B10调控成纤维细胞HA合成延缓EAM小鼠心肌纤维化进程的机制研究

Cardiac fibrosis is a critical process of cardiac function remodeling at the last stage of experimental autoimmune myocarditis (EAM); associated with fibroblasts activation, macrophage reprogramming and apoptosis of cardiac myocytes. It is of great significance to clarify the factors of fibrosis in the prevention and treatment of cardiovascular disease. Our date showed that the proportion of B10 was up-regulated in spleen of EAM mice. Meanwhile, B10 cell was infiltrated in heart tissue. It is noted that B10 cells modulated hyaluronan (HA) synthesis in cardiac fibroblasts and inhibited cardiac fibroblasts activation; furthermore, HA contributed to macrophage reprogramming and promoted cardiac myocytes proliferation; additionally fibroblast could promote B10 expansion. Therefore, we hypothesized that "B10 cell is involved in the regulation of HA synthesis in fibroblasts, inhibited it's activation and retarded EAM mice development." To confirm the hypothesis, the project will be carried out: in vitro, to clarify the mechanisms: (1) B10 modulated HA synthesis and inhibited cardiac fibroblasts activation; (2) HA contributed to macrophage reprogramming; (3) HA promoted cardiac myocytes proliferation; in vivo, wild type, IL-10-/-, FSP1-has-/-BALB/c and neutralizing antibody will be employed to clarify B10 cells roles in myocardial function remodeling following EAM. All the data will help us to comprehensive understanding of B10 cells roles in inflammatory diseases development and resolution.

心肌纤维化与成纤维细胞活化、Mφ再编程以及心肌细胞坏死等多因素相关，是EAM后期心肌功能不可逆转的核心事件。阐明影响纤维化发生的因素对于积极开展心血管疾病的防治具有重要意义。预实验发现EAM小鼠，脾脏B10比例增加、浸润心肌；体外B10能够抑制成纤维细胞活化、促进其HA合成；HA与Mφ的再编程以及心肌细胞的存活密切相关；而成纤维细胞能够促进B10扩增。由此提出：“B10通过抑制成纤维细胞活化、促进其HA的合成，延缓了EAM小鼠纤维化进程”的假说。为证实以上假说，课题拟从体内外两方面进行：体外阐明B10抑制成纤维细胞活化，促进其HA合成、调控心肌浸润Mφ再编程以及心肌细胞增殖的机制；体内通过使用WT、IL-10-/-、FSP1-has-/-BALB/c小鼠以及中和抗体进一步明确B10细胞在EAM小鼠心肌功能重构过程中的调控作用；为全面认识B10在炎症疾病发生及转归过程中的作用积累实验数据。

本项目旨在阐明B10分泌的IL-10直接或者间接调控成纤维细胞活化和巨噬细胞重编程的机制，及其缓解心肌病与心肌纤维化的作用，通过本项目的实施以期望为炎性心肌损伤的免疫干预治疗寻找到新的靶点。对照项目目标，本课题已圆满的完成了预期目标，证实B10能够通过分泌IL-10直接抑制成纤维细胞的迁移和活化，并促进成纤维细胞分泌HA来调控巨噬细胞重编程，缓解心肌纤维化与心肌病。在此基础之上，本课题还进行了拓展研究，主要在以下方面：①损伤心肌组织释放的PGE2促进B10细胞的扩增，并且抑制Th17细胞的分化，进而反馈性的缓解心脏炎症；②MI小鼠纤维化阶段中，心包脂肪组织来源的B10细胞也可迁移至受损心脏内维持B10细胞数量；③心肌炎模型小鼠心脏肌成纤维细胞诱导了巨噬细胞的M1表型并促进了M1型巨噬细胞的凋亡；④心肌炎小鼠心脏中CCR2+巨噬细胞在纤维化后转化为CX3CR1+巨噬细胞。这些工作的完成为心脏疾病中成纤维细胞、B10细胞、Th17细胞、巨噬细胞等非心肌细胞功能的阐明起到重要作用。