以 C2C12 小鼠肌肉前驅細胞模式評估山苦瓜萃取物與共軛脂肪酸對粒線體之影響

Abstract

目前有許多研究探討飲食調控肌肉生長或促進粒線體生合成。已有諸多文獻顯示，山苦瓜能透過多種機制進行血糖調節、改善脂肪代謝等問題。本實驗室先前研究指出，長期餵食山苦瓜之 C57BL/6J 小鼠，其骨骼肌粒線體生合成相關基因表現量增加。已知山苦瓜乙酸乙酯萃取物中含有多種活性化合物，其中共軛三烯亞麻油酸 (CLN) 的含量相當高。因此本研究之目標是以未分化的小鼠 C2C12 肌肉前驅細胞 (myoblasts) 模式更進一步探討山苦瓜萃取物促進粒線體活性之效果以及分析共軛脂肪酸 CLN 對粒線體的影響。 實驗先以1758品系之山苦瓜乙酸乙酯萃取物 (1758 EAE) 處理 C2C12肌肉前驅細胞 (myoblast) 0、1、6、24、48 小時，探討 1758 EAE 對粒線體的促進效果與時間之關係。結果顯示100 μg/mL 1758 EAE 處理細胞 24 小時後，可在不影響細胞數目的情況下，增加 MTT 讀值、促進粒線體功能指標 citrate synthase 活性、Mitrotracker 之相對螢光量。而 50 μg/mL 1758 EAE 處理細胞 6 小時後，可促進粒線體生合成相關基因之表現。此外，25 μM 的共軛脂肪酸 CLA亦可發現與 1758 EAE 有相似的效果，然而 CLN於此細胞模式下則無顯著之效果，且還發現 CLN 對於 C2C12 myoblasts 的毒性較另外兩者高，超過 10 μM 即造成細胞存活率降低。進一步以西方墨點法分析蛋白質表現量，結果顯示上述濃度之 1758 EAE 或 CLA 處理細胞 1 小時，可以促進粒線體生合成相關調控因子 AMPK 磷酸化表現量，而 CLN 於此細胞模式下無觀察到該現象。再者本研究以海馬生物能量代謝分析儀分析細胞之氧氣消耗速率 (OCR) 以及產酸速率 (ECAR)，結果顯示 100 μg/mL 1758 EAE 或 25 μM CLA 先處理細胞 24 小時後，細胞之基礎氧氣消耗速率與產酸速率明顯提升。而 CLN 則是皆無差異。 綜合以上，1758 EAE 與 CLA 於本細胞模式中，皆有促進粒線體活性的能力。然而 CLN 的部分，雖未觀察到明顯促進粒線體活性的效果，但是未來可藉由測試不同的處理條件，改善其對於 C2C12 myoblasts 毒性過高的問題後再進行粒線體之相關研究，或許可觀察到不同的結果。

Mitochondria play an important role in skeletal muscle metabolism. Impaired mitochondrial biogenesis is known to be associated with the development of metabolic diseases such as insulin resistance and type 2 diabetes. Previous studies showed that dietary wild bitter gourd can improve metabolic disorders such as insulin resistance and dysregulations of lipid metabolism through various mechanisms. Recent in vivo studies in our lab demonstrated that mice fed a 5% wild bitter gourd powder (BGP) diet up-regulated genes related to mitochondrial biogenesis. It is known that wild bitter gourd is rich in conjugated linolenic acid, which is one of the many bioactive compounds of this common tropical vegetable. This study aims to examine effects of wild bitter gourd extracts and conjugated fatty acid on mitochondria in C2C12 myoblasts. In this study, C2C12 myoblasts were cultivated in 0.1%BSA/DMEM and treated with cultivar-1758 wild bitter gourd ethyl acetate extract (1758 EAE) for 0, 1, 6, 24, 48 hours. Cell numbers were determined by counting as well as the MTT assay. Compared to the vehicle-treated cells, cells treated with 50 and 100 μg/mL 1758 EAE showed significantly higher optical density in the MTT assay but did not change the cell number from counting after 24 hours of treatment. In addition, 50 or 100 μg/mL 1758 EAE enhanced the activity of citrate synthase of the cells, a marker of mitochondrial function. In addition, the relative fluorescence of mitotracker staining was significantly higher in cells treated with these concentrations of 1758EAE. Cells treated with 100 μg/mL 1758 EAE for 6 hr showed up-regulated mitochondria-related gene expression. Cells treated with 25 μM conjugated linoleic acid (CLA) showed similar effects as the 1758 EAE. However, conjugated linolenic acid (CLN) showed significant cytotoxic effects at concentrations higher than 10μM in this system. No significant effects can be observed at this concentration. Western blot analysis further showed that 100 μg/mL 1758 EAE and 25 μM CLA increased AMPK phosphorylation of C2C12 myoblasts after one-hour treatment. Moreover, 100 μg/mL 1758 EAE and 25 μM CLA can also increase the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) after 24 hours of treatment. In conclusion, results of this study indicated that 50 or 100 μg/mL 1758 EAE and 25 μM CLA enhance the mitochondrial function in C2C12 myoblasts, and the regulation might be through the pathway involved AMPK and mitochondrial biogenesis.