miR-494调控自噬通路在运动延缓骨骼肌衰老中的作用与机制

Sarcopenia is associated with the loss of muscle mass and strength as well as the decline in physical functions, which results in the significant impact on the health and life activities of elderly people. It is well known that exercise is benefit to the prevention and treatment of sarcopenia; however, its mechanisms are still unclear. Based on the characteristics of relatively accelerated miR-494 and reduced autophagy during the development of sarcopenia, we would like to use SAMP8 mice as the experimental subjects. Following exercise training in different modes, the muscle atrophy rate of the aged rats will be determined; the size of muscle fiber cells will be evaluated after HE staining; the damage of mitochondria and endoplasmic reticulum (ER) and the number of autophagosomes will be analyzed by transmission electron microscope. The expression of miR-494 will be applied by real-time polymerase chain reaction (qRT-PCR). The proteins and genes associated with autophagy, apoptosis, senescence or energy metabolism will be subjected to Western blot, analysis, which will benefit for the exploration of the correlation between sarcopenia and miR-494 expression, the optimization of exercise intervention mode, and the confirmation of our hypothesis that exercise down-regulated expression of miR-494 can inhibit the development of sarcopenia, as well as the clarfication of its target protein and autophagic signal pathway network with optimized exercise intervention for the prevention, rehabilitation and treatments of sarcopenia. Meanwhile, we would like to apply aged mice subjected to down-regulation of miR-494 via antagomir with or without exercise intervention to explore the determinant role of miR-494/SIRT1/autophagy pathway in the prevention, rehabilitation and treatments of sarcopenia in the presence of optimal exercise intervention. This study will demonstrate that functional status of miR-494/SIRT1/autophagy pathway is a determinant role in modulating muscle homeostasis, improving mitochondrial quality control, and preventing and treating sarcopenia. All of these findings will be helpful to provide a theoretical guidance for reasonable exercise and scientific fitness training to reduce unnecessary injuries and accomplish the prevention and treatments of sarcopenia or some other diseases.

衰老性肌萎缩严重危害着老年人健康，运动干预有益于其预防与治疗，但作用机制还不明确。衰老性肌萎缩可能与miR-494过度表达和细胞自噬低下相关，本研究以SAMP8快速老化小鼠为研究对象，运动干预后测定肌萎缩率，电镜观察肌细胞内线粒体与自噬溶酶体，qRT-PCR检测miR-494表达，免疫印迹分析自噬、凋亡、衰老、能量代谢相关蛋白表达。探讨衰老性肌萎缩与miR-494及其下游靶标和自噬通路的相关性，确立其优化运动干预方式，初步探明运动调控miR-494诱导自噬激活的信号通路或靶点。利用antagomir-494抑制小鼠体内miR-494表达并辅以运动干预，阐明其诱导的自噬激活对防治衰老性肌萎缩的关键作用。本研究首次提出了运动通过调控miR-494/SIRT1/自噬通路在调节肌肉代谢稳态平衡，提高线粒体质量和防治肌萎缩中的决定性作用。

衰老性肌萎缩可能与miR-494过度表达和细胞自噬低下相关，尽管运动对于衰老性肌萎缩有明显的、积极的预防与治疗作用，然而其预防与干预性治疗肌萎缩的细胞或分子机制还不十分明确。（1）本研究以SAMP8快速老化小鼠为研究对象，相比R1对照组，P8小鼠骨骼肌湿重比及腓肠肌横截面积下降，HE染色和电镜结果表明肌细胞形态异常；qRT-PCR结果表明衰老骨骼肌miR-494表达水平升高，免疫印迹实验表明其下游AMPK/Sirtl 信号轴受到抑制进而影响机体能量代谢水平，骨骼肌能量代谢水平下降；自噬水平低下，TUNEL和免疫印迹均显示细胞凋亡过度激活。（2）三种运动干预方式均可不同程度改善衰老性肌萎缩，其中负重爬梯运动对逆转衰老骨骼肌湿重比以及横截面积低下效果较好，对miR-494的下调作用更加明显，且TUNEL和免疫印迹实验中表现出更优的抗凋亡效果，因此选择负重爬梯运动作为延缓衰老性肌萎缩的优化运动方式；跑台运动在逆转衰老引起的骨骼肌 AMPK/Sirtl 信号轴活性下降方面效果更优；自由转轮运动在改善衰老骨骼肌自噬水平和自噬流方面效果更优。（3）进一步通过antagomir-494联合抗阻运动，验证了运动协同antagomir-494对于骨骼肌衰老的抑制与相关信号通路、靶点的影响，结果表明下调miR-494在衰老小鼠骨骼肌的表达，能够改善衰老性肌萎缩，增加衰老骨骼肌的能量代谢与肌肉合成，减少细胞凋亡。由于miR-494下游靶基因广泛性，可能并非主要通过影响自噬来调节衰老骨骼肌的肌萎缩，其延缓衰老性肌萎缩的分子机制可能是通过调控多条途径共同完成。基于上述研究结果，miR-494参与衰老性肌萎缩的调控，通过合理的运动方式影响miRNA调控转录后基因表达，保持合适的细胞自噬和凋亡功能状态，对于有效的防治和延缓衰老性肌萎缩具有重要的意义与应用前景。