Periostin介导的巨噬细胞极化对PDGFs/PDGFRs信号通路参与心肌纤维化的调控及分子机制研究

Cardiac fibrosis is a common pathological manifestation of end-stage cardiovascular disease. Inhibition or reversal of myocardial fibrosis is helpful to improve the prognosis of cardiovascular diseases such as chronic heart failure.The mechanism of the occurrence and development of myocardial fibrosis is complex and the molecular mechanism is still unclear. Recent studies by our research group have shown that cardiac fibrosis is associated with over-activation of PDGFs/PDGFRs signal pathway on cardiac fibroblasts. Preliminary experiments have found that activation of PDGFs can promote the expression of periostin. However, how PDGFs/PDGFRs signal pathway regulate expression of periostin, as well as how Periostin induces cardiomyocyte fibroblast proliferation and transformation remains further research. Our study assumes that PDGFs regulate expression of periostin through intracellular PI3K/AKT signal pathway. Periostin mediates the polarization and aggregation of macrophages to M2 type. M2 macrophages release PDGFs and other factors, then induces fibroblasts proliferation and conversion into myofibroblasts through PDGFs/PDGFRs signal pathway. Moreover, extracellular matrix deposition is greater than degradation, which forms a vicious circle and eventually leads to myocardial fibrosis. On the basis of our primary studies, further research methods including of fibrosis model, gene knockout, cell coculture, plasmid construction, RT-PCR, Western blot, FCM, Edu proliferation assay and other means will be done to lucubrate the regulation and molecular mechanism of how periostin mediates macrophage polarization on the PDGFs/PDGFRs signal pathway involved in myocardial fibrosis.

心肌纤维化（MF）是多种心血管病终末期共同的病理表现，抑制MF有利于改善心衰等心血管病的预后。有关MF发生的机制复杂，分子机制尚不清楚。本课题组近年来的研究发现MF与心肌成纤维细胞表面PDGFs信号通路的过度激活有关，预实验发现PDGFs激活可促进Periostin表达，但如何调控Periostin表达及Periostin如何诱导心肌成纤维细胞增殖转化，均有待进一步明确。本项目假设PDGFs通过PI3K/AKT信号通路调控Periostin表达，Periostin介导巨噬细胞向M2型极化聚集，M2型巨噬细胞释放PDGFs等，通过PDGFs信号通路诱导成纤维细胞增殖转化，ECM沉积，形成恶性循环，导致MF。本项目建立MF模型并结合基因敲除、细胞共培养、质粒构建、RT-PCR、免疫印迹、FCM、Edu增殖检测等研究Periostin介导巨噬细胞极化对PDGFs信号通路参与MF的调控及分子机制。

心肌纤维化是多种心脏疾病，如冠心病、高血压性心脏病、心肌病等最终期共同的病理表现，它是心肌组织的间质中成纤维细胞过量增殖和心肌细胞外基质沉积与降解失衡的病理过程。心肌纤维化会引发心脏的收缩和舒张功能下降最终导致心力衰竭的发展加剧。心肌纤维化的原因复杂，其发生、发展的机制尚不清楚，是一个亟需解决、更需要深入研究的临床问题。本研究在前期研究基础上进一步探究了泛素羧基末端水解酶L1（UCH-L1）调节小鼠心肌纤维化中的分子机制。通过Ang II诱导建立小鼠心肌纤维化模型，应用UCHL1特异性抑制剂LDN57444处理小鼠，检测小鼠血压及心脏功能和结构变化，天狼猩红染色检测小鼠心肌纤维化程度。结果显示，与对照组相比，Ang II 灌注2周后小鼠收缩和平均动脉血压明显升高，心脏机构功能发生改变心脏顺应性降低，应用LDN57444 处理后小鼠收缩压没有明显降低，但是可明显抑制Ang II诱导的小鼠心功能降低，同时小鼠心肌纤维化程度明显降低。体外实验表明，与对照组相比，P组（细胞+培养基+20ng.ml-1 PDGF-DD）UCH-L1的mRNA和PDGFRβ/p-PDGFRβ、pPI3K/PI3K、UCH-L1、pRB/RB、ColⅠ的蛋白表达水平均显著升高。PL 组（细胞+培养基+20ng.ml-1 PDGF-DD +40uM LDN57444）中I型胶原蛋白的mRNA以及PDGFRβ/p-PDGFRβ、pPI3K/PI3K、Col I、pRB/RB 的蛋白表达量显著低于P组。染色质免疫共沉淀检测显示NF-κB蛋白调控下游UCH-L1基因转录。上诉结果表明，UCHL1抑制剂LDN57444处理后可改善Ang II诱导的小鼠心功能下降和心肌纤维化程度。PDGF-DD可以诱导心肌成纤维细胞增殖从而促进间质胶原蛋白合成，促进心肌纤维化。此外，PDGF-DD可能通过PDGFRβ/PI3K/AKT 信号通路影响心肌纤维化。PDGF-DD可刺激UCH-L1的mRNA和蛋白质表达。UCH-L1参与调控小鼠的心脏成纤维细胞增殖，机制可能是通过PI3K、AKT和NF-κB信号轴通路调节 UCH-L1表达，UCH-L1调控下游RB蛋白的表达，通过调控细胞周期，达到调控心肌成纤维细胞增殖作用。本项目有望揭示一种新的心肌纤维化分子机制，并为其的防治提供新的参考思路。