M2a型巨噬细胞外泌体的miR-511-3p在糖尿病肾病中抑制系膜基质分泌的作用及其机制

Diabetic nephropathy is a chronically progressive disease and effective treatment is needed to prevent its development. Restoring decreased M2 macrophages in kidney was associated with the improvement of diabetic nephropathy, however, the mechanism remains incompletely unknown. Our previous studies showed that exosomes secreted by M2a macrophages downregulated the extracellular matrix protein deposition. Meanwhile, the level of miR-511-3p selectively increased in M2a macrophage exosomes. These led us to hypothesize that M2a macrophages secrete exosomal miR-511-3p, which could inhibit the secretion of extracellular matrix protein to ameliorate diabetic nephropathy. Firstly, the aim of this project is to determine the role of M2a macrophage exosomes in inhibiting the secretion of extracellular matrix protein in diabetic nephropathy rats. And then, we will detect the levels of extracellular matrix proteins with up-regulation or down-regulation of miR-511-3p in high glucose-treated mesangial cells and in diabetic nephropathy mice respectively, aiming to characterize the role of miR-511-3p in diabetic nephropathy. Finally, bioinformatics technology was used to predict the downstream target genes of miR-511-3p, and the target gene Hic-5 was selected as the entry point. In the Hic-5 knockout model, we will illuminate the molecular mechanism by which miR-511-3p modulates Hic-5 to inhibit the secretion of extracellular matrix protein. This project will help to discover new intervention targets for diabetic nephropathy and provide experimental evidence for its control strategy optimization.

肾脏组织中M2型巨噬细胞增多可延缓糖尿病肾病进展，但机制尚不明确。我们前期研究发现，M2a型巨噬细胞外泌体可抑制肾小球系膜细胞基质蛋白分泌，其中miR-511-3p水平在M2a型巨噬细胞外泌体中特异性升高。我们推测，M2a型巨噬细胞外泌体可能通过释放miR-511-3p抑制系膜基质分泌以延缓糖尿病肾病发生发展。本项目拟首先在糖尿病肾病大鼠中，明确M2a型巨噬细胞外泌体抑制系膜基质蛋白分泌的作用；其次，观察上调或下调miR-511-3p对糖尿病肾病小鼠和高糖处理肾小球系膜细胞中基质蛋白分泌的影响，以确定miR-511-3p在糖尿病肾病中的作用；最后基于生物信息学预测miR-511-3p下游靶基因，拟选取靶基因Hic-5为研究切入点，在Hic-5敲除模型中，研究miR-511-3p调控Hic-5抑制系膜基质分泌的分子机制。本项目将有助于发现糖尿病肾病新的干预靶点，为其防治策略优化提供实验依据。

糖尿病肾病是一种进展性疾病，呈现不可逆性加重，亟待寻求从病理生理机制上遏制糖尿病肾病的治疗手段。糖尿病肾病M1型巨噬细胞增多，M2型巨噬细胞减少。国内外的研究和我们的工作基础均提示M2型巨噬细胞可以显著改善糖尿病肾病，但具体机制不明确。基于此，本项目通过明确M2a型巨噬细胞外泌体及其包含的miR-511-3p延缓糖尿病肾病的作用，和进一步阐明miR-511-3p在糖尿病肾病中抑制系膜基质分泌的作用机制。研究结果包括：首先，本项目通过验证了M2a型巨噬细胞可改善高糖处理的肾小球系膜细胞胶原蛋白分泌，并发现尾静脉注射M2a型巨噬细胞外泌体可抑制糖尿病肾病大鼠系膜基质分泌，明确了M2a型巨噬细胞外泌体抑制糖尿病肾病系膜基质的分泌。其次，验证了高通量筛选结果miR-511-3p在M2a型巨噬细胞外泌体中特异性高表达，并明确了miR-511-3p具有抑制高糖毒性肾小球系膜细胞分泌胶原蛋白进而延缓糖尿病肾病的作用。同时，通过检测尾静脉输注过表达/沉默miR-511-3p表达的慢病毒的糖尿病肾病小鼠肾脏组织中Hic-5以及Hic-5下游通路TGF-β/Smad的表达情况，明确了Hic-5是miR-511-3p的直接靶标，并参与了miR-511-3p抑制系膜基质分泌的作用机制。最后，在细胞实验中沉默Hic-5，检测Hic-5下游TGF-β/Smad通路及胶原蛋白分泌情况，阐明了miR-511-3p可降低Hic-5表达，进而抑制胶原蛋白分泌的作用机制。本项目更深入地理解M2型巨噬细胞改善糖尿病肾病的作用及机制，发现糖尿病肾病新的干预靶点，并为其防治策略优化提供实验依据。