“TGF-β/miRNA-433/多胺”反馈环路在糖尿病心肌纤维化中的作用及吡非尼酮干预研究

The TGF-β1/Smad3 pathway plays a major role in the development of diabetic myocardial fibrosis (DMF). MiRNA-433 not only is an important component of TGF-β1/Smad3-driven liver and renal fibrosis, but also an inhibitor of ornithine decarboxylase (ODC)/polyamine pathway. Moreover, polyamines play a fundamental role in myocardial cell structure and function, and their depletion may decrease cell growth and induce apoptosis. Also, depletion of intracellular polyamines induces the activation of TGF-β1/Smad3 pathway. Our previous studies indicated that TGF-β1 protein and miRNA-433 expressions were significantly increased and conversely, polyamines were found to be considerably decreased in diabetic db/db mice, which were accompanied by myocardial fibrosis. Pirfenidone, a new antifibrotic agent, attenuated these changes. In light of the previous studies and our findings, we hypothesize that there is a novel feedback circuits, “TGF-β1/Smad3 pathway-miRNA-433-ODC/polyamine pathway”, in the development of DMF, and pirfenidone maybe disturb this process by inhibiting miRNA-433 expression. To investigate our hypothesis, we will use a model of db/db mice to investigate this circuit loop in the development of DMF, and focus on the interventional effect of pirfenidone on the process. As a result, a new understanding of DMF mechanisms is put forward and miRNA-433 will be provided as a new target for DMF therapy. In addition, a novel anti-fibrosis mechanism of pirfenidone also will be clarified.

TGF-β1/Smad3通路活化是糖尿病心肌纤维化（DMF）发生的重要机制。miRNA-433不仅是该通路下游靶基因可被其激活促进肝、肾纤维化，还抑制鸟氨酸脱羧酶（ODC）/多胺通路、引起多胺耗竭，而多胺耗竭影响心肌细胞结构与功能并可放大TGF-β1/Smad3通路活性。我们前期研究发现，DMF动物心肌TGF-β1高表达并伴有miRNA-433上调和多胺减少，而吡非尼酮（Pir）可下调miRNA-433、改善DMF。由此推测，DMF过程中存在“TGF-β1/Smad3通路-miRNA-433-ODC/多胺通路”反馈环路，Pir通过下调miRNA-433抑制环路、改善DMF。项目拟以心肌成纤维细胞和db/db小鼠为对象，研究上述环路，并证明Pir通过下调miRNA-433抑制环路、改善DMF。项目的完成将提出DMF发生新机制及DMF防治新靶点，并为Pir临床应用提供新依据。

本研究以心肌成纤维细胞（CF，体外实验）和db/db小鼠（体内实验）为研究体系，以miRNA-433、TGF-β1/Smad3通路以及ODC/多胺通路为研究目标，观察相关基因及蛋白在高糖诱导CF转化、增殖及胶原合成过程中以及db/db小鼠糖尿病心肌纤维化（DMF）发生过程中的变化。同时观察吡非尼酮对上述病理过程的改善及对相关蛋白表达的影响。体外研究发现，高糖处理CF后细胞增殖活性增加、CF向肌成纤维细胞转化能力增强，胶原及I、III型胶原合成增加；TGF-β1、Smad3、miRNA-433表达增加；同时，AZI、ODC 表达减少，多胺含量减少。与高糖处理CF相比，Ly364947、anti-miRNA-433、AZI过表达质粒转染及吡非尼酮预处理能够抑制细胞增殖，抑制CF向肌成纤维细胞转化、抑制胶原及I、III型胶原合成；同时，TGF-β1、Smad3和miRNA-433表达减少，AZI、ODC表达及多胺含量增加。研究结果表明“TGF-β1/Smad3通路-miRNA-433-ODC/多胺通路” 反馈环路在高糖刺激CF损伤过程中发挥重要作用。吡非尼酮下调miRNA-433、抑制上述环路发挥高糖刺激的CF保护作用。体内研究发现，db/db小鼠DMF发生过程中，伴有心功能障碍及心肌纤维化病变；心肌组织TGF-β1、Smad3及miRNA-433表达增加，AZI、ODC蛋白表达减少，多胺合成减少。与DMF组相比，TGF-β1抑制剂Ly364947、miRNA-433 shRNAd转染、AZI过表达质粒转染及吡非尼酮给予db/db小鼠可改善心功能及心肌纤维化病变；同时，心肌组织TGF-β1、Smad3、miRNA-433表达减少，AZI、ODC表达增加，多胺合成增加。研究表明“TGF-β1/Smad3通路-miRNA-433-ODC/多胺通路”反馈环路介导了DMF病理过程，吡非尼酮下调miRNA-433、抑制上述环路、改善DMF。本项目的完成不仅提出DMF发生新机制、为DMF防治提供新靶点（miRNA-433），还揭示了吡非尼酮抗纤维化作用新机制，为其临床应用提供新的理论依据。