NonO在动脉粥样硬化斑块易损性中的作用及机制研究

Atherosclerotic plaque vulnerable to rupture is the major cause of acute coronary syndrome (ACS), which is identified by a thin, weakened fibrous cap, a large lipid core, accumulation of inflammatory cells and the imbalance between extracellular matrix (ECM) synthesis and degradation. Among them, collagen metabolism and inflammatory response are the main factors of plaque vulnerability..NonO, originally identified as a non-POU-domain-containing, octamer-binding protein, is 55 kDa ubiquitously expressed protein which contains two kinds of nucleic acid binding domains which could bind to both DNA and RNA. NonO could regulate the expression of many genes not only through directly binding to DNA as a transcriptional factor but also through binding to other protein as a cofactor. So, NonO could regulate many genes through different ways, among which some genes are in close relationship with atherosclerosis. NonO acts as a component of the cAMP-signaling pathway and is necessary for the activation of cAMP response element binding protein (CREB) target genes which contains TNF-α, IL-2 and IL-6, COX-2 and so on, suggesting that NonO may participate in promoting inflammation. NonO can bind to the auxiliary upstream sequence elements of cyclooxygenase-2 (COX-2) and regulate its expression which also participate in the progression of AS. So, these studies suggests that NonO may take part in inflammation and atherosclerosis. Especially in recent years, Zhang C et al found that in HASMCs, NonO silencing dramatically attenuated TNF-α suppression of P4Hα1 gene expression and collagen synthesis. Ren Z et al found that aortic P54(nrb) /NonO protein and mRNA were decreased in aortic dissection (AD) patients, compared with controls. Decreased P54(nrb) /NonO mRNA correlated significantly with increased collagen deposition and fibrosis in AD aortas, suggesting the effect of NonO in collagen metabolism in vivo. However, the exact role of NonO in vulnerable plaque is unclear. The present study was undertaken to test the hypothesis that NonO may play an important role in the formation of vulnerable plaque and gene silencing of NonO could increase the stability of plaque. In addition, the molecular mechanisms of NonO in plaque vulnerability were also explored. A series of in vitro and in vivo experiments were performed to validate this hypothesis. NonO may provided a new intervention target for the AS plaque stabilization.

为了明确NonO在动脉粥样硬化(AS)斑块易损性中的作用及其分子机制，本课题首先在ApoE敲基因小鼠中通过颈动脉套管+高脂喂养的方法构建易损斑块模型，转染NonO过表达和干扰的慢病毒载体，再行精神应激4周，从正反两方面观察NonO对斑块破裂率及易损指数的影响，并通过激光共聚焦、免疫组化、实时定量RT-PCR、Western blot等技术检测颈动脉斑块内胶原含量、炎症反应、细胞凋亡及氧化应激水平。体外实验部分血管平滑肌细胞或巨噬细胞转染NonO过表达和干扰的慢病毒载体后给予TNF-α刺激，观察NonO对胶原合成和降解、炎症反应、细胞凋亡、氧化应激以及相关信号通路的影响，明确NonO对细胞的作用及其机制，从而为稳定AS斑块提供新的干预靶点。

NonO 是一个大小为 54KDa 并在体内广泛表达的蛋白，NonO可直接与 DNA 结合参与转录调控，也可与其他转录蛋白相互作用调节基因表达。既往研究提示 NonO 可能参与调节胶原代谢和炎症反应，但是NonO在动脉粥样硬化斑块稳定性中的作用尚未明确。我们预实验发现ApoE-/-小鼠易损斑块模型中，颈动脉斑块内NonO的表达与Control组正常小鼠的颈动脉组织相比显著升高，强烈提示NonO在AS易损斑块的形成中发挥作用。因此，我们提出如下假说： NonO 通过促进炎症反应，抑制胶原合成进而增加 AS 斑块的易损性。为了验证这一假说，我们设计了一系列的体内体外实验。在体内实验中，我们通过颈动脉套管，高脂饮食和精神应激的方法诱导不稳定斑块的形成，然后通过在颈动脉斑块局部过表达和基因沉默 NonO 观察 NonO 对易损斑块破裂率的影响以及斑块内成分的改变；在体外实验中，通过过表达和基因沉默 NonO 探讨 NonO 作用的分子机制。我们首次发现 NonO 在动脉粥样硬化斑块中高表达；在 ApoE-/-小鼠易损斑块模型中， NonO 降低斑块稳定性，增加斑块的破裂率，而 NonO 基因沉默后斑块稳定性增强，破裂率降低；NonO 降低斑块稳定性的机制为增加 MMP-2 和 MMP-9 的生成，减少P4Hα1 的表达以及抑制 NF-κB 介导的局部炎症反应。这一发现为干预动脉粥样硬化进展提供了新的治疗靶点。