山苦瓜調節骨骼肌粒線體活性與預防肌少症之應用

Wild bitter gourd regulates mitochondrial activity in skeletal muscles and its application to the prevention of sarcopenia

汪芝翎(Chih-Ling Wang)

指導教授 ： 黃青真

國立臺灣大學/生命科學院/生化科技學系/博士(2019年)

https://doi.org/10.6342/NTU201900162

摘要

肌少症為骨骼肌流失伴隨功能退化之現象，為老齡衰弱症重要成因。已知雄激素可促進肌肉質量增加，但其伴隨的副作用限制了臨床上之應用，因此選擇性雄激素受體調節劑(selective androgen receptor modulator, SARM)乃成為研發之焦點。此外，可促進粒腺體生合成之PGC1亦為治療肌肉萎縮研發標的之一。過去研究暗示苦瓜具有促進粒腺體活性與肌肉功能之潛力，本研究旨在藉由高脂飲食誘導小鼠肥胖、睪丸剔除誘導小鼠肌肉流失與老齡鼠之動物模式分別探討山苦瓜是否具有SARM的特質及其應用於減緩肌肉流失的保護效用。
首先以高脂飲食誘導C57BL/6J小鼠肥胖與代謝異常之動物模式初步探討山苦瓜暨其水解樣品對不同肌群的影響。結果顯示山苦瓜暨其水解樣品添加於高脂飲食中能顯著改善飲食所造成的肥胖與代謝異常，且可上調小鼠後肢比目魚肌與腓腸肌中粒線體生合成相關基因表現。其中山苦瓜對骨骼肌中粒線體相關基因的影響優於水解處理後之山苦瓜樣品，因此後續研究選用未經處理之山苦瓜全果凍乾粉，進一步探討山苦瓜補充於骨骼肌流失之動物模式中對肌肉質量與功能的保護效用。
於細胞轉錄活性分析研究中觀察到山苦瓜萃物可略微活化雄激素受體(AR)之轉錄活性。而當山苦瓜萃物與雄性激素(5α-Dihydrotestosterone)共處理下則可抑制AR轉錄活性，此特性與已知SARM相似。進一步使用睪丸剔除(Castration)誘導肌肉萎縮之小鼠模式，評估山苦瓜對肌肉萎縮的保護效用。實驗使用C57BL/6J公鼠分別進行假手術或睪丸剔除(Castration)手術。假手術組(Sham group)餵食以AIN-93G為基礎進行修改之高蔗糖飲食(HS diet)。剔除組(Cast group)則隨機分為三組，分別給予於高蔗糖飲食不添加(Cast)或添加5%山苦瓜全果凍乾粉 (Cast+BGP)或睪固酮(7 mg Testosterone propionate/kg diet, Cast+TP)之飲食餵食23週。實驗結果顯示，山苦瓜補充未影響血清睪固酮濃度及前列腺、精囊之重量，而肌肉組織中具雄激素高敏感之球海綿體肌(BC)與提肛肌(LA)的重量均顯著高於Cast組(p<0.05)。與Cast組相比，山苦瓜補充亦可上調腓腸肌(GAS)、球海綿體肌及提肛肌Pgc1α與Ucp2基因表現。骨骼肌組織切片結果顯示，於脛骨間前肌 (TA)與腓腸肌中，Cast組中表現高琥珀酸脫氫酶(SDH)活性之肌纖維比例顯著低於Sham組(p<0.05)，而山苦瓜補充於睪丸剔除小鼠後，於TA與GAS中表現高琥珀酸脫氫酶(SDH)活性之肌纖維比例在Sham組及Cast+TP組間未有顯著的差異(p>0.05)。運動表現方面，Cast+BGP組於前肢抓力與rotarod表現上也均與Cast+TP組無顯著差異(p>0.05)。上述結果顯示山苦瓜具SARM之特質可改善因睪丸剔除手術所導致之肌肉質量、粒線體功能與運動表現降低。
最後以老齡小鼠進一步確認山苦瓜對老齡肌肉功能之保護效用及評估其應用於預防肌少症之潛力。18月齡C57BL/6J公鼠隨機分為兩組，分別給予高蔗糖飲食不添加(Aging)或添加5%山苦瓜全果凍乾粉(Aging+BGP)之飲食餵食22週，此外實驗同時設計一組8月齡年輕公鼠餵食HS diet作為對照組。實驗結果顯示，山苦瓜補充未影響老年鼠血清睪固酮濃度但可減緩老化所致前肢抓力表現降低。骨骼肌組織切片結果也觀察到肱三頭肌中Aging+BGP組肌纖維(cross-sectional area, CSA)較Aging組大(p<0.05)，同時山苦瓜補充組表現高SDH活性之肌纖維比例也顯著高於Aging組(p<0.05)。與Aging組相比，山苦瓜補充亦可上調肱三頭與腓腸肌中Ucp2基因表現(p<0.05)。顯示山苦瓜可減緩老化所致前肢肌肉肌纖維的萎縮與粒線體活性降低，利於肌肉功能的維持。
綜合以上，山苦瓜可作為選擇性雄激素受體調節劑，選擇性調節骨骼肌粒線體活性與肌肉功能，同時不會對生殖器官前列腺/精囊有不良之影響之特質，使山苦瓜具應用於預防肌肉流失之潛力。

關鍵字

山苦瓜 ； 選擇性雄激素受體調節劑 ； 骨骼肌 ； 睪丸剔除 ； 肌少症 ； 粒線體

並列摘要

Sarcopenia, a gradual loss of skeletal muscle mass and function, is a major contribution factor of frailty in elderly and wasting syndrome. Androgen is an anabolic steroid that can increase muscle mass, but side effects have limited its wide applications. Selective androgen receptor modulators (SARMs) are thus regarded as alternative targets. On the other hand, PGC1α is known to regulate mitochondria biogenesis in skeletal muscle, hence also become a molecular target for developing therapeutics for sarcopenia. Previous studies suggested that supplementation of wild bitter gourd (Mommordica charantia, BGP) might improve mitochondrial activity and muscle function. This study thus aims to examine the potential of wild bitter gourd as a SARM and its application to prevent sarcopenia by using diet-induced obesity, castration and aged mice models.
In the diet-induced obesity model, five groups of C57BL/6J male mice were fed an high-fat diet (HF, modified from AIN-93G) for 15 weeks, followed by supplementing with or without 3% BGP, 5% BGP, 3% hydrolyzed BGP (BGPa) or 5% BGPa respectively in the HF diet and fed for 20 weeks. Both BGP and BGPa supplementation improved metabolic disorders induced by the high-fat diet. Furthermore, BGP and BGPa mainly up-regulated the mRNA expression levels of mitochondrial biogenic and its target genes in soleus (SOL) and gastrocnemius (GAS) muscle. Noticeably, the BGP showed better effects than the BGPa.
In a cell-based transactivation assay, the BGP ethyl acetate extract showed a weak agonistic and an antagonistic activity, resembled those of some SARM in development. To examine the potential of wild bitter gourd as a SARM and its application to prevent muscle wasting induced by orchiectomy, male C57BL/6J mice were sham-operated (Sham group) or castrated (Cast groups) and fed a high sucrose diet (modified from AIN-93G, HS diet). Three groups of castrated mice were respectively fed: the HS diet supplemented without (Cast) or with 5% BGP (Cast+BGP) or with testosterone propionate (7mg TP/kg diet, Cast+TP) for 23 weeks. In contrast to the Cast+TP group, the BGP supplementation did not affect serum testosterone concentration, prostate and seminal vesicle mass. Both TP and BGP supplementation increased the weight of androgen responsive muscles, bulbocavernosus (BC) and levator ani (LA) (p<0.05). The grip strength and the performance on rotarod of the Cast+BGP group were comparable to those of the Cast+TP group (p>0.05). The number of succinate dehydrogenase (SDH)-positive fibers in tibialis anterior (TA) and gastrocnemius (GAS) muscle of the Cast+BGP group was not significantly different from that of Sham and Cast+TP groups (p>0.05). The BGP supplementation up-regulated the Pgc1α and Ucp2 gene expression in GAS, BC and LA muscles of castrated mice (p<0.05). Thus, the BGP showed characteristics of SARM and might improve skeletal muscle function through the up-regulation of mitochondria biogenic genes and oxidative capacity, and ameliorated the castration-induced decline of skeletal muscle function in mice.
In the aged mice model, 18-month old C57BL/6J mice were respectively fed the HS diet supplemented without (Aging group) or with 5% BGP (Aging+BGP group) for 22 weeks. Another group of 8-month old mice were also fed the HS diet (Younger control group) along with the aged mice and serve as the control group. Supplementation of the BGP did not affect the serum testosterone levels in aged mice but ameliorated the age-related decline of forelimb grip strength. The cross-sectional area of the triceps brachii (TRI) muscle of the Aging+BGP group was also larger than that of the Aging group (p<0.05). Likewise, the number of SDH-positive fibers of the TRI muscle of the Aging+BGP group was also significantly higher than that of the Aging group (p<0.05). Moreover, BGP treatment up-regulated the Ucp2 gene expression in TRI and GAS. These results suggested that BGP may be beneficial for maintaining muscle function, at least in part, in aged mice through the up-regulation of skeletal muscle Ucp2 expression, oxidative capacity and prevent the age-associated decline of muscle fiber size and muscle function. An anabolic effect of BGP is thus implicated.
In summary, results of these studies provide evidences supporting the potential of BGP as an SARM for the amelioration of muscle wasting.

並列關鍵字

wild bitter gourd ； selective androgen receptor modulator ； skeletal muscle ； castration ； sarcopenia ； mitochondria

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