State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Avenida da Universidade, Taipa, Macau, China.
Institute of Medicinal Plant Development, Peking Union Medical College, Chinese Academy of Medical Science, Beijing, 100193, China.
J Cachexia Sarcopenia Muscle. 2019 Apr;10(2):429-444. doi: 10.1002/jcsm.12393. Epub 2019 Feb 21.
Muscle atrophy and weakness are adverse effects of high dose or the sustained usage of glucocorticoids. Loss of mitochondria and degradation of protein are highly correlated with muscle dysfunction. The deacetylase sirtuin 1 (SIRT1) plays a vital role in muscle remodelling. The current study was designed to identify myricanol as a SIRT1 activator, which could protect skeletal muscle against dexamethasone-induced wasting.
The dexamethasone-induced atrophy in C2C12 myotubes was evaluated by expression of myosin heavy chain, muscle atrophy F-box (atrogin-1), and muscle ring finger 1 (MuRF1), using western blots. The mitochondrial content and oxygen consumption were assessed by MitoTracker staining and extracellular flux analysis, respectively. Muscle dysfunction was established in male C57BL/6 mice (8-10 weeks old, n = 6) treated with a relatively high dose of dexamethasone (25 mg/kg body weight, i.p., 10 days). Body weight, grip strength, forced swimming capacity, muscle weight, and muscle histology were assessed. The expression of proteolysis-related, autophagy-related, apoptosis-related, and mitochondria-related proteins was analysed by western blots or immunoprecipitation.
Myricanol (10 μM) was found to rescue dexamethasone-induced muscle atrophy and dysfunction in C2C12 myotubes, indicated by increased expression of myosin heavy chain (0.33 ± 0.14 vs. 0.89 ± 0.21, *P < 0.05), decreased expression of atrogin-1 (2.31 ± 0.67 vs. 1.53 ± 0.25, *P < 0.05) and MuRF1 (1.55 ± 0.08 vs. 0.99 ± 0.12, **P < 0.01), and elevated ATP production (3.83 ± 0.46 vs. 5.84 ± 0.79 nM/mg protein, **P < 0.01), mitochondrial content (68.12 ± 10.07% vs. 116.38 ± 5.12%, *P < 0.05), and mitochondrial oxygen consumption (166.59 ± 22.89 vs. 223.77 ± 22.59 pmol/min, **P < 0.01). Myricanol directly binds and activates SIRT1, with binding energy of -5.87 kcal/mol. Through activating SIRT1 deacetylation, myricanol inhibits forkhead box O 3a transcriptional activity to reduce protein degradation, induces autophagy to enhance degraded protein clearance, and increases peroxisome proliferator-activated receptor γ coactivator-1α activity to promote mitochondrial biogenesis. In dexamethasone-induced muscle wasting C57BL/6 mice, 5 mg/kg myricanol treatment reduces the loss of muscle mass; the percentages of quadriceps and gastrocnemius muscle in myricanol-treated mice are 1.36 ± 0.02% and 0.87 ± 0.08%, respectively (cf. 1.18 ± 0.06% and 0.78 ± 0.05% in dexamethasone-treated mice, respectively). Myricanol also rescues dexamethasone-induced muscle weakness, indicated by improved grip strength (70.90 ± 4.59 vs. 120.58 ± 7.93 g, **P < 0.01) and prolonged swimming exhaustive time (48.80 ± 11.43 vs. 83.75 ± 15.19 s, **P < 0.01). Myricanol prevents dexamethasone-induced muscle atrophy and weakness by activating SIRT1, to reduce muscle protein degradation, enhance autophagy, and promote mitochondrial biogenesis and function in mice.
Myricanol ameliorates dexamethasone-induced skeletal muscle wasting by activating SIRT1, which might be developed as a therapeutic agent for treatment of muscle atrophy and weakness.
高剂量或持续使用糖皮质激素会导致肌肉萎缩和无力。线粒体丧失和蛋白质降解与肌肉功能障碍高度相关。去乙酰化酶 SIRT1（沉默调节蛋白 1）在肌肉重塑中起着至关重要的作用。本研究旨在鉴定杨梅素作为 SIRT1 激活剂，可防止地塞米松诱导的骨骼肌萎缩。
通过 Western blot 检测肌球蛋白重链、肌肉萎缩 F 盒（atrogin-1）和肌肉环指 1（MuRF1）的表达，评估 C2C12 肌管中的地塞米松诱导萎缩。通过 MitoTracker 染色和细胞外通量分析分别评估线粒体含量和耗氧量。使用相对较高剂量的地塞米松（25mg/kg 体重，腹腔注射，10 天）处理雄性 C57BL/6 小鼠（8-10 周龄，n=6）建立肌肉功能障碍模型。评估体重、握力、强迫游泳能力、肌肉重量和肌肉组织学。通过 Western blot 或免疫沉淀分析来分析与蛋白水解、自噬、凋亡和线粒体相关的蛋白表达。
杨梅素（10μM）可挽救 C2C12 肌管中地塞米松诱导的肌肉萎缩和功能障碍，表现为肌球蛋白重链表达增加（0.33±0.14 对 0.89±0.21，*P<0.05），atrogin-1 表达减少（2.31±0.67 对 1.53±0.25，*P<0.05）和 MuRF1 表达减少（1.55±0.08 对 0.99±0.12，**P<0.01），以及 ATP 产生增加（3.83±0.46 对 5.84±0.79nM/mg 蛋白，**P<0.01），线粒体含量增加（68.12±10.07% 对 116.38±5.12%，*P<0.05），线粒体耗氧量增加（166.59±22.89 对 223.77±22.59pmol/min，**P<0.01）。杨梅素直接结合并激活 SIRT1，结合能为-5.87kcal/mol。通过激活 SIRT1 的去乙酰化，杨梅素抑制叉头框 O3a 转录活性以减少蛋白质降解，诱导自噬以增强降解蛋白的清除，并增加过氧化物酶体增殖物激活受体 γ 共激活因子 1α 的活性以促进线粒体生物发生。在地塞米松诱导的肌肉消耗 C57BL/6 小鼠中，5mg/kg 杨梅素治疗可减少肌肉质量损失；杨梅素处理小鼠的股四头肌和比目鱼肌百分比分别为 1.36±0.02%和 0.87±0.08%（分别为地塞米松处理小鼠的 1.18±0.06%和 0.78±0.05%）。杨梅素还可挽救地塞米松诱导的肌肉无力，表现为握力提高（70.90±4.59 对 120.58±7.93g，**P<0.01）和游泳时间延长（48.80±11.43 对 83.75±15.19s，**P<0.01）。杨梅素通过激活 SIRT1 减少肌肉蛋白降解，增强自噬，促进线粒体生物发生和功能，从而预防地塞米松诱导的肌肉萎缩和无力。
杨梅素通过激活 SIRT1 改善地塞米松诱导的骨骼肌萎缩，可能被开发为治疗肌肉萎缩和无力的药物。
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