外泌体miR-106a诱导内皮细胞LDL-LDLR代谢异常在硬皮病纤维化中作用及机制研究

Endothelial cell dysfunction is the initiating factor of scleroderma fibrosis. The applicant's previous study found that the number of plasma exosomes in systemic sclerosis patients (SSc-Exo) were increased. Additionally, SSc-Exo induced a variety of abnormal behaviors in endothelial cells, including inflammation and endothelial-mesenchymal transition (EndMT). Furthermore, transcriptomics revealed that SSc-Exo are rich in miR-106a. In a mouse model of scleroderma fibrosis, miR-106a expression was also significantly elevated. Further studies showed that the imbalance of LDL-LDLR metabolism impelled by miR-106a promoted pathological changes of gene regulatory networks, which in turn induced adaptive remodeling of endothelial cells in function and phenotype, aberrantly secreting fibrotic factors, and eventually activating downstream fibrosis response. Therefore, in order to systematic understanding of the molecular mechanism and achieving the goal of effectively treatment of scleroderma fibrosis, we intend to clarify the role of miR-106a, LDL and LDLR in endothelial cells injury and subsequently fibrosis by using miR-106 overexpressing and LDLR deficient cell and mice models. We will also evaluate miR-106a inhibitor-containing exosomes and LDL-LDLR metabolic-based medicine in the treatment of scleroderma fibrosis by targeting miR-106a-LDLR-LDL regulatory axis. All of these will provide a theoretical basis for further clinical transformation.

内皮细胞功能紊乱是硬皮病纤维化的始动因素。申请人前期研究发现，硬皮病患者血浆外泌体数量增多，且诱导内皮细胞炎症反应、间质转化等多种异常行为；转录组学发现，硬皮病外泌体富含miR-106a。小鼠硬皮病纤维化模型中，miR-106a表达也显著升高。进一步研究表明，miR-106a靶向诱导LDL-LDLR代谢失衡、促使细胞表达调控网络发生病理性改变，继而导致细胞在功能与表型上发生适应性重塑并异常分泌纤维化因子，激活下游纤维化反应。因此，立足于系统理解硬皮病纤维化分子机制及实现有效干预的角度，我们拟利用miR-106a过表达、LDLR敲除小鼠和细胞模型，研究miR-106a、LDL、LDLR在内皮细胞损伤及纤维化中作用，并以miR-106a-LDLR-LDL轴为靶点，评估miR-106a抑制剂-外泌体复合物和LDL-LDLR代谢调节药物在治疗硬皮病纤维化中的可行性，为进一步临床转化提供理论基础。

硬皮病，又称系统性硬化症。它是一种以皮肤增厚和纤维化为特征并累及内脏器官，同时伴有微血管系统损伤的自身免疫性疾病。临床上，超过70%的 SSc患者有明显肺部改变，其中以间质性肺病为主，现已成为SSc死亡的首要原因。然而，硬皮病相关肺纤维化发病机制迄今仍未明确。因此，深入开展硬皮病肺纤维化的机理研究、寻找安全有效的治疗措施尤为重要。.项目申请人从临床样本、小鼠肺纤维化模型出发，通过肺组织、血液等样本进行检测，并利用体内动物实验、体外细胞实验深入阐释了表观遗传因子miR-106a表达异常致代谢基因改变，继而驱动代谢重编程的机制；系统评估了不同类型的代谢异常（低密度脂蛋白LDL水平异常和铁代谢异常）在肺纤维化中作用，并基于代谢干预策略评估了其在肺纤维化中的治疗效果，为临床转化提供依据。主要结果如下：1）发现SSc患者血浆外泌体数量显著高于正常人，且SSc来源外泌体具有明显促炎、促纤维化效果；2）通过外泌体转录组学研究发现SSc来源外泌体富含miR-106a。miR-106a高表达促进博来霉素诱导的小鼠肺纤维化进程。3）发现表观修饰因子miR-106a在调节低密度脂蛋白受体（LDLR）以及膜铁转运蛋白（FPN1）两种关键脂代谢和铁代谢基因中的重要作用；4）发现肺纤维化中LDLR表达缺失诱发低密度脂蛋白LDL过度沉积，并揭示LDL-LDLR代谢轴通过诱导内皮、上皮凋亡和转化以及成纤维细胞活化等细胞功能紊乱继而加重肺纤维化；基于该代谢轴的他汀+PCSK9抗体联用策略可修复LDL-LDLR代谢失调，并有效缓解肺纤维化；5）发现新发铁代谢基因FPN1在调节铁超载、铁死亡中作用及机制，基于铁代谢的干预策略能够有效缓解肺纤维化发生发展。.通过本项目的实施，加深了对硬皮病肺纤维化发病机制的认知，为肺纤维化治疗靶标的开发开辟了新的方向，对临床转化具有重要参考价值和借鉴意义。