心肌梗死后心脏损伤重构过程中Wnt3a表达调控及其效应的研究

Wnt3a is found to play a detrimental role in cardiac injury and remodeling, especially in myocardial infarction. However, the expression mechanism of Wnt3a gene during cardiac injury and remodeling remains obscure. In our initial study, we found there exist two evolutionally conserved sites for the binding of STAT and Smad transcription factors on promoter of Wnt3a, respectively, HMGB1-JAK/STAT and TGF-β/Smad signaling pathways plays vital role in myocardial apoptosis and fibrosis in cardiac remodeling after infarction. Therefore, we hypothesize that JAK/STAT and TGF-β/Smad signaling pathways may exert crucial effects in the expression regulation of Wnt3a under different pathological circumstances. Probably, JAK/STAT pathway in response to direct damage and inflammatory stimuli affects the early-stage induction of Wnt3a. While the TGF-β/Smad pathway may activate the expression of Wnt3a during cardiac remodeling and fibrosis. In this study, we will establish the cellular and rat myocardial infarction to ascertain our scientific hypothesis. We will use different methods including luciferase assay, EMSA, ChIP, gain-of-function, and RNAi, to demonstrate the effect of JAK/STAT and TGF-β/Smad pathways on Wnt3a expression. And we will further test the temporal pattern of wnt3a expression and the association with the two signaling pathways in the rat following myocardial infarction. Furthermore, we will investigate the therapeutic effectiveness of disturbing Wnt3a expression via manipulating JAK/STAT and TGF-β/Smad pathways. Collectively, this study will focus on the mechanism of Wnt3a expression in cardiac pathophysiology, and offer theoretical and experimental evidence for the treatment targeting Wnt3a expression or function.

Wnt3a在心脏病理性损伤重构中发挥了重要作用，但其在心梗后心脏损伤重构中的活化机制尚不明确。前期研究发现Wnt3a基因启动子有2个进化保守的STAT和Smad转录因子结合位点，而HMGB1-JAK/STAT及TGF-β/Smad分别在急性损伤期与修复期发挥着重要作用，因此推测JAK/STAT及TGF-β/Smad信号通路在调控Wnt3a基因表达中发挥了重要作用，可分别介导早期损伤刺激和后期纤维修复刺激对Wnt3a的诱生，以调控心肌细胞和成纤维细胞的凋亡和增殖。本课题将在细胞水平及大鼠心梗模型中验证Wnt3a在心脏病理损伤重构中的表达时序及其效应，及其与JAK/STAT、TGF-β/Smad信号通路的相关性，探讨封闭HMGB1-JAK/STAT、TGF-/Smad信号通路以干扰Wnt3a病理效应的潜在治疗价值，为以Wnt3a为靶点的心脏保护和重构逆转提供实验及理论依据。

Wnt3a在心脏损伤重构特别是纤维化重构中发挥了重要作用，但其在心脏损伤及病理重构中的活化机制尚未得到阐明。本项目阐述了缺氧和TGF-1刺激条件下，Wnt3a的上调机制。我们发现在乳鼠心肌细胞中，缺氧刺激诱导Wnt3a上调，机制研究发现缺氧诱导转录因子HIF-1α结合于Wnt3a启动子，而Wnt3a上调降低了乳鼠心肌细胞存活，上调了caspase-3表达和PARP-1蛋白剪切，细胞色素C释放。而且Wnt3a上调抑制了Sirt3表达，干扰了心肌细胞的线粒体稳定。我们还发现TGF-诱导Wnt3a上调，介导c-Myc等增殖和肥大基因表达，诱导心肌肥大。TGF-1经由ALK5-Smad2/3通路诱导Wnt3a表达，导致Wnt/β-catenin通路激活，促进胞浆β-catenin稳定并入核，发挥转录激活功能。β-catenin诱导c-Myc上调，促进心肌细胞肥大。因此，Wnt3a抑制可拮抗TGF-1的促心肌肥大效应。总之，本课题阐明Wnt3a病理性表达的调控模式，为以Wnt3a为靶点的心脏保护和重构逆转提供实验及理论依据。