and Management. Gastroenterol Clin North Am 45, 639-652.
Chalasani, N., Younossi, Z., Lavine, J. E., Diehl, A. M., Brunt, E. M., Cusi, K., Charlton, M. & Sanyal, A. J. (2012). The diagnosis and management of non-alcoholic fatty liver disease: practice Guideline by the American Association for the Study of Liver Diseases, American College of Gastroenterology, and the American Gastroenterological Association. Hepatology 55, 2005-2023.
Charrez, B., Qiao, L. & Hebbard, L. (2016). Hepatocellular carcinoma and non-alcoholic steatohepatitis: The state of play. World J Gastroenterol 22, 2494-2502.
Chen, C. H., Huang, M. H., Yang, J. C., Nien, C. K., Yang, C. C., Yeh, Y. H. & Yueh, S. K. (2006). Prevalence and risk factors of nonalcoholic fatty liver disease in an adult population of taiwan: metabolic significance of nonalcoholic fatty liver disease in nonobese adults. J Clin Gastroenterol 40, 745-752.
Chen, F. & Hu, X. (2005). Study on red fermented rice with high concentration of monacolin K and low concentration of citrinin. Int J Food Microbiol 103, 331-337.
Chen, Z., Lei, Y. L., Wang, W. P., Lei, Y. Y., Liu, Y. H., Hei, J., Hu, J. & Sui, H. (2017). Effects of Saponin from Trigonella Foenum-Graecum Seeds on Dyslipidemia. Iran J Med Sci 42, 577-585.
Cho, Y. S., Lee, J. I., Shin, D., Kim, H. T., Jung, H. Y., Lee, T. G., Kang, L. W., Ahn, Y. J., Cho, H. S. & Heo, Y. S. (2010). Molecular mechanism for the regulation of human ACC2 through phosphorylation by AMPK. Biochem Biophys Res Commun 391, 187-192.
Day, C. P. & James, O. F. (1998). Steatohepatitis: a tale of two "hits"? Gastroenterology 114, 842-845.
Donnelly, K. L., Smith, C. I., Schwarzenberg, S. J., Jessurun, J., Boldt, M. D. & Parks, E. J. (2005). Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease. J Clin Invest 115, 1343-1351.
Egan, M. & Prasad, S. (2011). PURLs: statins for patients with nonalcoholic fatty liver? J Fam Pract 60, 536-538.
Endo, A. (1979). Monacolin K, a new hypocholesterolemic agent produced by a Monascus species. J Antibiot (Tokyo) 32, 852-854.
Feldstein, A. E., Canbay, A., Angulo, P., Taniai, M., Burgart, L. J., Lindor, K. D. & Gores, G. J. (2003). Hepatocyte apoptosis and fas expression are prominent features of human nonalcoholic steatohepatitis. Gastroenterology 125, 437-443.
Fuller, S. & Stephens, J. M. (2015). Diosgenin, 4-hydroxyisoleucine, and fiber from fenugreek: mechanisms of actions and potential effects on metabolic syndrome. Adv Nutr 6, 189-197.
Guicciardi, M. E., Malhi, H., Mott, J. L. & Gores, G. J. (2013). Apoptosis and necrosis in the liver. Compr Physiol 3, 977-1010.
Guilherme, A., Virbasius, J. V., Puri, V. & Czech, M. P. (2008). Adipocyte dysfunctions linking obesity to insulin resistance and type 2 diabetes. Nat Rev Mol Cell Biol 9, 367-377.
Hardie, D. G., Ross, F. A. & Hawley, S. A. (2012). AMPK: a nutrient and energy sensor that maintains energy homeostasis. Nat Rev Mol Cell Biol 13, 251-262.
Hassan, K., Bhalla, V., El Regal, M. E. & HH, A. K. (2014). Nonalcoholic fatty liver disease: a comprehensive review of a growing epidemic. World J Gastroenterol 20, 12082-12101.
Ito, M., Suzuki, J., Tsujioka, S., Sasaki, M., Gomori, A., Shirakura, T., Hirose, H., Ito, M., Ishihara, A., Iwaasa, H. & Kanatani, A. (2007). Longitudinal analysis of murine steatohepatitis model induced by chronic exposure to high-fat diet. Hepatol Res 37, 50-57.
Kanda, T., Matsuoka, S., Yamazaki, M., Shibata, T., Nirei, K., Takahashi, H., Kaneko, T., Fujisawa, M., Higuchi, T., Nakamura, H., Matsumoto, N., Yamagami, H., Ogawa, M., Imazu, H., Kuroda, K. & Moriyama, M. (2018). Apoptosis and non-alcoholic fatty liver diseases. World J Gastroenterol 24, 2661-2672.
Kohjima, M., Enjoji, M., Higuchi, N., Kato, M., Kotoh, K., Yoshimoto, T., Fujino, T., Yada, M., Yada, R., Harada, N., Takayanagi, R. & Nakamuta, M. (2007). Re-evaluation of fatty acid metabolism-related gene expression in nonalcoholic fatty liver disease. Int J Mol Med 20, 351-358.
Kumar, P., Bhandari, U. & Jamadagni, S. (2014). Fenugreek seed extract inhibit fat accumulation and ameliorates dyslipidemia in high fat diet-induced obese rats. Biomed Res Int 2014, 606021.
Laurin, J., Lindor, K. D., Crippin, J. S., Gossard, A., Gores, G. J., Ludwig, J., Rakela, J. & McGill, D. B. (1996). Ursodeoxycholic acid or clofibrate in the treatment of non-alcohol-induced steatohepatitis: a pilot study. Hepatology 23, 1464-1467.
Lavine, J. E., Schwimmer, J. B., Van Natta, M. L., Molleston, J. P., Murray, K. F., Rosenthal, P., Abrams, S. H., Scheimann, A. O., Sanyal, A. J., Chalasani, N., Tonascia, J., Unalp, A., Clark, J. M., Brunt, E. M., Kleiner, D. E., Hoofnagle, J. H. & Robuck, P. R. (2011). Effect of vitamin E or metformin for treatment of nonalcoholic fatty liver disease in children and adolescents: the TONIC randomized controlled trial. JAMA 305, 1659-1668.
Lewis, G. F., Carpentier, A., Adeli, K. & Giacca, A. (2002). Disordered fat storage and mobilization in the pathogenesis of insulin resistance and type 2 diabetes. Endocr Rev 23, 201-229.
Lin, C. C., Li, T. C. & Lai, M. M. (2005). Efficacy and safety of Monascus purpureus Went rice in subjects with hyperlipidemia. Eur J Endocrinol 153, 679-686.
Liss, K. H. & Finck, B. N. (2017). PPARs and nonalcoholic fatty liver disease. Biochimie 136, 65-74.
Liu, J., Han, L., Zhu, L. & Yu, Y. (2016). Free fatty acids, not triglycerides, are associated with non-alcoholic liver injury progression in high fat diet induced obese rats. Lipids Health Dis 15, 27.
Liu, J., Zhang, J., Shi, Y., Grimsgaard, S., Alraek, T. & Fonnebo, V. (2006). Chinese red yeast rice (Monascus purpureus) for primary hyperlipidemia: a meta-analysis of randomized controlled trials. Chin Med 1, 4.
Lopez-Jimenez, M., Valenca, M. M., Moreira, A. C. & Antunes-Rodrigues, J. (1989). Ether and immobilization stress effects on pituitary adrenal function in hemidecorticate rats. Braz J Med Biol Res 22, 779-782.
Mandard, S., Muller, M. & Kersten, S. (2004). Peroxisome proliferator-activated receptor alpha target genes. Cell Mol Life Sci 61, 393-416.
McGarry, J. D. & Foster, D. W. (1980). Regulation of hepatic fatty acid oxidation and ketone body production. Annu Rev Biochem 49, 395-420.
Nagulapalli Venkata, K. C., Swaroop, A., Bagchi, D. & Bishayee, A. (2017). A small plant with big benefits: Fenugreek (Trigonella foenum-graecum Linn.) for disease prevention and health promotion. Mol Nutr Food Res 61.
Oh, T. S., Jeon, Y., Kim, S. & Kim, E. K. (2016). Hypothalamic AMP-activated Protein Kinase as a Regulator of Food Intake and Energy Balance. CNS Neurol Disord Drug Targets 15, 896-909.
Peverill, W., Powell, L. W. & Skoien, R. (2014). Evolving concepts in the pathogenesis of NASH: beyond steatosis and inflammation. Int J Mol Sci 15, 8591-8638.
Postic, C. & Girard, J. (2008). Contribution of de novo fatty acid synthesis to hepatic steatosis and insulin resistance: lessons from genetically engineered mice. J Clin Invest 118, 829-838.
Promrat, K., Kleiner, D. E., Niemeier, H. M., Jackvony, E., Kearns, M., Wands, J. R., Fava, J. L. & Wing, R. R. (2010). Randomized controlled trial testing the effects of weight loss on nonalcoholic steatohepatitis. Hepatology 51, 121-129.
Reddy, J. K. & Rao, M. S. (2006). Lipid metabolism and liver inflammation. II. Fatty liver disease and fatty acid oxidation. Am J Physiol Gastrointest Liver Physiol 290, G852-858.
Roberts, K. T. (2011). The potential of fenugreek (Trigonella foenum-graecum) as a functional food and nutraceutical and its effects on glycemia and lipidemia. J Med Food 14, 1485-1489.
Romero-Gomez, M., Zelber-Sagi, S. & Trenell, M. (2017). Treatment of NAFLD with diet, physical activity and exercise. J Hepatol 67, 829-846.
Ruderman, N. B., Carling, D., Prentki, M. & Cacicedo, J. M. (2013). AMPK, insulin resistance, and the metabolic syndrome. J Clin Invest 123, 2764-2772.
Rusu, E., Enache, G., Jinga, M., Dragut, R., Nan, R., Popescu, H., Parpala, C., Homentcovschi, C., Nitescu, M., Stoian, M., Costache, A., Posea, M., Rusu, F., Jinga, V., Mischianu, D. & Radulian, G. (2015). Medical nutrition therapy in non-alcoholic fatty liver disease--a review of literature. J Med Life 8, 258-262.
Smith, U. (2015). Abdominal obesity: a marker of ectopic fat accumulation. J Clin Invest 125, 1790-1792.
Stefan, N., Kantartzis, K. & Haring, H. U. (2008). Causes and metabolic consequences of Fatty liver. Endocr Rev 29, 939-960.
Steinberg, G. R., Michell, B. J., van Denderen, B. J., Watt, M. J., Carey, A. L., Fam, B. C., Andrikopoulos, S., Proietto, J., Gorgun, C. Z., Carling, D., Hotamisligil, G. S., Febbraio, M. A., Kay, T. W. & Kemp, B. E. (2006). Tumor necrosis factor alpha-induced skeletal muscle insulin resistance involves suppression of AMP-kinase signaling. Cell Metab 4, 465-474.
Tilg, H. & Moschen, A. R. (2010). Evolution of inflammation in nonalcoholic fatty liver disease: the multiple parallel hits hypothesis. Hepatology 52, 1836-1846.
Timlin, M. T. & Parks, E. J. (2005). Temporal pattern of de novo lipogenesis in the postprandial state in healthy men. Am J Clin Nutr 81, 35-42.
Van Herck, M. A., Vonghia, L. & Francque, S. M. (2017). Animal Models of Nonalcoholic Fatty Liver Disease-A Starter's Guide. Nutrients 9.
Viollet, B., Horman, S., Leclerc, J., Lantier, L., Foretz, M., Billaud, M., Giri, S. & Andreelli, F. (2010). AMPK inhibition in health and disease. Crit Rev Biochem Mol Biol 45, 276-295.
Wang, T. H. & Lin, T. F. (2007). Monascus rice products. Adv Food Nutr Res 53, 123-159.
Xiong, X., Wang, P., Li, X., Zhang, Y. & Li, S. (2017). The effects of red yeast rice dietary supplement on blood pressure, lipid profile, and C-reactive protein in hypertension: A systematic review. Crit Rev Food Sci Nutr 57, 1831-1851.
Younossi, Z. M., Koenig, A. B., Abdelatif, D., Fazel, Y., Henry, L. & Wymer, M. (2016). Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology 64, 73-84.
Zargar, S. (2014). Protective effect of Trigonella foenum-graecum on thioacetamide induced hepatotoxicity in rats. Saudi J Biol Sci 21, 139-145.
Zhou, Y., Ding, Y. L., Zhang, J. L., Zhang, P., Wang, J. Q. & Li, Z. H. (2018). Alpinetin improved high fat diet-induced non-alcoholic fatty liver disease (NAFLD) through improving oxidative stress, inflammatory response and lipid metabolism. Biomed Pharmacother 97, 1397-1408.