炎症微环境在骨骼肌损伤和再生中的作用机制

The unbalance of injury and regeneration in skeletal muscle leads to skeletal muscle atrophy. Utilizing cardiotoxin-induced muscle injury model, our preliminary studies suggested that immune related gene expression significantly up-regulated; complement was activated; T cell promote macrophage infiltration. The phagocytose, autophagy and differentiation of macrophage play an important role in development of skeletal muscle injury and regeneration. However, the underlined mechanism is remained unclear. The goal of this study is to clarify that how inflammatory microenvironment is formed and how communication of T cells and macrophage causes skeletal muscle regeneration. Specifically, the specific aims are: 1) to explore how injured muscle activates complement and initiates inflammatory response using C3aR-/- and C5aR-/- mice; 2) to determine how the cell-cell interaction between T cells and macrophages to amplify inflammation using T cell depletion/transfer and proteomics technique; 3) to explore the molecular mechanisms of SRBI and Cathepsin S affect macrophage phagocytose and autophagy using SRBI-/-, Cats-/- mice and siRNA knockdown; 4) to determine the production IFN-γ and its role in macrophage differentiation using CD4-/-, IFN-γ -/- mice and recombinant IFN-γ treatment; 5) to determine the critical roles of CX3CR1 in macrophage function using CR3CR1-/- mice and RNA-Seq technique; 6) to clarify how the inflammatory cells and cytokines affect the proliferation and differentiation of satellite cell and induce skeletal muscle injury and regeneration using cell-cell coculture. Our research will shed light on how inflammatory microenviroment participates the pathogenesis of skeletal muscle injury and regeneration, and provide the theoretical and experimental basis for intervention of skeletal muscle atrophy .

骨骼肌损伤和修复的失衡导致骨骼肌萎缩。我们前期发现：1）骨骼肌损伤中炎症相关基因表达上调，2）激活补体促进炎症细胞浸润，3）T细胞加剧巨噬细胞招募 4）巨噬细胞表型转换参与骨骼肌再生。但该过程中炎症微环境如何形成、放大，炎症细胞间如何相互作用，功能如何被调控进而影响肌卫星干细胞活化和骨骼肌再生修复等科学问题尚不清楚。本课题利用补体基因缺陷小鼠，阐明受损骨骼肌激活补体系统启动炎症的机制；利用T细胞回输\敲除和蛋白质组学，明确T细胞介导巨噬细胞招募，放大炎症的机制；利用SRBI基因敲除和siRNA干扰，明确其调控巨噬细胞吞噬、自噬功能的机制；利用IFN-r基因缺陷小鼠、重组细胞因子及mRNA转录组测序明确细胞因子对巨噬细胞表型转化及功能的调控机制；利用体外共培养系统，明确炎症微环境影响骨骼肌损伤修复的机制，从而寻找预防骨骼肌萎缩或调控骨骼肌再生的新靶点。

骨骼肌萎缩是多种慢性疾病的共同现象，并与这些疾病的死亡率和预后转归密切相关，骨骼肌损伤再生是复杂的病理生理过程，深入探索和验证某些关键炎症微环境成份及其分子作用机制对于寻找预防骨骼肌萎缩或调控骨骼肌损伤修复的策略具有至关重要的意义。为此我们运用多种分子生物学、细胞生物学、免疫学、基因组学等手段，利用多种关键炎症细胞、炎症因子和细胞表面受体敲除小鼠，结合细胞回输和细胞共培养体系等方法，主要发现如下：1）发现了损伤骨骼肌激活补体系统替代途径产生分子C3a促进巨噬细胞产生趋化因子，IL-33/IL1RL1通路刺激FAP产生趋化因子，进而共同启动损伤后骨骼肌炎症反应；2）发现浸润的CD8+T细胞一方面与巨噬细胞相互作用促进MCP-1的释放，招募CCR2+巨噬细胞浸润至损伤部位，一方面释放IFN-促进巨噬细胞分化为IFN反应性巨噬细胞，分泌细胞因子CXCL10促进肌卫星干细胞的增殖；3）明确了巨噬细胞SRB1和miR-223-3p分别通过介导吞噬反应和靶向抑制IL-6的表达调控自身表型分化的分子机制；4）发现其他促肌卫星干细胞分化的分子（Psmb8和KLF15）对骨骼肌损伤后再生的重要作用。总之，以上的一系列发现阐明了炎症微环境在骨骼肌损伤、再生、修复过程中的作用机制，有助于了解在慢性疾病过程中病理因素通过影响系统和局部炎症环境抑制骨骼肌再生、修复的病理过程，为预防骨骼肌萎缩和提高老年人生活质量的新方法提供重要理论指导。