Tribble3调控COP1与FoxO3介导的泛素化参与重症监护病房获得性肌无力的机制研究

Intensive care unit acquired muscle weakness (ICUAW) is manifested as acute amyotrophy and flaccid paralysis of skeletal muscle in ICU patients. ICUAW can significantly prolong the ICU stay and recovery. Increased protein decomposition mediated by ubiquitination is the main pathophysiological mechanism leading to ICUAW. Previous studies found that Tribble3 plays a pivotal role in insulin resistance and regulating protein metabolism. Pre-experimental results confirm that Tribble3 expression and ubiquitination mediated by COP1 and FoxO3 is increased significantly in ICUAW human and mouse skeletal muscle samples. Moreover, knockout of Tribble3 alleviate amyotrophy in ICUAW mice. Thus, we propose that Tribble3 may regulate the pathogenesis of ICUAW by affecting the COP1 and FoxO3 mediated ubiquitination. Based on the previous work, this study intends to explore whether Tribble3 may participate in COP1 and FoxO3 mediated ubiquitination by means of CRISPR-Cas9 and ubiquitination-modifies proteomics，clarifies the effect and mechanism of ICUAW. The results will provide a new target for the prevention and treatment of ICUAW.

重症监护病房获得性肌无力（ICUAW）表现为ICU患者骨骼肌的急性肌量减少与弛缓性瘫痪，显著延长患者住院与康复时间。胰岛素抵抗时泛素化介导的肌蛋白降解增多是导致ICUAW的主要机制。既往研究发现Tribble3基因是调节胰岛素抵抗与蛋白代谢的重要分子，预实验结果证实ICUAW人体与小鼠骨骼肌中Tribble3表达显著增加，COP1与FoxO3介导的泛素化增多，敲除Tribble3可以减少ICUAW小鼠肌萎缩。由此，我们提出了Tribble3可能通过影响COP1与FoxO3介导的泛素化参与骨骼肌蛋白代谢调控ICUAW发病过程的假说。本课题拟在前期工作的基础上运用CRISPR-Cas9、泛素化修饰蛋白质组学等技术深入探讨Tribble3在COP1与FoxO3介导的泛素化过程中的作用，阐明其对ICUAW患者骨骼肌的影响及分子位点，研究结果将为防治ICUAW提供新的靶点。

本课题在临床患者、细胞及整体动物水平验证Trib3在调节脓毒症危重症情况(包括衰老、心衰）下诱发ICUAW的作用及其信号转导机制。⑴ 明确了ICU患者肌肉Trib3表达与ICUAW发病率与疾病负担的相关性；⑵ 发现炎症、衰老、心衰等疾病调节下Trib3对骨骼肌细胞萎缩、纤维分型的影响；⑶ 阐明Trib3对骨骼肌自噬、线粒体自噬、泛素化与间质纤维化信号通路的作用及机制；⑷ 证明小鼠在ICUAW疾病条件下，敲除Trib3后减轻骨骼肌细胞萎缩、肌萎缩，改善骨骼肌功能。本研究成果将促进对Trib3生物学功能的认识，更重要的是有助于阐明危重症环境下ICUAW患者自噬、泛素化依赖的肌蛋白降解条件过程与Trib3分子链接机制，为防治ICUAW病变，开发针对Trib3及相应通路的小分子药物，减少危重症患者ICU治疗时间与花费，实现早日重返正常生活提供新的靶点。