簡易檢索 / 詳目顯示

研究生: 郭彥廷
Kuo, Yen-Ting
論文名稱: 海藻糖芭樂汁對第二型尿病大鼠之腎臟及胰的保護效應
Protective Effects Effects of Guava (Psidium guajava) Juice Combination with Trehalose in Kidney and Pancreas in T2DM rats
指導教授: 鄭劍廷
Chien, Chiang-Ting
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2015
畢業學年度: 103
語文別: 英文
論文頁數: 88
中文關鍵詞: 芭樂海藻糖第二型糖尿病氧化壓力發炎
英文關鍵詞: Guava, Trehalose, Type II diabetes, oxidative stress, inflammation
論文種類: 學術論文
相關次數: 點閱:169下載:10
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報
  • 第二型糖尿病(Type 2 diabetes, T2DM)是目前廣泛流行的代謝症候群。研究預測第二型糖尿病的人口數,在西元2030年時會攀升至3.66億。芭樂 (Psidium guajava),被發現具有抗氧化(anti-oxidation)、抗發炎(anti-inflammation)及抗糖尿病(anti-diabetes)的特性。並且也被發現在第一型糖尿病的大鼠上,可以保護胰臟的β細胞,免於受到氧化壓力損害。然而,很少研究是利用芭樂的可食部位探討其對第二型糖尿病之間的作用機制。所以我們利用芭樂可食部位製成芭樂汁(40%),以研究此主題。為了提升芭樂汁的適口性,我們在果汁當中添加了海藻糖(Trehalose)。海藻糖是一種雙醣,目前已被應用為細胞冷凍保存的工具。先前的研究指出它可以防止阿茲海默症中,類澱粉蛋白的累積。以及亨丁頓氏舞蹈症中,多聚谷氨酰胺的形成而產生抗氧化之作用。本研究之主要目的在評估芭樂汁(40%)添加海藻糖(5%)對於第二型糖尿病大鼠在腎臟與胰臟的保護作用效應。
    利用Nicotinamide (NA)及Streptozotocin (STZ)腹腔注射以誘發 Wistar品系雌性大鼠第二型糖尿病。誘發成功後,分為六組進行實驗,分別為CON, DM, T1, T2, T5和B1。每日灌餵芭樂汁,連續四周。灌餵之劑量如下: T1, T2, T5: 4, 8, 20 ml/kg BW芭樂汁含5%海藻糖。 B1: 4 ml/kg BW芭樂汁不含5% 海藻糖。紀錄葡萄糖耐受性試驗(OGTT)、血清胰島素、糖化血色素與換算之胰島素阻抗和分泌量。並測量腎臟活體自由基,而後進行犧牲,收取組織進行免疫組織染色、螢光染色、西方墨點法及離體血清自由基測試。我們亦以LC/MS的方式定量芭樂有效成份。
    結果顯示,芭樂汁中含有高量的槲皮素,且槲皮素與芭樂汁可清除H2O2 and HOCl。而本研究也發現,海藻糖可清除H2O2,但無法清除HOCl。第二型糖尿病(DM組)會增加大鼠之氧化壓力及發炎反應。相較之下,T1, T2, T5組在灌餵海藻糖芭樂汁之後,表現出較低程度的氧化壓力及發炎指標,如IL-1β, Caspase 3及4-HNE。第二型糖尿病(DM組)增加胰島素阻抗和降低胰島素分泌量。相較之下,T1, T2, T5組在灌餵海藻糖芭樂汁之後,會降低第二型糖尿病(DM組)所增加之腎臟和胰臟氧化壓力及發炎反應包括降低IL-1β, Caspase 3 及4-HNE之表現。而且會降低胰島素阻抗和部份增加胰島素分泌量。此外,我們發現在離體血清自由基測試中,B1組的血清自由基較T1, T2, T5組高,其中T2及T5組統計達顯著差異(P < 0.05)。免疫組織染色及螢光染色結果也有相同趨勢。這結果表示海藻糖芭樂汁對於腎臟及胰臟的保護功效,較單獨芭樂汁佳。
    總結,海藻糖可以提升芭樂汁在第二型糖尿病中的保護功效。將兩者合併攝取,可降低腎臟及胰臟的氧化壓力及發炎反應。

    Type II diabetes is one of the most epidemic metabolic syndrome. It was predicted that people with T2DM would rise to 366 million in 2030. Guava (Psidium guajava) has been reported to provide anti-oxidation, anti-inflammation, and anti-diabetes. Besides, it protected β cells from the damage of oxidative stress in type I DM rats. However, there were few studies which report the mechanism between the edible proportion of guava and the T2DM. Therefore, we utilized guava juice to investigate its effect on T2DM. To improve the palatability, we added one kind of sugar, trehalose. Trehalose is a disaccharide, which has been used in cryopreservation of cells. In addition, it was found to avoid β-amyloid formation and polyglutamine (polyQ) in Alzheimer disease and Huntington’s disease indicating its protective function. This study is to discover the protective mechanism of guava juice (40%) combination with trehalose (5%) on the pathophysiology of kidney and pancreas in T2DM rats.
    T2DM was induced in female Wistar rats by intraperitoneal administration of nicotinamide and streptozotocin and combination with high fructose diets for 8 weeks. After successful induction (> 230 mg/dL), the rats were divided into 6 groups, CON, DM, T1, T2, T5, B1, and were fed with different dosage of guava juice combination with or without trehalose for 4 weeks (Dose: T1, T2, T5: 4, 8, 20 ml/kg BW guava juice with 5% trehalose; B1: 4 ml/kg BW guava juice without trehalose).OGTT, plasma insulin, HbA1c, Homeostasis model assessment of IR (HOMA-IR, an indicator of insulin resistance) and HOMA-β (an index of the function of β cell in pancreas and insulin secretion) were determined. We also measured the kidney reactive oxygen species (ROS) in vivo. The oxidative and inflammatory degrees were measured by immunohistochemistry stain, fluorescent stain, serum free radical value and western blotting. We also measured the active component of guava juice with LC/MS analysis.
    We found high content of quercetin existing in the guava juice. Quercetin and guava juice could scavenge H2O2 and HOCl, whereas trehalose can selectively reduce H2O2, not HOCl in the in vitro study. The results showed that the rats in group DM had elevated the degree of oxidative stress and inflammatory levels. In contrast, rats treated with oral intake of trehalose and guava juice in group T1, T2, T5 showed less expression of oxidative and inflammatory indicators, such as IL-1β, Caspase 3 and 4-HNE compared to DM group. Consistently, in the measurement of serum free radical levels, we found that rats in T1, T2 and T5 have significantly (P < 0.05) lower free radicals counts than B1 and DM groups. The results of immunohisotchemic and fluorescent stain also showed that oral intake of guava juice with trehalose in T1, T2, T5 rats had less (P < 0.05) oxidative damage, autophagy and apoptosis in the kidney and pancreas than B1 rats.
    In conclusion, trehalose supplement seems to provide the additively protective effect of guava juice in T2DM. Combination with trehalose and guava juice not only increases palatability, but also protects pancreas and kidney against oxidative and proinflammatory damages in T2DM.

    中文摘要 1 Abstract 4 Abbreviations 8 1. Introduction 10 1-1 Type 2 diabetes (T2DM) 10 1-2 Insulin insensitivity 10 1-3 Diabetic nephropathy 11 1-4 Reactive oxygen species (ROS) 11 1-5 Guava (Psidium guajava) 12 1-6 Trehalose 13 1-7 Goal 14 2. Methods and materials 15 2-1 Animals 15 2-2 Induction of T2DM 15 2-3 Animal Treatment 16 2-4 Determine Blood Glucose 17 2-5 Determine Insulin Levels 17 2-6 HOMA-IR and HOMA-β 18 2-7 Glucose Tolerance Test 18 2-8 Determine HbA1c 19 2-9 Metabolic Parameter 19 2-10 Assay of ROS Level 20 2-11 Immunohistochemistry (IHC) 21 2-12 Masson’s Stain 22 2-13 TUNEL Stain 23 2-14 Fluorescent Stain 25 2-15 Western Blot 26 2-16 Guava Extraction 27 2-17 LC/MS 28 3. Results 29 3-1 Guava Juice in vitro ROS Levels 29 3-2 Trehalose in vitro ROS Levels 29 3-3 Quercetin Content In Guava Extraction 29 3-4 Guava Extraction in vitro ROS Levels 30 3-5 Oral Guava Juice Tolerance Test 30 3-6 Intra Venous Glucose and Trehalose Tolerance Test 30 3-7 Oral Glucose Tolerance Test 31 3-8 Blood Glucose Changes 31 3-9 Insulin Levels 32 3-10 HOMA-IR 33 3-11 HOMA-β 33 3-12 HbA1c Levels 33 3-13 Metabolic Parameter 34 3-14 Renal in vivo ROS Levels 34 3-15 Serum in vitro ROS Levels 34 3-16 HE Stain 35 3-17 Masson’s Stain 35 3-18 Fluorescent Stain 36 3-19 IHC Stain 37 3-20 TUNEL Stain 38 3-21 Western Blot 39 4. Discussion 40 5. References 46 6. Figures and Tables 51

    Bazzano, L. A., Li, T. Y., Joshipura, K. J., & Hu, F. B. (2008). Intake of fruit, vegetables, and fruit juices and risk of diabetes in women. Diabetes care.
    Bieger, W. P., Michel, G., Barwich, D., Biehl, K., & Wirth, A. (1984). Diminished insulin receptors on monocytes and erythrocytes in hypertriglyceridemia. Metabolism, 33(11), 982-987.
    Butler, A. E., Janson, J., Bonner-Weir, S., Ritzel, R., Rizza, R. A., & Butler, P. C. (2003). β-cell deficit and increased β-cell apoptosis in humans with type 2 diabetes. Diabetes, 52(1), 102-110.
    Calcutt, N. A., Cooper, M. E., Kern, T. S., & Schmidt, A. M. (2009). Therapies for hyperglycaemia-induced diabetic complications: from animal models to clinical trials. Nature Reviews Drug Discovery, 8(5), 417-430.
    Chen, Q., & Haddad, G. G. (2004). Role of trehalose phosphate synthase and trehalose during hypoxia: from flies to mammals. Journal of Experimental Biology, 207(18), 3125-3129.
    Sarah, W., Gojka, R., Anders, G., Richard, S., & Hilary, K. (2004). Global prevalence of diabetes. Diabetes care, 27(5), 1047-1053.
    Chen, H. Y., & Yen, G. C. (2007). Antioxidant activity and free radical-scavenging capacity of extracts from guava (Psidium guajava L.) leaves. Food Chemistry, 101(2), 686-694.
    Chien, C. T., Lee, P. H., Chen, C. F., Ma, M. C., Lai, M. K., & Hsu, S. M. (2001). De novo demonstration and co-localization of free-radical production and apoptosis formation in rat kidney subjected to ischemia reperfusion.Journal of the American Society of Nephrology, 12(5), 973-982.
    Eidenberger, T., Selg, M., & Krennhuber, K. (2013). Inhibition of dipeptidyl peptidase activity by flavonol glycosides of guava (Psidium guajava L.): A key to the beneficial effects of guava in type II diabetes mellitus. Fitoterapia, 89, 74-79.
    Eroglu, A., Russo, M. J., Bieganski, R., Fowler, A., Cheley, S., Bayley, H., & Toner, M. (2000). Intracellular trehalose improves the survival of cryopreserved mammalian cells. Nature biotechnology, 18(2), 163-167.
    Flores, G., Dastmalchi, K., Wu, S. B., Whalen, K., Dabo, A. J., Reynertson, K. A., Foronjy, R. F., D’Armiento, J.M. & Kennelly, E. J. (2013). Phenolic-rich extract from the Costa Rican guava (Psidium friedrichsthalianum) pulp with antioxidant and anti-inflammatory activity. Potential for COPD therapy. Food chemistry, 141(2), 889-895.
    Huang, C. S., Yin, M. C., & Chiu, L. C. (2011). Antihyperglycemic and antioxidative potential of Psidium guajava fruit in streptozotocin-induced diabetic rats. Food and Chemical Toxicology, 49(9), 2189-2195.
    Hunyadi, A., Martins, A., Hsieh, T. J., Seres, A., & Zupkó, I. (2012). Chlorogenic acid and rutin play a major role in the in vivo anti-diabetic activity of Morus alba leaf extract on type II diabetic rats. PLOS ONE, e50619.
    Jha, J. C., Gray, S. P., Barit, D., Okabe, J., El-Osta, A., Namikoshi, T., Thallas-Bonke, V., Wingler, K., Szyndralewiez, C., Heitz, F., Touyz, R. M., Cooper, M. E., Schmidt, H. H. W. & Jandeleit-Dahm, K. A. (2014). Genetic targeting or pharmacologic inhibition of NADPH oxidase Nox4 provides renoprotection in long-term diabetic nephropathy. Journal of the American Society of Nephrology, ASN-2013070810.
    Kakehi, T., & Yabe-Nishimura, C. (2008, July). NOX enzymes and diabetic complications. In Seminars in immunopathology (Vol. 30, No. 3, pp. 301-314). Springer-Verlag.
    Kang, R., Zeh, H. J., Lotze, M. T., & Tang, D. (2011). The Beclin 1 network regulates autophagy and apoptosis. Cell Death & Differentiation, 18(4), 571-580.
    Liu, R., Barkhordarian, H., Emadi, S., Park, C. B., & Sierks, M. R. (2005). Trehalose differentially inhibits aggregation and neurotoxicity of beta-amyloid 40 and 42. Neurobiology of disease, 20(1), 74-81.
    Katsuki, A., Sumida, Y., Gabazza, E. C., Murashima, S., Furuta, M., Araki-Sasaki, R.,Hori, Y., Yano. Y. & Adachi, Y. (2001). Homeostasis model assessment is a reliable indicator of insulin resistance during follow-up of patients with type 2 diabetes. Diabetes care, 24(2), 362-365.
    Masiello, P., Broca, C., Gross, R., Roye, M., Manteghetti, M., Hillaire-Buys, D., Novelli, M. & Ribes, G. (1998). Experimental NIDDM: development of a new model in adult rats administered streptozotocin and nicotinamide. Diabetes, 47(2), 224-229.
    Masiello, P., Broca, C., Gross, R., Roye, M., Manteghetti, M., Hillaire-Buys, D., Novelli, M. & Ribes, G. (1998). Experimental NIDDM: development of a new model in adult rats administered streptozotocin and nicotinamide. Diabetes, 47(2), 224-229.
    Masini, M., Bugliani, M., Lupi, R., Del Guerra, S., Boggi, U., Filipponi, F., Marselli, L., Masiellom, P. & Marchetti, P. (2009). Autophagy in human type 2 diabetes pancreatic beta cells. Diabetologia, 52(6), 1083-1086.
    Molitch, M. E., Defronzo, R. A., Franz, M. J., Keane, W. F., Mogensen, C. E., Parving, H. H., & Steffes, M. W. (2004). Nephropathy in diabetes. Diabetes care, 27, S79-83.
    Nakamura, T., Terajima, T., Ogata, T., Ueno, K., Hashimoto, N., Ono, K., & Yano, S. (2006). Establishment and pathophysiological characterization of type 2 diabetic mouse model produced by streptozotocin and nicotinamide.Biological and Pharmaceutical Bulletin, 29(6), 1167-1174.
    Srinivasan, K., Viswanad, B., Asrat, L., Kaul, C. L., & Ramarao, P. (2005). Combination of high-fat diet-fed and low-dose streptozotocin-treated rat: a model for type 2 diabetes and pharmacological screening. Pharmacological Research, 52(4), 313-320.
    Sarkar, S., Davies, J. E., Huang, Z., Tunnacliffe, A., & Rubinsztein, D. C. (2007). Trehalose, a novel mTOR-independent autophagy enhancer, accelerates the clearance of mutant huntingtin and α-synuclein. Journal of Biological Chemistry, 282(8), 5641-5652.
    Stumvoll, M., Goldstein, B. J., & van Haeften, T. W. (2005). Type 2 diabetes: principles of pathogenesis and therapy. The Lancet, 365(9467), 1333-1346.
    Tas, S., Sarandol, E., Ayvalik, S. Z., Serdar, Z., & Dirican, M. (2007). Vanadyl sulfate, taurine, and combined vanadyl sulfate and taurine treatments in diabetic rats: effects on the oxidative and antioxidative systems. Archives of medical research, 38(3), 276-283.
    Xu, C., Li, X., Wang, F., Weng, H., & Yang, P. (2013). Trehalose prevents neural tube defects by correcting maternal diabetes-suppressed autophagy and neurogenesis. American Journal of Physiology-Endocrinology and Metabolism,305(5), E667-E678.
    Yadav, H., Jain, S., & Sinha, P. R. (2007). Antidiabetic effect of probiotic dahi containing Lactobacillus acidophilus and Lactobacillus casei in high fructose fed rats. Nutrition, 23(1), 62-68.
    Yoon, K. H., Lee, J. H., Kim, J. W., Cho, J. H., Choi, Y. H., Ko, S. H., ... & Son, H. Y. (2006). Epidemic obesity and type 2 diabetes in Asia. The Lancet,368(9548), 1681-1688.

    QR CODE