臺灣博碩士論文加值系統

肥胖的盛行率正逐年上升，它也被認為是代謝症候群的主要原因之一。番石榴葉含有豐富的酚類化合物，過去的研究顯示番石榴葉多酚 (Guava leaves polyphenol, GLP) 具有抗氧化、降血壓等功效。但目前尚未有GLP用於治療全身代謝性疾病的研究成果發表。因此，本研究之目的是探討GLP改善肥胖及全身性代謝性疾病的潛在機制。以正常小鼠肝細胞 (FL83B細胞) 利用油酸刺激72小時誘導脂質積累，然後給予GLP治療24小時，透過西方墨點法、油紅染色、螢光染色以及流式細胞儀評估GLP是否具有減少脂質積累並調節脂肪肝細胞的脂質代謝之能力。以小鼠纖維母細胞 (C2C12 細胞) 加入GLP共同培養48小時、再利用棕櫚酸刺激24小時誘導肌肉萎縮，透過西方墨點法評估GLP是否具有改善肌肉質量之作用。以5週齡C57BL/6小鼠適應環境2週，餵食高脂飼料4週誘導肥胖後，每週2次透過腹腔注射劑量為10及30 mg/kg 之GLP治療12週，經由西方墨點法、生化分析、免疫組織染色以及免疫螢光染色來評估GLP對改善肥胖、代謝性疾病和全身健康的效果及作用機轉。結果顯示，GLP減少油酸誘導的脂肪肝細胞之脂質合成，增加脂肪分解和脂肪酸β-氧化作用。動物實驗的結果顯示，GLP顯著減緩高脂飲食引起體重增加的速度並且改善肥胖小鼠代謝性脂肪肝病 (metabolic-associated fatty liver disease, MAFLD) 之程度，促進白色脂肪米色化及提升粒線體之功能。同時，GLP透過調節胰島素的葡萄糖轉運蛋白 (glucose transporters, GLUT) 之表現，促進全身組織對葡萄糖的利用和儲存來降低血液中葡萄糖的濃度、並且改善肥胖造成的肌肉質量衰退。此外，GLP經由恢復腸道黏膜的完整性與腸壁厚度，改善高脂飲食所造成的腸壁屏障損傷。

關鍵字：番石榴葉多酚、腸道屏障、代謝性脂肪肝、肥胖型肌少症、脂肪組織發炎反應

The prevalence of obesity is increasing year by year, and it is considered one of the main causes of metabolic syndrome. Guava leaves are rich in phenolic compounds and previous studies have shown that Guava leaves polyphenols (GLP) has antioxidant effects. However, there is no research results have been published on the use of GLP in the treatment of systemic metabolic diseases. Therefore, the aim of this study is to investigate the GLP potential mechanisms of improving systemic metabolic diseases. In vitro, normal mouse liver cell line (FL83B cells) are pre-stimulated with oleic acid for 72 hours to induce lipid accumulation and then treated with GLP for 24 hours, to assess GLP reduced lipid accumulation and regulated the lipids metabolism by western blot, oil red o stain, fluorescent staining, and flow cytometry. Mouse myoblast cell line (C2C12 cells) are co-cultured with GLP for 48 hours and then stimulated with palmitic acid for 24 hours to induce muscle atrophy, to assess GLP promoted muscle mass quality by western blot. In vivo, 5 weeks of C57BL/6 mice were adapted to the environment for 2 weeks, using high-fat diet (HFD) feed to induce obesity for 4 weeks, giving GLP treatment at the doses of 10 and 30 mg/kg by I.P twice a week for 12 weeks. Using the western blot, biochemistry, immunohistochemistry stain, and immunofluorescence stain to assess the effects of GLP on improving obesity、metabolic diseases and systemic health improvements. The results showed that GLP reduced lipid synthesis, increased lipolysis, and fatty acid β-oxidation in oleic acid-induced FL83B cells. In vivo, GLP significantly inhibited the rate of weight gain caused by high-fat diet and improved metabolic-associated fatty liver disease in obese mice, promoted the beigeization of white adipose and improved mitochondrial function. We also observed that GLP increased glucose transporters (GLUT) expression to reduce blood glucose concentration and improve muscle mass loss in HFD-induced mice. Furthermore, GLP improved intestinal barrier damage, restoring the integrity of the intestinal mucosa and intestinal wall thickness in HFD-induced obese mice.

Key words: Guava leaves polyphenols (GLP), Intestinal mucosal barrier, Metabolic-associated fatty liver disease (MAFLD), Sarcopenic Obesity, Adipose tissue inflammation

碩士學位論文指導教授推薦書
論文口試委員會審定書
誌謝 ii
英文縮寫 i
Abstract vi
目錄 viii
圖目錄 xiv
附圖目錄 - 1 -
附表目錄 - 2 -
第一節 前言 - 3 -
壹、肥胖 (Obesity) 的定義與盛行率 - 3 -
貳、代謝症候群 (Metabolic Syndrome) - 4 -
參、代謝性脂肪肝病 (Metabolic-associated fatty liver disease, MAFLD) - 5 -
肆、肝臟脂質合成 (Lipogenesis) - 6 -
伍、肝臟脂質分解 (Lipolysis) - 7 -
陸、肝臟脂肪酸β-oxidation - 7 -
柒、代謝性疾病與脂肪組織的影響 - 8 -
捌、腸道菌叢失調與腸漏症 (Gut Dysbiosis and Leaky Gut Syndrome) - 9 -
玖、肥胖型肌少症 (Sarcopenic Obesity) - 10 -
拾、骨骼肌萎縮與蛋白質合成 (Skeletal muscle atrophy and protein synthesis) - 12 -
拾壹、骨骼肌分化 (Skeletal muscle myogenesis) - 12 -
拾貳、番石榴葉多酚 (Guava leaves polyphenol) - 13 -
第二節 研究動機與目的 - 14 -
壹、研究動機 - 14 -
貳、研究目的 - 14 -
第二章 材料與方法 - 16 -
第一節細胞實驗 - 16 -
壹、細胞株 - 16 -
貳、續代培養 - 16 -
參、冷凍細胞 - 17 -
肆、番石榴葉多酚Guava leaves polyphenols (GLP) 配製 - 17 -
伍、油酸Oleic Acid (OA) 配製 - 17 -
陸、 棕櫚酸Palmitic acid (PA) 配置 - 17 -
柒、肝臟細胞實驗流程 - 18 -
捌、肝臟細胞實驗項目 - 18 -
玖、肌肉細胞實驗流程 - 19 -
拾、肌肉細胞實驗項目 - 19 -
第二節 動物實驗 - 19 -
壹、實驗動物品系 - 19 -
貳、動物實驗項目 - 20 -
參、動物實驗流程 - 20 -
第三節 實驗方法 - 21 -
壹、西方墨點法 (Western Blot) - 21 -
貳、油紅染色 (Oil-Red O stain) - 23 -
參、螢光染色 (Fluorescent staining) - 24 -
肆、細胞免疫螢光染色 (Immunofluorescence staining) - 25 -
伍、流式細胞儀 (Flow cytometry) - 26 -
陸、犧牲小鼠 - 27 -
柒、血清生化分析 - 27 -
捌、酵素結合免疫吸附分析法 (Enzyme-Linked Immunosorbent - 27 -
玖、萃取組織蛋白質 - 28 -
拾、免疫組織化學染色 (Immunohistochemical stain, IHC stain) - 28 -
拾壹、免疫組織螢光染色 (Immunofluorescence stain, IF stain) - 29 -
拾貳、蘇木精-伊紅染色 (Hematoxylin and Eosin Stain, H&E) - 29 -
拾參、肝醣染色試驗 (Periodic acid-Schiff stain, PAS stain) - 30 -
拾肆、腸道微生物群定序分析 - 30 -
拾伍、統計分析 - 31 -
第三章 結果 - 32 -
第一節 番石榴葉多酚調節油酸誘導FL83B肝細胞的脂質代謝作用 - 32 -
第二節 番石榴葉多酚減少肝細胞對油酸的攝入和脂質堆積 - 33 -
第三節 番石榴葉多酚改善高脂飲食誘導小鼠的肥胖程度以及血液生化分析數值 - 34 -
第四節 番石榴葉多酚改善高脂飲食誘導的代謝性脂肪肝病 - 35 -
第五節 番石榴葉多酚改善高脂飲食誘導肥胖小鼠肝臟葡萄糖代謝異常 - 36 -
第六節 番石榴葉多酚抑制高脂飲食誘導肥胖小鼠之脂肪組織的發炎反應 - 37 -
第七節 番石榴多酚促進肥胖小鼠脂肪組織的能量代謝功能 - 38 -
第八節 番石榴葉多酚改善C2C12肌母細胞的蛋白質合成作用 - 40 -
第九節 番石榴葉多酚改善C2C12肌母細胞的分化作用 - 40 -
第十節 番石榴葉多酚改善C2C12肌母細胞之萎縮作用 - 41 -
第十一節 番石榴葉多酚改善C2C12肌母細胞的再生作用 - 42 -
第十二節 番石榴葉多酚改善高脂飲食誘導肥胖小鼠的骨骼肌肉質量 - 42 -
第十三節 番石榴葉多酚改善高脂飲食誘導肥胖小鼠的骨骼肌肉萎縮 - 43 -
第十四節 番石榴葉多酚改善高脂飲食誘導肥胖小鼠的骨骼肌肉能量代謝 - 44 -
第十五節 番石榴葉多酚改善高脂飲食誘導肥胖小鼠的腸道屏障損傷及腸道菌失衡 - 45 -
第四章 討論 - 47 -
第一節 番石榴葉多酚改善高脂飲食誘導肥胖小鼠之體重增加 - 47 -
第二章 番石榴葉多酚改善肥胖引起的代謝性脂肪肝病 - 48 -
第三節 番石榴葉多酚改善肥胖小鼠組織對葡萄糖的攝入與利用 - 49 -
第四節 番石榴葉多酚改善因肥胖誘導的肌肉萎縮 - 50 -
第五節 番石榴葉多酚改善高脂飲食誘導肥胖小鼠的腸道損傷及腸道菌相F/B比值 - 51 -
第五章 結論 - 52 -
參考文獻 - 53 -
結果圖 - 58 -
附圖 - 111 -
附表 - 122 -

1.世界肥胖聯盟. 世界肥胖日. 2023; Available from: https://www.worldobesityday.org.
2.Kasper, P., et al., NAFLD and cardiovascular diseases: a clinical review. Clin Res Cardiol, 2021. 110(7): p. 921-937.
3.衛生福利部. 112年國人死因統計結果. 2024; Available from: https://www.mohw.gov.tw/cp-16-79055-1.html.
4.Saklayen, M.G., The Global Epidemic of the Metabolic Syndrome. Curr Hypertens Rep, 2018. 20(2): p. 12.
5.衛生福利部國民健康署, 代謝症候群. 2024.
6.Engin, A., The Definition and Prevalence of Obesity and Metabolic Syndrome. Adv Exp Med Biol, 2017. 960: p. 1-17.
7.Trefts, E., M. Gannon, and D.H. Wasserman, The liver. Curr Biol, 2017. 27(21): p. R1147-r1151.
8.Zhang, P., et al., Similarities and Differences: A Comparative Review of the Molecular Mechanisms and Effectors of NAFLD and AFLD. Front Physiol, 2021. 12: p. 710285.
9.Younossi, Z., et al., Global Perspectives on Nonalcoholic Fatty Liver Disease and Nonalcoholic Steatohepatitis. Hepatology, 2019. 69(6): p. 2672-2682.
10.Fernando, D.H., et al., Development and Progression of Non-Alcoholic Fatty Liver Disease: The Role of Advanced Glycation End Products. Int J Mol Sci, 2019. 20(20).
11.Fang, C., et al., The AMPK pathway in fatty liver disease. Front Physiol, 2022. 13: p. 970292.
12.Sánchez-García, F.J., et al., The Role of Tricarboxylic Acid Cycle Metabolites in Viral Infections. Front Cell Infect Microbiol, 2021. 11: p. 725043.
13.Kohjima, M., et al., SREBP-1c, regulated by the insulin and AMPK signaling pathways, plays a role in nonalcoholic fatty liver disease. Int J Mol Med, 2008. 21(4): p. 507-11.
14.Hofer, P., et al., The Lipolysome-A Highly Complex and Dynamic Protein Network Orchestrating Cytoplasmic Triacylglycerol Degradation. Metabolites, 2020. 10(4).
15.Wang, M., et al., Carnitine Palmitoyltransferase System: A New Target for Anti-Inflammatory and Anticancer Therapy? Front Pharmacol, 2021. 12: p. 760581.
16.Clifford, B.L., et al., FXR activation protects against NAFLD via bile-acid-dependent reductions in lipid absorption. Cell Metab, 2021. 33(8): p. 1671-1684.e4.
17.Chiang, J.Y.L. and J.M. Ferrell, Bile acid receptors FXR and TGR5 signaling in fatty liver diseases and therapy. Am J Physiol Gastrointest Liver Physiol, 2020. 318(3): p. G554-g573.
18.Cheng, L., et al., Brown and beige adipose tissue: a novel therapeutic strategy for obesity and type 2 diabetes mellitus. Adipocyte, 2021. 10(1): p. 48-65.
19.Hachemi, I. and U.D. M, Brown Adipose Tissue: Activation and Metabolism in Humans. Endocrinol Metab (Seoul), 2023. 38(2): p. 214-222.
20.Zhang, P., et al., Factors Associated with White Fat Browning: New Regulators of Lipid Metabolism. Int J Mol Sci, 2022. 23(14).
21.Li, X., et al., Adipose tissue macrophages as potential targets for obesity and metabolic diseases. Front Immunol, 2023. 14: p. 1153915.
22.Mills, C.D., et al., M-1/M-2 macrophages and the Th1/Th2 paradigm. J Immunol, 2000. 164(12): p. 6166-73.
23.Mily, A., et al., Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection. J Vis Exp, 2020(163).
24.Chae, Y.R., et al., Diet-Induced Gut Dysbiosis and Leaky Gut Syndrome. J Microbiol Biotechnol, 2024. 34(4): p. 747-756.
25.Aleman, R.S., M. Moncada, and K.J. Aryana, Leaky Gut and the Ingredients That Help Treat It: A Review. Molecules, 2023. 28(2).
26.Brunner, J., S. Ragupathy, and G. Borchard, Target specific tight junction modulators. Adv Drug Deliv Rev, 2021. 171: p. 266-288.
27.Stojanov, S., A. Berlec, and B. Štrukelj, The Influence of Probiotics on the Firmicutes/Bacteroidetes Ratio in the Treatment of Obesity and Inflammatory Bowel disease. Microorganisms, 2020. 8(11).
28.Frontera, W.R. and J. Ochala, Skeletal muscle: a brief review of structure and function. Calcif Tissue Int, 2015. 96(3): p. 183-95.
29.Akhmedov, D. and R. Berdeaux, The effects of obesity on skeletal muscle regeneration. Front Physiol, 2013. 4: p. 371.
30.Altajar, S. and G. Baffy, Skeletal Muscle Dysfunction in the Development and Progression of Nonalcoholic Fatty Liver Disease. J Clin Transl Hepatol, 2020. 8(4): p. 414-423.
31.Byun, K.A., et al., Dieckol-Attenuated High-Fat Diet Induced Muscle Atrophy by Modulating Muscular Deposition of Lipid Droplets. Nutrients, 2021. 13(9).
32.Richter, E.A. and M. Hargreaves, Exercise, GLUT4, and skeletal muscle glucose uptake. Physiol Rev, 2013. 93(3): p. 993-1017.
33.Yusuke, N., et al., Ubiquitin E3 ligases Atrogin-1 and MuRF1 protein contents are differentially regulated in the rapamycin-sensitive mTOR-S6K1 signaling pathway in C2C12 myotubes. bioRxiv, 2021: p. 2021.10.15.463676.
34.Jack, V.S., Effect of acute low oxygen exposure on the proliferation rate, viability and gene expression of C₂C₁₂ myoblasts in vitro. bioRxiv, 2020: p. 2020.07.09.162123.
35.Kumar, M., et al., Guava (Psidium guajava L.) Leaves: Nutritional Composition, Phytochemical Profile, and Health-Promoting Bioactivities. Foods, 2021. 10(4).
36.Prabu, G.R., A. Gnanamani, and S. Sadulla, Guaijaverin -- a plant flavonoid as potential antiplaque agent against Streptococcus mutans. J Appl Microbiol, 2006. 101(2): p. 487-95.
37.Verdam, F.J., et al., Human intestinal microbiota composition is associated with local and systemic inflammation in obesity. Obesity (Silver Spring), 2013. 21(12): p. E607-15.
38.Wong, M.L., et al., Inflammasome signaling affects anxiety- and depressive-like behavior and gut microbiome composition. Mol Psychiatry, 2016. 21(6): p. 797-805.
39.Ley, R.E., et al., Obesity alters gut microbial ecology. Proc Natl Acad Sci U S A, 2005. 102(31): p. 11070-5.
40.Turnbaugh, P.J., et al., An obesity-associated gut microbiome with increased capacity for energy harvest. Nature, 2006. 444(7122): p. 1027-31.
41.van der Heijden, R.A., et al., High-fat diet induced obesity primes inflammation in adipose tissue prior to liver in C57BL/6j mice. Aging (Albany NY), 2015. 7(4): p. 256-68.
42.Cutolo, M., et al., The Role of M1/M2 Macrophage Polarization in Rheumatoid Arthritis Synovitis. Front Immunol, 2022. 13: p. 867260.
43.Cheng, L., et al., Emodin Improves Glucose and Lipid Metabolism Disorders in Obese Mice via Activating Brown Adipose Tissue and Inducing Browning of White Adipose Tissue. Front Endocrinol (Lausanne), 2021. 12: p. 618037.
44.Argentato, P.P., et al., Programming mediated by fatty acids affects uncoupling protein 1 (UCP-1) in brown adipose tissue. Br J Nutr, 2018. 120(6): p. 619-627.
45.Geng, L., et al., Exercise Alleviates Obesity-Induced Metabolic Dysfunction via Enhancing FGF21 Sensitivity in Adipose Tissues. Cell Rep, 2019. 26(10): p. 2738-2752.e4.
46.Bodine, S.C., et al., Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo. Nat Cell Biol, 2001. 3(11): p. 1014-9.
47.Horiike, M., Y. Ogawa, and S. Kawada, Effects of hyperoxia and hypoxia on the proliferation of C2C12 myoblasts. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 2021. 321.
48.Xu, M., et al., FoxO1: a novel insight into its molecular mechanisms in the regulation of skeletal muscle differentiation and fiber type specification. Oncotarget, 2017. 8(6): p. 10662-10674.
49.Bodine, S.C. and L.M. Baehr, Skeletal muscle atrophy and the E3 ubiquitin ligases MuRF1 and MAFbx/atrogin-1. Am J Physiol Endocrinol Metab, 2014. 307(6): p. E469-84.
50.Sasaki, T., et al., The exercise-inducible bile acid receptor Tgr5 improves skeletal muscle function in mice. J Biol Chem, 2018. 293(26): p. 10322-10332.
51.Lauritzen, H.P. and J.D. Schertzer, Measuring GLUT4 translocation in mature muscle fibers. Am J Physiol Endocrinol Metab, 2010. 299(2): p. E169-79.
52.Kociszewska, D., et al., The Link between Gut Dysbiosis Caused by a High-Fat Diet and Hearing Loss. Int J Mol Sci, 2021. 22(24).
53.Organization, W.H., Obesity and overweight. 2024.
54.Corrêa, T.A. and M.M. Rogero, Polyphenols regulating microRNAs and inflammation biomarkers in obesity. Nutrition, 2019. 59: p. 150-157.
55.Ramaiah, P., et al., Dietary polyphenols and the risk of metabolic syndrome: a systematic review and meta-analysis. BMC Endocr Disord, 2024. 24(1): p. 26.
56.Zheng, Y., S.H. Ley, and F.B. Hu, Global aetiology and epidemiology of type 2 diabetes mellitus and its complications. Nat Rev Endocrinol, 2018. 14(2): p. 88-98.
57.Chatterjee, S., K. Khunti, and M.J. Davies, Type 2 diabetes. Lancet, 2017. 389(10085): p. 2239-2251.
58.Rohm, T.V., et al., Inflammation in obesity, diabetes, and related disorders. Immunity, 2022. 55(1): p. 31-55.
59.Matveyenko, A.V. and P.C. Butler, Relationship between beta-cell mass and diabetes onset. Diabetes Obes Metab, 2008. 10 Suppl 4(0 4): p. 23-31.
60.Alam, F., et al., Metabolic Control of Type 2 Diabetes by Targeting the GLUT4 Glucose Transporter: Intervention Approaches. Curr Pharm Des, 2016. 22(20): p. 3034-49.
61.Aldahish, A., et al., Elucidating the Potential Inhibitor against Type 2 Diabetes Mellitus Associated Gene of GLUT4. J Pers Med, 2023. 13(4).
62.Thorens, B., GLUT2, glucose sensing and glucose homeostasis. Diabetologia, 2015. 58(2): p. 221-32.
63.Iwai, S., et al., Branched Chain Amino Acids Promote ATP Production Via Translocation of Glucose Transporters. Invest Ophthalmol Vis Sci, 2022. 63(9): p. 7.
64.Lombardo, M., et al., Sarcopenic obesity: etiology and lifestyle therapy. Eur Rev Med Pharmacol Sci, 2019. 23(16): p. 7152-7162.
65.Merz, K.E. and D.C. Thurmond, Role of Skeletal Muscle in Insulin Resistance and Glucose Uptake. Compr Physiol, 2020. 10(3): p. 785-809.
66.Tu, H. and Y.L. Li, Inflammation balance in skeletal muscle damage and repair. Front Immunol, 2023. 14: p. 1133355.
67.Xie, Y., et al., Impact of a high‑fat diet on intestinal stem cells and epithelial barrier function in middle‑aged female mice. Mol Med Rep, 2020. 21(3): p. 1133-1144.
68.Quesada-Vázquez, S., et al., Microbiota Dysbiosis and Gut Barrier Dysfunction Associated with Non-Alcoholic Fatty Liver Disease Are Modulated by a Specific Metabolic Cofactors' Combination. Int J Mol Sci, 2022. 23(22).
69.Magne, F., et al., The Firmicutes/Bacteroidetes Ratio: A Relevant Marker of Gut Dysbiosis in Obese Patients? Nutrients, 2020. 12(5).