抑制聚二磷酸腺苷核糖聚合酶1对血管紧张素II诱导的ApoE-/-小鼠腹主动脉瘤的保护作用及机制研究

Abdominal aortic aneurysm (AAA) is a chronic vascular degenerative disease associated with a high mortality among elderly people in the world. It is characterized by the deterioration of the aortic wall leading to progressive segmental dilation with a lethal risk of aortic rupture. Unfortunately, the pathogenesis of AAA remains poorly understood and the therapeutic approach is limited to surgical repair. However, surgery is not suitable for asymptomatic patients or those with a small AAA or contraindications for surgery. Thus, a safe and effective pharmacological treatment is in demond urgently for the prevention of AAA formation and progression..Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme which takes part in the repair of DAN damage. Excessive activation of this en zyme results in the intracellular depletion both of its substrate NAD and of the precursor ATP, thereby causing a cellular energy crisis and irreversible cytotoxicity and cell death. PARP has also been suggested to regulate the expression of a variety of key inflammatory genes including MCP-1, inducible nitric oxide synthase, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, all of which are regulated by nuclear factor-κB (NF-κB). It has been demonstrated that PARP-1 plays a critical role in the pathogenesis of endothelial dysfunction, atherosclerosis and diabetes..However, in the present, there is little known about the role and mechanism of PARP-1 in angiotensin II (AngII) induced abdominal aortic aneurysm (AAA), which needs further investigation. Thus, we assumed that PARP-1 inhibition played protective effect on AngII induced AAA. To validate the assumption and investigate the underlying mechanism, we designed in vivo and vitro experiments. In vitro, after PARP-1 was inhibited by siRNA, aortic endothelial cells, smooth muscle cells, and macrophages were stimulated by AngII; in vivo, after PARP-1 was inhibited by gene knockout or inhibitor TIQ-A, ApoE gene knockout mice were pumped AngII and fed high fat diet, then real-time PCR, western blot, immunofluorescence, immunohistochemistry, TUNEL, DHE and comet assay were used to observe the effect of PARP-1 on AngII induced AAA and investigate the mechanism in the aspects of molecule, cells, tissue and animal. Our experiments will shed light on the pathogenesis of AAA and provide new approach for the treatment.

聚二磷酸腺苷核糖聚合酶1(PARP-1)是一种DNA损伤修复酶，在动脉粥样硬化、糖尿病等发挥重要的作用。迄今关于PARP-1对血管紧张素II（AngII）诱导的腹主动脉瘤（AAA）形成的作用及机制还知之甚少，我们预实验发现动脉瘤部位PARP-1表达和活性升高。为此我们提出假设：抑制PARP-1对AngII诱导的ApoE-/-小鼠腹主动脉瘤的形成具有保护作用。体外实验应用PARP-1 siRNA后，应用AngII刺激主动脉内皮细胞、平滑肌细胞和巨噬细胞，体内实验高脂喂养的ApoE-/-小鼠泵入AngII，应用PARP-1敲除或其抑制剂TIQ-A，然后采用RT-PCR、western blot、免疫荧光、免疫组化等方法，从分子、细胞、组织及动物整体等观察PARP-1在AAA形成中的作用并阐明可能的机制。本研究将从PARP-1这个新视点为揭示AAA的发生机制奠定基础，为AAA的防治提供新思路。

背景：腹主动脉瘤（AAA）是一种常见的血管退行性变，经常导致老龄患者的猝死。聚二磷酸腺苷核糖聚合酶1（PARP-1）是一种核蛋白，能够被损伤的DNA激活并在各种疾病中发挥重要的作用。我们假设PARP-1在AAA的发生中发挥重要的作用，抑制PARP-1能够减少AAA的发生。.材料与方法：在小鼠体内，血管紧张素II（Ang II）通过微泵持续泵入；在体外，利用Ang II (1μM)刺激人主动脉内皮细胞和人血管平滑肌细胞24小时；.结果：在 ApoE-/-小鼠中，Ang II持续泵入能够增加PARP-1的表达和活性，并成功诱导AAA的发生；基因敲除PARP-1能够减少AAA的发生率，腹主动脉内径，巨噬细胞的趋化，细胞间粘附分子1和血管粘附分子1的表达，以及基质金属蛋白酶的表达，但是增加了AAA中平滑肌细胞的表达；在主动脉内皮细胞中，Ang II诱导了氧化应激和DNA损伤，从而增加了PARP-1的表达和活性；与正常对照组相比，Ang II增加了TNF-ɑ和IL-6的分泌、THP-1粘附细胞的数量以及细胞间粘附分子1的表达，而利用siRNA抑制PARP-1通过ERK、AKT和NF-κB通路减少了这些炎症反应。在人血管平滑肌细胞中，Ang II刺激减少了胶原合成和增加了基质金属蛋白酶的表达，而抑制PARP-1通过NF-κB/MMP信号通路增加了胶原合成并减少了基质金属蛋白酶的表达。.结论：我们的研究揭示了PARP-1在AAA发生中的重要作用，提示了抑制PARP-1是治疗AAA的潜在靶点。