Effect of Teaghrelin on Dexamethasone-Induced Skeletal Muscle Atrophy

惡病質(Cachexia)為癌症及多種慢性病患者(如糖尿病、心臟衰竭、腎衰竭、慢性阻塞性肺病等)主要的併發症，主要病徵為肌肉量及脂肪組織的減少，造成體重大幅下降，嚴重影響病患的治療品質。飢餓素(Ghrelin) 主要是胃部分泌的多肽類荷爾蒙，於近年研究發現，其具有促進肌肉細胞分化及抑制骨骼肌肉萎縮的生理活性，為治療惡病質的方向之一。在青心烏龍中含有一種獨特的醯化黃酮醇配糖稱為-茶飢素(Teaghrelin)，於本實驗室先前的研究中已證明該成分和飢餓素有相似的生理活性，故推測茶飢素具有治療惡病質的潛力。本研究將茶飢素作用於小鼠骨骼肌肉細胞C2C12。首先透過未分化的C2C12，觀察細胞型態及檢測兩個分化相關蛋白之表達量，判斷茶飢素對於肌肉分化之效果。藉由計算細胞的延長指數(Elongation index)可表示細胞之分化程度。結果發現茶飢素(2.5x10-4 M)組別可促使細胞的延長指數增加20%，且顯著促進肌細胞生成素(Myogenin)與肌球蛋白重鍊(Myosin heavy chain)之表達，顯示茶飢素能夠有效促進C2C12的分化。另一方面，利用人工合成的糖皮質激素-地塞米松(Dexamethasone)使已分化的C2C12萎縮，建立肌肉萎縮的模式，透過觀察細胞型態及檢測兩個肌肉特異性泛素連接酶的表達量，作為肌肉萎縮嚴重程度之依據。發現茶飢素(2.5x10-5 M)組別可顯著恢復30%的肌肉束直徑大小，且有效抑制Atrogin-1及MuRF 1兩者泛素連接酶之表達。結果顯示，茶飢素有緩解肌肉萎縮的能力。根據上述實驗結果認為茶飢素有機會成為治療惡病質的策略之一。飢餓素對於許多臨床疾病的治療有重要的生理功效，而茶飢素也許會有相似的作用，可同樣應用於疾病治療上，為具有開發價值的天然飢餓素類似物之來源。Cachexia is a common complication during cancer, diabetes, cardiac failure, renal failure, liver failure, chronic obstructive pulmonary disorder, and several other disease state. Primarily symptom is losing of skeletal muscle and body fat, which leads to substantial weight loss. It also causes difficulties on cancer treatment. Ghrelin is a peptide hormone mainly released from stomach. Recent studies have demonstrated that ghrelin is a potential therapeutic way for cachexia. It promotes skeletal muscle cell differentiation and prevents lose muscle mass. Previously, our lab have discovered a unique acylated flavonoid tetraglycosides, Teaghrelin, in Chin-shin oolong tea and demonstrated this Ghrelin-like compound can mimic the bioactivity in vivo. Because ghrelin and Teaghrelin shows similar bioactivity, therefore the potential of Teaghrelin on cachexia treatment is worth evaluating. In this study, Teaghrelin was treated on mouse skeletal muscle cells, C2C12. Teaghrelin (2.5x10-4M) induce C2C12 myoblast differentiation in growth medium. The extent of cells differentiation was measured by elongation index under light microscopy. The elongation index of myoblast increased about 20% in Teaghrelin treatment group. The expression of two differentiation-related proteins, myogenin and myosin heavy chain also increased after 72h incubation. These results indicate that Teaghrelin significantly stimulate C2C12 skeletal myoblast cell differentiation. Moreover, we discovered Teaghrelin can attenuate dexamethasone-induced muscle atrophy from preliminary experiment. The fully differentiated C2C12 co-incubated with Teaghrelin and dexamethasone for 24h. Teaghrelin (2.5x10-5M) recovered myotube diameters about 30% and reduced the expression of two ubiquitin ligase, Atrogin-1 and MuRF1, which drive muscle protein degradation. We considered that Teaghrelin has a positive effect to prevent muscle atrophy and it may have potential to be a new treatments of cachexia.中文摘要 ...........................................i Abstract...........................................ii 目錄 ...........................................iii 圖目錄 ...........................................vi 表目錄 ...........................................vii 第一章 緒論........................................... 1 第二章 文獻回顧..........................................3 第一節 肌肉細胞的來源與生成 (Myogenesis).................3 一、骨骼肌 (Skeletal muscle).............................5 第二節 骨骼肌肉萎縮 (Skeletal muscle atrophy)............8 一、萎縮 (Atrophy).......................................8 二、肌肉降解路徑 (Muscle degradation pathway).............9 三、引發肌肉萎縮之模式藥物-地塞米松 (Dexamethasone)........11 第三節 飢餓素 (Ghrelin).................................13 一、飢餓素發現的歷史......................................13 二、飢餓素的結構與功能....................................13 三、飢餓素與飢餓素衍生物生理功能的影響......................15 四、人工合成藥物之發展.....................................16 第四節 茶葉主要功效性成分.................................17 一、茶葉的營養成分........................................17 二、茶葉的藥用成分........................................19 三、茶飢素的發現..........................................21 第三章 材料與方法........................................24 第一節 實驗試劑與材料....................................24 一、細胞株來源...........................................24 二、實驗藥品.............................................24 三、抗體.................................................26 四、實驗儀器..............................................27 第二節 實驗方法..........................................28 一、細胞培養常用溶劑......................................28 二、細胞培養步驟..........................................28 三、細胞繼代 (Subculture)................................29 四、細胞分化測試 (Cell differentiation)..................29 五、肌肉萎縮測試 (Muscle atrophy)........................30 六、西方墨點法 (Western blot)............................31 七、免疫染色法 (immunostaining)..........................33 八、統計分析.............................................34 第四章 結果..............................................35 第一節 肌肉生成..........................................35 一、顯微鏡檢測茶飢素對於C2C12細胞分化型態之改變.............35 二、西方墨點法分析茶飢素對於C2C12細胞分化標誌蛋白Myogenin和MHC之影響...................................................36 三、螢光免疫染色分析茶飢素對於C2C12細胞分化標誌蛋白MHC之影響..38 第二節 肌肉萎縮..........................................39 一、顯微鏡鏡檢測茶飢素對於C2C12肌管萎縮模式型態之改變........39 二、西方墨點法分析茶飢素對於萎縮的C2C12肌管萎縮標誌蛋白之影響 .........................................................40 第五章 討論...............................................41 第六章 圖表...............................................48 第七章 附錄...............................................59 第八章 引用文獻...........................................6