- 學位論文
限制飲食調控粒線體品質以預防骨骼肌老化
Dietary restriction attenuates skeletal muscle aging via manipulating mitochondrial quality control
摘要
骨骼肌佔體組成40-50%，且負責身體能量代謝與運動收縮功能，其中粒線體品質 (mitochondrial quality control, MQC) 調控了骨骼肌功能正常運作。MQC包括生合成 (biogenesis)、融合 (fusion)、分裂 (fission) 與粒線體自噬 (mitophagy) 等動態平衡 (dynamics)，但是隨著肌肉老化，MQC將逐漸下降，如粒線體動態失衡，堆積過多破碎化 (fragmentation) 粒線體造成肌纖維結構異常以及粒線體能量代謝速率下降等，這些因素會造成骨骼肌收縮能力降低與萎縮 (antrophy)。過去限制飲食 (dietary restriction, DR) 之研究雖證實能增加MQC，但DR可否藉改善骨骼肌MQC以減緩老化卻付之闕如，因此本研究將探討DR對老化過程骨骼肌MQC之影響，研究分為三部分。 第一階段試驗目的為了解骨骼肌老化過程之變化，試驗針對7 (青年)、14 (中年) 及19 (老年) 月齡C57BL/6J小鼠之股四頭肌 (quadriceps) 進行比較。試驗首先發現老年組相較另兩組之血糖與骨骼肌纖維化皆顯著上升 (p< 0.05)，而在電子顯微鏡下觀察到肌纖維結構異常與肌膜內粒線體趨向破碎化的現象，因此，實驗進一步比較老年與中年在MQC之差異，老年組之粒線體動態分裂蛋白指標 (Drp1) 下降 (p< 0.05)，融合蛋白指標 (Mfn2, Opa1) 也降低 (p< 0.05)，而粒線體總量 (VDAC) 則為顯著上升 (p< 0.05)。推論老化過程發生粒線體動態失衡與破碎化粒線體累積，進而引發骨骼肌纖維化與肌纖維異常。 第二階段試驗目的為探討DR的長度對於延緩骨骼肌老化之影響。C57BL/6J小鼠於3月齡開始為期11或16個月40% DR或任食 (ad libtium, AL)，發現DR可有效降低中年及老年組血糖、骨骼肌纖維化 (p< 0.05)。在中年組中，DR藉促進粒線體自噬 (PINK1, Parkin, LC3I) (p< 0.05)。而老年組中，DR能顯著降低氧化壓力 (TBARS)，減少骨骼肌內粒線體總量 (VDAC) 並提高粒線體動態平衡蛋白指標 (Drp1, Opa1) 與粒線體體積 (p< 0.05)。推測DR在不同年齡階段所調控MQC的方式不同，在中年以提升粒線體自噬為主，而老年則由維持粒線體動態平衡，使兩個階段保留代謝功能較佳之粒線體以延緩骨骼肌纖維化。 第三階段建立體外骨骼肌老化模型，進一步探討DR對老化肌肉粒線體功能之可能機制。試驗首先將C2C12肌原母細胞分化為肌管後藉氧化壓力誘發成為老化體外模型，後續添加薑黃素 (curcumin) 作為DR模擬物，探討DR如何調控細胞以延緩老化之分子機制。目前已知薑黃素能達到減緩細胞凋亡蛋白 (p53, p21)，進而減緩骨骼肌受老化之影響。 綜上所述，經由活體試驗，推測DR能透過促進粒線體自噬與粒線體動態平衡，使骨骼肌減少老化萎縮之影響，而DR調控粒線體之詳細機制仍待未來體外試驗證實。
並列摘要
Skeletal muscle is responsible for energy metabolism of body and exercise contraction, is which mitochondrial functions and their quality control (MQC). MQC are highly associated with muscles functionality, refers to the balance of mitochondrial biogenesis, fusion, fission and mitophagy, namely mitochondrial dynamics. During aging, MQC imbalance, such as dysfuction of mitochodonrial dynamics and accumulation of fragmented mitochondria, are related to decreased contraction of muscle fiber and antrophy. Past studies showed that dietary restriction (DR) cloud increases MQC, but the underlying associtation between aging and MQC by DR in skeletal muscle is still unknown. Therefore, this study explored the effect of DR on MQC in skeletal muscle along the progression of aging. The study is divided into three parts. The first experiment explored the changes of skeletal muscle with aging process, by comparing quadriceps femoris muscle of 7 (young), 14 (middle-aged) and 19 (elderly)-month old C57BL/6J mice levels. The elderly group exhibited a significant increase of blood glucose, fibrosis of skeletal muscle, when compared with the other two groups (p< 0.05). Besides, a tendency to accumulate abnormal structures of myofibrils and fragmentation of mitochondria in the sarcolemma of elderly group were observed. Therefore, the differences in MQC between the middle-aged and elderly mice were further analyzed. The mitochondrial dynamic fission (Drp1) and fusion (Mfn2, Opa1) protein were decreased in the elderly group (p< 0.05), whereas the total mitochondrial volume (VDAC) was significantly increased (p<0.05), suggesting that an imbalance of mitochondrial dynamics and accumulation of fragmented mitochondria occurre with aging, leading to fibrosis of skeletal muscle and myofibril abnormalities. The second experiment investigated the effect of DR on attenuating aging of skeletal muscle. C57BL/6J mice at 3 months of age were treated with middle (11 months) or long (16 months) period of DR (60% of ad libtium (AL). DR effectively reduced blood glucose levels and fibrosis of skeletal muscle in both middle-aged and elderly groups (p< 0.05). DR promoted mitophagy (PINK1, Parkin, LC3I) in the middle-aged mice (p< 0.05). Otherwise, DR significantly reduced oxidative stress (TBARS) and total mitochondrial volume (VDAC), but increased mitochondrial dynamics (Drp1, Opa1) and mitochondrial area (p< 0.05) in the skeletal muscle of elderly mice. These results suggest that DR exhibits different actions on MQC during the progress of aging. DR mainly promots mitophagy in middle-aged whereas maintained mitochondrial dynamics in elderly-age, and therefore, DR sustains functional mitochondria to delay fibrosis of skeletal muscle. The third experiment aimed to establish in vitro aging model of skeletal muscle, and explore the mechanism of DR on MQC of the aging muscles. C2C12 myoblasts were differentiated into myotubes, and induced for aging by oxidative stress reduction. Curcumin was used as DR mimic to explore the molecular mechanism of DR on regulating aging. Results showed that curcumin decreased the expression of apoptotic proteins (p53, p21) to attenuate aging of skeletal muscle. In summary, in vivo experiments suggested that DR reduced the effects of aging and atrophy on skeletal muscle via promoting mitophagy and mitochondrial dynamics. The detailed mechanisms of DR in regulating mitochondrial functionality further studies.
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