糖皮质激素提高力竭运动应激耐受力的非基因组机制

Glucocorticoids (GCs) are a class of steroid hormones, which play a pivotal role in stress response and enhance tolerance and adaption to stress. It is believed traditionally that the effects are mediated by genomic mechanism that takes at leaset 30 min. As well known, the secretion of GC to acute exercese stress is very fast. And then there is a time lag between rapid secretion of GC during acute stress and classic effects, which is difficult to explain by genomic mechanism. Apart from the well-known classic genomic mechanism, mounting evidence suggests that GC affect various functions via rapid nongenomic mechanisms. However, there still lacks powerful direct basis of enhancing tolerance to stress by nongenomic mechanism. We ever found that GC could prolong the exhaustion time and protect the skeletal muscle from ultrastructural damage in a weight-loaded forced swimming model using adrenalectomized mice in less than 30 min, which couldn't be inhibited by RU486, an antagonist of GC receptor. Therefore, to investigate the effects and its rapid nongenomic mechanism of GC on enhancing acute stress tolerance to exhaustive exercise, we will take acute weight-loaded forced swimming test as the model of exhaustive exercise using adrenalectomized mice, study the skeletal muscle and GC receptor knockout cell line using gene targeting, from the whole, organ and tissue, cellular and molecular level. The work may contribute to understanding the physiological significance of nongenomic effects of GC.

糖皮质激素(GC) 是应激反应枢纽，可提高机体对应激刺激的耐受和适应能力，通常认为是通过至少30分钟才显效的经典基因组机制实现的。但运动应激早期即大量快速分泌GC与其基因组作用显效间的时间窗难以用基因组机制解释。GC作用除基因组机制外还存在非基因组机制，但GC非基因组作用研究主要集中在神经、免疫调节及临床应用上，缺乏提高应激耐受力的直接有力证据。我们曾用去肾上腺小鼠负荷游泳力竭模型，发现应激浓度GC在30分钟内即可延长小鼠力竭时间，减轻骨骼肌超微结构损伤变化，且不能被GC受体拮抗剂所阻断。本项目拟在原工作基础上，应用负荷强迫游泳的力竭运动应激模型，以骨骼肌为研究对象，应用基因打靶技术建立骨骼肌GC受体基因敲除细胞株，从整体、器官和组织、细胞和亚细胞及分子水平展开系列研究，以期明确GC在提高机体对急性力竭运动应激耐受力中的早期快速非基因组机制，揭示GC非基因组机制的内在生理意义的冰山一角。

本研究以负荷强迫游泳为力竭运动应激模型，采用系列分子生物学、细胞生物学、电镜、激光共聚焦和流式细胞仪等方法，在整体、组织、细胞和分子水平，研究糖皮质激素（GC）的非基因组机制。结果显示：GC可以通过其非基因组作用快速提高机体的应激耐受力，减少骨骼肌损伤，其机制与维持血糖浓度和降低血浆一氧化氮（NO）有关；体外构建的C2C12肌管细胞收缩模型用于模拟急性运动应激，GC能够通过非基因组机制快速抑制肌管细胞收缩引起的AMPK、CaMKII磷酸化以及胞内钙离子浓度升高，抑制Glut4的膜转位，进而降低肌管收缩引起的葡萄糖摄取，从而维持血糖浓度，保障心脑等重要器官的能量供应；GC在短时间内并不能显著影响收缩肌管细胞的肌酸激酶活性、乳酸含量、活性氧（ROS）生成以及肌球蛋白轻链的磷酸化水平；通过低氧条件模拟急性运动应激下的心脏环境，低氧能够促进H9C2心肌细胞生成NO和ROS，而GC能够快速抑制NO和ROS的产生。结果表明，GC能够通过非基因组机制快速提高机体对急性运动应激的耐受力，完善了我们提出的GC非基因组机制存在的生理意义，也为有效应对急性应激提供了新思路。